U.S. patent application number 13/377498 was filed with the patent office on 2012-06-14 for compositions and methods for the prevention and treatment of hypertension.
This patent application is currently assigned to Generex Pharmaceuticals, Inc.. Invention is credited to Ming Li, Peng Peng.
Application Number | 20120148691 13/377498 |
Document ID | / |
Family ID | 43308481 |
Filed Date | 2012-06-14 |
United States Patent
Application |
20120148691 |
Kind Code |
A1 |
Li; Ming ; et al. |
June 14, 2012 |
COMPOSITIONS AND METHODS FOR THE PREVENTION AND TREATMENT OF
HYPERTENSION
Abstract
Disclosed herein are compounds, extracts, and active fractions
of the plant Geum japonicum and methods for preventing or treating
hypertension. The compounds provided herein can be formulated into
pharmaceutical compositions and medicaments that are useful in the
disclosed methods. Also provided are the use of the compounds and
extracts in preparing pharmaceutical formulations and
medicaments.
Inventors: |
Li; Ming; (New Territories,
HK) ; Peng; Peng; (Hunan, CN) |
Assignee: |
Generex Pharmaceuticals,
Inc.
|
Family ID: |
43308481 |
Appl. No.: |
13/377498 |
Filed: |
June 11, 2010 |
PCT Filed: |
June 11, 2010 |
PCT NO: |
PCT/IB10/01412 |
371 Date: |
February 23, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61186709 |
Jun 12, 2009 |
|
|
|
61187905 |
Jun 17, 2009 |
|
|
|
Current U.S.
Class: |
424/765 |
Current CPC
Class: |
A61K 36/23 20130101;
A61P 9/00 20180101; A61P 29/00 20180101; A61P 3/10 20180101; A61P
25/16 20180101; A61K 31/7028 20130101; A61P 9/12 20180101; A61P
27/02 20180101; A61K 36/73 20130101; A61K 31/7032 20130101; A61P
9/04 20180101; A61P 7/02 20180101; A61P 9/10 20180101; A61K 31/56
20130101; A61K 31/7034 20130101; A61P 25/28 20180101; A61P 33/06
20180101; A61P 17/02 20180101; A61K 2300/00 20130101; Y02A 50/411
20180101; A61P 43/00 20180101; A61K 31/565 20130101; A61P 7/00
20180101; Y02A 50/30 20180101; A61K 36/00 20130101; A61P 3/00
20180101; A61P 9/06 20180101; A61P 25/00 20180101; A61K 31/56
20130101; A61K 2300/00 20130101; A61K 31/565 20130101; A61K 2300/00
20130101; A61K 31/7028 20130101; A61K 2300/00 20130101; A61K
31/7032 20130101; A61K 2300/00 20130101; A61K 31/7034 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/765 |
International
Class: |
A61K 36/73 20060101
A61K036/73; A61P 9/12 20060101 A61P009/12 |
Claims
1. A method for treating or preventing hypertension in mammalian
subjects, the method comprising administering to a subject in need
thereof an effective amount of an organic extract of Geum japonicum
(OEGJ).
2. The method of claim 1, wherein the peripheral resistance of
small arteries is systemically decreased in a subject administered
the OEGJ compared to a subject not administered the OEGJ.
3. The method of claim 2, wherein the peripheral resistance of
small arteries is decreased by collateral vessel formation in
organs or tissues.
4. The method of claim 1, wherein OEGJ is administered in an amount
ranging from about 0.01 mg to about 10,000 mg of the extract per
kilogram of body weight per day.
5. The method of claim 1, wherein OEGJ is administered in a dosage
unit form.
6. The method of claim 1, wherein OEGJ is administered in a dosage
unit form comprising a pharmaceutically acceptable carrier.
7. The method of claim 1, wherein OEGJ stimulates collateral vessel
formation in organs or tissues with increased resistance of small
arteries.
8. The method of claim 1, wherein the subject's blood pressure is
reduced compared to a subject not administered with the OEGJ.
9. The method of claim 1, wherein OEGJ is administered orally.
10. The method of claim 1, wherein OEGJ is administered by
subcutaneous injection, intramuscular injection, or intravenous
infusion.
11. The method of claim 1, wherein the OEGJ is a lower alkyl
alcohol solvent extract of Geum japonicum.
12. The method of claim 11, wherein lower alkyl alcohol has 1-6
carbons atoms.
13. The method of claim 11, wherein the solvent is ethanol.
14. The method of claim 11, wherein the solvent is methanol.
15. The method of claim 1, wherein said mammalian subject is a
human.
16. A pharmaceutical composition for treating or preventing
hypertension in mammalian subjects, comprising an effective amount
of an organic extract of Geum japonicum (OEGJ) and a
pharmaceutically acceptable excipient.
17. A kit comprising the pharmaceutical composition of claim 16, a
container and instructions indicating that the pharmaceutical
composition is beneficial to a human suffering from hypertension or
high blood pressure.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/186,709, filed Jun. 12, 2009, and U.S.
Provisional Application No. 61/187,905, filed Jun. 17, 2009, the
entire contents of which are hereby incorporated by reference in
their entirety.
BACKGROUND
[0002] The following description is provided to assist the
understanding of the reader. None of the information provided or
references cited is admitted to be prior art to the present
invention.
[0003] The American Heart Association estimates high blood pressure
affects approximately one in three adults in the United States--73
million people. According to the report of Journal of the American
Medical Association, high blood pressure is also estimated to
affect about two million American teens and children. Hypertension
is clearly a major public health problem. Uncomplicated high blood
pressure usually occurs without any symptoms and so hypertension
has been labeled "the top silent killer." Hypertension can progress
silently to finally develop any one or more of the several
potentially fatal complications, such as heart attack or stroke,
which are the leading causes of death among Western countries and
now many Asian countries. Hypertension is already a highly
prevalent cardiovascular risk factor worldwide because of
increasing longevity and prevalence of contributing factors, such
as obesity.
[0004] Despite the rapid advances of therapeutic technologies
today, the currently available drugs and treatment modalities, such
as vessel dilators, .beta.-blockers, diuretics, etc., for
hypertension can only provide modest control of blood pressure
under continuous medication and symptomatic improvement, and none
of the drugs or therapeutic strategies is considered to have
disease-modifying effects that can provide a curative effect on
hypertension. They can only relieve the symptoms or slow down the
pace of getting worse. Hypertension remains incurable and
inadequately managed everywhere.
SUMMARY
[0005] In one aspect, the present invention provides a method for
treating or preventing hypertension in mammalian subjects, the
method comprising administering to a subject in need thereof an
effective amount of an organic extract of Geum japonicum (OEGJ). In
one embodiment, the peripheral resistance of small arteries is
systemically decreased in a subject administered the OEGJ compared
to a subject not administered the OEGJ. In one embodiment, the
peripheral resistance of small arteries is decreased by collateral
vessel formation in organs or tissues. In one embodiment, OEGJ
stimulates collateral vessel formation in organs or tissues with
increased resistance of small arteries so that the peripheral
resistance will be decreased. In one embodiment, the subject's
blood pressure is reduced compared to a subject not administered
with the OEGJ. In one embodiment, the mammalian subject is a
human.
[0006] In one embodiment, OEGJ is administered in an amount ranging
from about 0.01 mg to about 10 g of the extract per kilogram of
body weight per day. In one embodiment, OEGJ is administered in a
dosage unit form. In one embodiment, OEGJ is administered in a
dosage unit form comprising a pharmaceutically acceptable carrier.
In one embodiment, OEGJ is administered orally. In one embodiment,
OEGJ is administered by subcutaneous injection, intramuscular
injection, or intravenous infusion.
[0007] In one embodiment, the OEGJ is a lower alkyl alcohol solvent
extract of Geum japonicum. In one embodiment, the lower alkyl
alcohol has 1-6 carbons atoms. In one embodiment, the lower alkyl
alcohol is ethanol. In one embodiment, the lower alkyl alcohol is
methanol.
[0008] In another aspect, the present invention provides a
pharmaceutical composition for treating or preventing hypertension
in mammalian subjects, comprising an effective amount of an organic
extract of Geum japonicum (OEGJ) and a pharmaceutically acceptable
carrier.
[0009] In another aspect, the present invention provides a kit
comprising an effective amount of an organic extract of Geum
japonicum (OEGJ) and a pharmaceutically acceptable carrier, a
container and instructions indicating that the pharmaceutical
composition is beneficial to a human suffering from hypertension or
high blood pressure.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1 is micrograph showing OEGJ-induced differentiation of
vessel endothelial cells (HUVEC). Panel (a): The vehicle treated
cells show proliferation and no sign of differentiation. Panel (b):
OEGJ (30 .mu.g/ml) treatment enhanced proliferation of the cells
(30-50% more cells according to MTT assay). Panel (c): OEGJ (60
.mu.g/ml) treated cells show elongated and refractive cells, which
are characteristic of differentiation of vessel endothelial cells.
Panel (d): OEGJ (180 .mu.g/ml) treated cells display thin and
elongated phenotype, forming tube-like structures.
[0011] FIG. 2 presents data showing that OEGJ treatment decreased
the blood pressure in a 2VO rat model. Panel (a): The blood
pressure was elevated (154.25.+-.3.95 mmHg) after 2VO ligation
measured 6 weeks post-2VO ligation in vehicle treated rats. Panel
(b): By contrast, the elevated blood pressure due to 2VO was
reduced (135.+-.2.14 mmHg) measured 6 weeks post-ligation in
OEGJ-treated rats.
[0012] FIG. 3 shows ultrasound Doppler evaluation of the reduced
peripheral resistance of blood vessels in 2VO brain 2 weeks after
termination of OEGJ treatment. The blood flow volume was evaluated
by measuring basal artery of the experimental animals. Frequency is
plotted vertically and time horizontally. Each signal corresponds
to one cardiac cycle. A, Real time two dimensional image showing
the basal artery in longitudinal section (upper panel) and Doppler
shift signals recorded from basal artery (lower panel) in vehicle
treated animals. B, Real time two dimensional image showing the
basal artery in longitudinal section (upper panel) and Doppler
shift signals recorded from basal artery (lower panel) in OEGJ
treated animals. Although OEGJ treated animals had lower blood
pressure (128 mmHg) compared with the significantly elevated blood
pressure (148 mmHg) in vehicle treated, the cerebral blood flow
volume was significantly higher (21.1 ml/min) in OEGJ treated group
than that (14.6 ml/min) in vehicle treated. Based on the formula
blood pressure (BP)/blood flow volume (BF)=blood vessel peripheral
resistance, almost no difference of the peripheral resistance was
observed between normal and control group, however, the resistance
of OEGJ treated group is 43% lower, an indication of growth of new
collateral arteries that increased the sectional area of peripheral
arteries in brain by 37.4% obtained from vessel counting and
brought about 30.8% more blood supply to the brain compared to the
vehicle-treated 2VO animals.
[0013] FIG. 4 is a representative image of the cortex of the
frontal lobe in OEGJ-treated 2VO rats showing significantly more
vessels (.about.61.7.+-.20.3/HPF) compared with that
(38.5.+-.12.6/HPF) of vehicle treated control 2VO rats (C).
[0014] FIG. 5 is a graph showing blood pressure and cerebral blood
flow in a 2VO animal model. BP, denotes blood pressure. Basal CBFV,
denotes the cerebral blood flow volume through basal artery. Nor,
Normal control rats. Mod, Vehicle treated 2VO rats. OEGJ, OEGJ
treated 2VO rats.
[0015] FIG. 6 is a graph showing blood pressure and cerebral blood
flow in partial ligation (60%) of the right carotid artery in SD
rats. (BP) blood pressure; (CBFV) the total cerebral blood flow
volume; (Ctr) vehicle treated model rats; (Treated) OEGJ treated
model rats. OEGJ treatment significantly increased cerebral blood
flow through basal artery while the normal blood pressure was
maintained. In comparison, the cerebral blood flow through basal
artery in vehicle treated rats is significantly lower than that in
OEGJ treated, while the blood pressure was elevated.
[0016] FIG. 7 is a graph showing blood pressure in APP mice. (OEGJ)
OEGJ treated APP mice; (Ctr) vehicle treated APP mice. OEGJ
treatment decreased the blood pressure by about 10% to a normal
level in OEGJ treated APP mice compared with that in vehicle
treated APP control mice.
DETAILED DESCRIPTION
[0017] In various aspects, the present invention provides
compounds, extracts, and methods for preventing or treating
hypertension. The compounds provided herein can be formulated into
pharmaceutical compositions and medicaments that are useful in the
disclosed methods. Also provided are the use of the compounds and
extracts in preparing pharmaceutical formulations and
medicaments.
[0018] It is to be appreciated that certain aspects, modes,
embodiments, variations and features of the invention are described
below in various levels of detail in order to provide a substantial
understanding of the present invention. The following terms are
used throughout as described below, unless context clearly
indicates otherwise.
[0019] As used herein, the "administration" of an agent or drug to
a subject or subject includes any route of introducing or
delivering to a subject a compound to perform its intended
function. Administration can be carried out by any suitable route,
including orally, intranasally, parenterally (intravenously,
intramuscularly, intraperitoneally, or subcutaneously), rectally,
or topically. Administration includes self-administration and the
administration by another.
[0020] As used herein, the term "effective amount" or
"pharmaceutically effective amount" or "therapeutically effective
amount" of a composition, is a quantity sufficient to achieve a
desired therapeutic and/or prophylactic effect, e.g., an amount
which results in the prevention of, or a decrease in, the symptoms
associated with a disease that is being treated. The amount of a
composition administered to the subject will depend on the type and
severity of the disease and on the characteristics of the
individual, such as general health, age, sex, body weight and
tolerance to drugs. It will also depend on the degree, severity and
type of disease. The skilled artisan will be able to determine
appropriate dosages depending on these and other factors. The
compositions of the present invention can also be administered in
combination with one or more additional therapeutic compounds.
[0021] The abbreviation "OEGJ" used in the invention, without
specific indication, means an extract of the plant Geum japonicum
Thunb. var. by an organic solvent described below.
[0022] As used herein, the term "disease" or "medical condition"
are used interchangeably and includes, but is not limited to, any
condition or disease manifested as one or more physical and/or
psychological symptoms for which treatment and/or prevention is
desirable, and includes previously and newly identified diseases
and other disorders. For example, a medical condition may be
hypertension.
[0023] As used herein, the term "subject" includes any mammalian
subject, such as a human, but can also be an animal, e.g., domestic
animals (e.g., dogs, cats and the like), farm animals (e.g., cows,
sheep, pigs, horses and the like) and laboratory animals (e.g.,
monkey, rats, mice, rabbits, guinea pigs and the like).
[0024] As used herein, the terms "treating" or "treatment" or
"alleviation" refers to both therapeutic treatment and prophylactic
or preventative measures, wherein the object is to prevent or slow
down (lessen) the targeted pathologic condition or disorder. A
subject is successfully "treated" for a disorder if, after
receiving a therapeutic agent according to the methods of the
present invention, the subject shows observable and/or measurable
reduction in or absence of one or more signs and symptoms of a
particular disease or condition.
[0025] As used herein, "prevention" or "preventing" of a disorder
or condition refers to a compound that, in a statistical sample,
reduces the occurrence of the disorder or condition in the treated
sample relative to an untreated control sample, or delays the onset
or reduces the severity of one or more symptoms of the disorder or
condition relative to the untreated control sample.
Compositions of the Invention
[0026] The present disclosure provides methods of treating or
preventing hypertension with agents and/or extracts and compounds,
and derivatives of such compounds from a variety of plants
including Geum japonicum. In some embodiments, the agent is an
extract, e.g., an organic extract, of Geum japonicum. In a
particular embodiment, the agent is a methanol or ethanol extract
of Geum japonicum or an active fraction thereof. An agent of the
invention may be part of a pharmaceutical composition containing
one or more excipients, carriers, or fillers. In one embodiment,
the pharmaceutical composition is packaged in unit dosage form. The
unit dosage form is effective in improving various diseases or
medical conditions when administered to a subject in need
thereof.
[0027] A method for preparing an organic extract from Geum
japonicum is provided. This method comprises the step of (a)
extracting the plant of Geum japonicum with alcohol selected from
the group consisting of C1-C4 alcohols. This step maybe repeated
3-6 times, typically 5 times, at room temperature. Before
performing step (a), the plant material may be powdered or cut into
small pieces. The C1-C4 alcohols include methanol, ethanol,
n-propanol, iso-propanol, n-butanol, iso-butanol, and ter-butanol.
Typically, alcohol is added in 1-10 times by weight of the amount
of the Geum japonicum to be extracted.
[0028] The methods may further comprise the step of (b) drying the
extract obtained from the step of (a) into a dried powder; and (c)
successively extracting the powder obtained from the step of (b)
with C6 alkane, EtOAc and an alcohol selected from the group
consisting of C1-C4 alcohols. The C6 alkane includes cyclic and
non-cyclic alkane having 6 carbon atoms,1 including, for example,
cyclohexane, n-hexane, and neo-hexane, etc. The C1-C4 alcohols
include methanol, ethanol, n-propanol, iso-propanol, n-butanol,
iso-butanol, and ter-butanol. The amount of organic solvent to be
used is typically 1-10 times by weight of the amount of the powders
to be further extracted.
[0029] The method as recited above may also include filtering the
extract to remove any insoluble powders therein. A drying step may
be completed under reduced pressure at a temperature higher than
room temperature, for example, at 50.degree. C.
[0030] To purify the OEGJ, the method may further comprise the
steps of applying the powder to a chromatographic column; and
eluting the column with an aqueous solution with increasing
concentration of an alcohol selected from the group consisting of
C1-C4 alcohols. For example, a Sephadex or reverse phase column may
be used. The alcohol used may be any one selected from the group
consisting of methanol, ethanol, n-propanol, iso-propanol,
n-butanol, iso-butanol, and ter-butanol.
[0031] By NMR analysis, it is found that the OEGJ typically
contains mainly tannins including Gemin A, B, C, D, E and F and
triterpenes including 2-hydroxyoleanolic acid, 2-hydroxylursolic
acid, 2,19-dihydroxy-ursolic acid,
2-.alpha.,19-.alpha.-dihydroxy-3-oxo-12-ursen-28-oic acid, ursolic
acid, epimolic acid, maslinic acid, euscaphic acid, tormentic acid,
28-.beta.-D-glucoside of tormentic acid.
[0032] In one embodiment, the extracts, fractions, and compounds of
the invention are obtained by extraction, using water and/or of an
organic solvent, from crude plant material comprises the following
stages: [0033] 1. Extraction by addition to the plant material, of
water and/or of organic solvent(s), by subjecting the whole to a
treatment such as maceration/lixiviation, ultrasonics or
microwaves; [0034] 2. Delipidation before or after the extraction
stage using a solvent of petroleum ether, hexane or chloroform
type; [0035] 3. Optionally, additional extraction of the extract
recovered by an organic solvent of ethyl acetate or ethyl ether
type, [0036] 4. Optionally, concentration of the crude extract
obtained, and, if desired, its lyophilization.
[0037] According one aspect, considering the enrichment that it
allows to be attained, the crude extract may be subjected to a
purification stage by chromatography. In one embodiment,
centrifugal partition chromatography (CPC) is used. This technique
is in particular described by A. P. FOUCAULT, Ed., Centrifugal
Partition Chromatography, Chromatographic Science Series, Marcel
Dekker Inc., 1995, 68, or W. D. CONWAY, Ed., Countercurrent
Chromatography apparatus theory and applications, VCH Publishers
Inc., 1990. CPC is based on the partition of the solutes between
two non-miscible liquid phases prepared by the mixture of two or
more solvents or solutions. One of the two phases is kept
stationary by a centrifugal force. The solvents, their proportions
and the flow rate chosen closely depend both on the stability of
the stationary phase within the CPC column and the actual
pressure.
[0038] A person skilled in the art will therefore choose the most
appropriate solvent or solvents depending on the nature of the
purified extract desired. These different extracts, namely crude or
enriched also fall within the scope of the invention. The
implementation of additional separation stages allows isolation of
these extracts enriched with one or more compounds. These
separations can be carried out on fractions enriched from a crude
extract or on the crude extract itself by using mixtures of
appropriate solvents according to the proportions which are
suitable for the sought separation.
Methods for the Prevention and Treatment of Hypertension
[0039] Systolic blood pressure is the maximum pressure in the
arteries when the heart contracts and pushes blood out into the
body. The diastolic blood pressure is the minimum pressure in the
arteries between beats when the heart relaxes to fill with blood.
Hypertension is defined as an average systolic blood pressure above
140-150 mm Hg, a diastolic blood pressure above 90-95 mm Hg, or
both. An elevation of the systolic and/or diastolic blood pressure
increases the risk of developing heart disease, kidney disease,
hardening of the arteries (atherosclerosis or arteriosclerosis),
eye damage, and stroke (brain damage). These complications of
hypertension are often referred to as end-organ damage because
damage to these organs is the end result of chronic high blood
pressure. For this reason, the diagnosis and early treatment of
high blood pressure is important to normalize blood pressure and
prevent complications.
[0040] Although current advanced treatment strategies can slow down
the progress of the disease, it can at the same time develop severe
complications, for example, ACE inhibitors stop the production of a
hormone called angiotensin II that makes the blood vessels narrow;
Angiotensin-II receptor antagonists work in a similar way as ACE
inhibitors; Beta-blockers block the effect of the hormone
adrenaline and the sympathetic nervous system on the body relaxing
the heart; Alpha-blockers cause the blood vessels to relax and
widen; and calcium-channel blockers reduce muscle tension in the
arteries.
[0041] The nature of blood pressure is the force of blood as it is
pumped through the arteries. The more blood the heart pumps and the
narrower the arteries are, the higher the blood pressure. To
maintain sufficient amount of blood supply to all organs and
tissues to support the necessary requirement of normal activities
of the body, the heart is required to pump certain volume of blood
around the body through the arteries. Therefore, the best solution
to reduce blood pressure without negatively affecting normal
activities of the body is to permanently increase the
cross-sectional area of the total arteries of the whole body.
[0042] The present inventors have discovered an organic extract of
Geum japonicum that can significantly decrease the elevated blood
pressure of subjects after about a two-week treatment with the
extract. In some embodiments, treatment for four to eight weeks
with the extract can permanently increase the cross-sectional area
of the arteries in subjects, which substantially reduces the
resistance of the peripheral arteries. As a result, the blood
pressure decreases without compromising functional performance of
the whole body.
[0043] The present invention is related to the use of an organic
extract of Geum japonicum (OEGJ) and a method of treating
hypertension in humans or animals and diseases associated with
hypertension. Particularly, it relates to a pharmaceutical
composition and method for reducing systemic blood pressure.
Without wishing to be limited by theory, the OEGJ may act by
stimulating the growth of new collateral capillaries, arterioles
and micro-vessels systemically in the subject with increased
peripheral resistance that substantially improves blood perfusion
to important organs and tissues and increases cross-sectional area
of the small blood vessels at different levels. As a result, the
increased peripheral resistance of the small arterioles in
hypertension is rectified due to the compensation of the newly
grown collateral vessels to the narrowed arteries, thereby leading
to a decrease in blood pressure. Therefore, the methods provide a
substantial treatment modality that addresses the underlying
pathological cause of hypertension.
[0044] In accordance with one aspect, the invention provides
methods of treating or preventing hypertension in a subject in need
thereof, which comprises administering to the subject an effective
amount of a compound, composition, fraction, or extract described
herein. In one aspect, the methods for the prevention or treatment
of hypertension include administering to a mammal in need thereof
fractions and/or extracts from a variety of plants including Geum
japonicum. In some embodiments, the extract is an organic extract
obtained from the plant Geum japonicum.
[0045] In another aspect, an agent for the treatment or prevention
of hypertension is part of a pharmaceutical composition containing
one or more excipients, carriers, or fillers. In one embodiment,
the pharmaceutical composition is packaged in unit dosage form. The
unit dosage form is effective in improving (i.e., lowering) blood
pressure in the subject.
[0046] In various embodiments of the invention, suitable in vitro
or in vivo assays are performed to determine the effect of an agent
(extracts, fractions, and compounds) of the invention and whether
its administration is indicated for the treatment or prevention of
hypertension in a subject. In some embodiments, in vivo models of
hypertension are used to assess the effects of an agent on a
subject. The effects of the agent in mediating the hypertension in
the animal subject are investigated and compared to suitable
controls.
[0047] In another embodiment, plants, extracts, active fractions,
and/or compounds of the invention may be administered as part of a
combination therapeutic with another cardiovascular agent. Examples
of cardiovascular agents include vasodilators, for example,
hydralazine; angiotensin converting enzyme inhibitors, for example,
captopril; anti-anginal agents, for example, isosorbide nitrate,
glyceryl trinitrate and pentaerythritol tetranitrate;
anti-arrhythmic agents, for example, quinidine, procainaltide and
lignocaine; cardioglycosides, for example, digoxin and digitoxin;
calcium antagonists, for example, verapamil and nifedipine;
diuretics, such as thiazides and related compounds, for example,
bendrofluazide, chlorothiazide, chlorothalidone,
hydrochlorothiazide and other diuretics, for example, fursemide and
triamterene, and sedatives, for example, nitrazepam, flurazepam and
diazepam.
[0048] Other exemplary cardiovascular agents include, for example,
a cyclooxygenase inhibitor such as aspirin or indomethacin, a
platelet aggregation inhibitor such as clopidogrel, ticlopidene or
aspirin, fibrinogen antagonists or a diuretic such as
chlorothiazide, hydrochlorothiazide, flumethiazide,
hydroflumethiazide, bendroflumethiazide, methylchlorthiazide,
trichloromethiazide, polythiazide or benzthiazide as well as
ethacrynic acid ticrynafen, chlorthalidone, furosemide, muzolimine,
bumetanide, triamterene, amiloride and spironolactone and salts of
such compounds, angiotensin converting enzyme inhibitors such as
captopril, zofenopril, fosinopril, enalapril, ceranopril,
cilazapril, delapril, pentopril, quinapril, ramipril, lisinopril,
and salts of such compounds, angiotensin II antagonists such as
losartan, irbesartan or valsartan, thrombolytic agents such as
tissue plasminogen activator (tPA), recombinant tPA, streptokinase,
urokinase, prourokinase, and anisoylated plasminogen streptokinase
activator complex (APSAC, Eminase, Beecham Laboratories), or animal
salivary gland plasminogen activators, calcium channel blocking
agents such as verapamil, nifedipine or diltiazem, thromboxane
receptor antagonists such as ifetroban, prostacyclin mimetics, or
phosphodiesterase inhibitors. Such combination products if
formulated as a fixed dose employ the compounds of this invention
within the dose range described above and the other
pharmaceutically active agent within its approved dose range.
[0049] Yet other exemplary cardiovascular agents include, for
example, vasodilators, e.g., bencyclane, cinnarizine, citicoline,
cyclandelate, cyclonicate, ebumamonine, phenoxezyl, flunarizine,
ibudilast, ifenprodil, lomerizine, naphlole, nikamate, nosergoline,
nimodipine, papaverine, pentifylline, nofedoline, vincamin,
vinpocetine, vichizyl, pentoxifylline, prostacyclin derivatives
(such as prostaglandin E1 and prostaglandin I2), an endothelin
receptor blocking drug (such as bosentan), diltiazem, nicorandil,
and nitroglycerin. Examples of the cerebral protecting drug include
radical scavengers (such as edaravone, vitamin E, and vitamin C),
glutamate antagonists, AMPA antagonists, kainate antagonists, NMDA
antagonists, GABA agonists, growth factors, opioid antagonists,
phosphatidylcholine precursors, serotonin agonists,
Na.sup.+/Ca.sup.2+ channel inhibitory drugs, and K.sup.+ channel
opening drugs. Examples of the brain metabolic stimulants include
amantadine, tiapride, and gamma-aminobutyric acid. Examples of the
anticoagulant include heparins (such as heparin sodium, heparin
potassium, dalteparin sodium, dalteparin calcium, heparin calcium,
pamaparin sodium, reviparin sodium, and danaparoid sodium),
warfarin, enoxaparin, argatroban, batroxobin, and sodium citrate.
Examples of the antiplatelet drug include ticlopidine
hydrochloride, dipyridamole, cilostazol, ethyl icosapentate,
sarpogrelate hydrochloride, dilazep hydrochloride, trapidil, a
nonsteroidal antiinflammatory agent (such as aspirin),
beraprostsodium, iloprost, and indobufene. Examples of the
thrombolytic drug include urokinase, tissue-type plasminogen
activators (such as alteplase, tisokinase, nateplase, pamiteplase,
monteplase, and rateplase), and nasaruplase. Examples of the
antihypertensive drug include angiotensin converting enzyme
inhibitors (such as captopril, alacepril, lisinopril, imidapril,
quinapril, temocapril, delapril, benazepril, cilazapril,
trandolapril, enalapril, ceronapril, fosinopril, imadapril,
mobertpril, perindopril, ramipril, spirapril, and randolapril),
angiotensin II antagonists (such as losartan, candesartan,
valsartan, eprosartan, and irbesartan), calcium channel blocking
drugs (such as aranidipine, efonidipine, nicardipine, bamidipine,
benidipine, manidipine, cilnidipine, nisoldipine, nitrendipine,
nifedipine, nilvadipine, felodipine, amlodipine, diltiazem,
bepridil, clentiazem, phendilin, galopamil, mibefradil,
prenylamine, semotiadil, terodiline, verapamil, cilnidipine,
elgodipine, isradipine, lacidipine, lercanidipine, nimodipine,
cinnarizine, flunarizine, lidoflazine, lomerizine, bencyclane,
etafenone, and perhexiline), .beta.-adrenaline receptor blocking
drugs (propranolol, pindolol, indenolol, carteolol, bunitrolol,
atenolol, acebutolol, metoprolol, timolol, nipradilol, penbutolol,
nadolol, tilisolol, carvedilol, bisoprolol, betaxolol, celiprolol,
bopindolol, bevantolol, labetalol, alprenolol, amosulalol,
arotinolol, befunolol, bucumolol, bufetolol, buferalol,
buprandolol, butylidine, butofilolol, carazolol, cetamolol,
cloranolol, dilevalol, epanolol, levobunolol, mepindolol,
metipranolol, moprolol, nadoxolol, nevibolol, oxprenolol, practol,
pronetalol, sotalol, sufinalol, talindolol, tertalol, toliprolol,
xybenolol, and esmolol), .alpha.-receptor blocking drugs (such as
amosulalol, prazosin, terazosin, doxazosin, bunazosin, urapidil,
phentolamine, arotinolol, dapiprazole, fenspiride, indoramin,
labetalol, naftopidil, nicergoline, tamsulosin, tolazoline,
trimazosin, and yohimbine), sympathetic nerve inhibitors (such as
clonidine, guanfacine, guanabenz, methyldopa, and reserpine),
hydralazine, todralazine, budralazine, and cadralazine. Examples of
the anti anginal drug include nitrate drugs (such as amyl nitrite,
nitroglycerin, and isosorbide), .beta.-adrenaline receptor blocking
drugs (such as propranolol, pindolol, indenolol, carteolol,
bunitrolol, atenolol, acebutolol, metoprolol, timolol, nipradilol,
penbutolol, nadolol, tilisolol, carvedilol, bisoprolol, betaxolol,
celiprolol, bopindolol, bevantolol, labetalol, alprenolol,
amosulalol, arotinolol, befunolol, bucumolol, bufetolol, buferalol,
buprandolol, butylidine, butofilolol, carazolol, cetamolol,
cloranolol, dilevalol, epanolol, levobunolol, mepindolol,
metipranolol, moprolol, nadoxolol, nevibolol, oxprenolol, practol,
pronetalol, sotalol, sufinalol, talindolol, tertalol, toliprolol,
andxybenolol), calcium channel blocking drugs (such as aranidipine,
efonidipine, nicardipine, bamidipine, benidipine, manidipine,
cilnidipine, nisoldipine, nitrendipine, nifedipine, nilvadipine,
felodipine, amlodipine, diltiazem, bepridil, clentiazem,
phendiline, galopamil, mibefradil, prenylamine, semotiadil,
terodiline, verapamil, cilnidipine, elgodipine, isradipine,
lacidipine, lercanidipine, nimodipine, cinnarizine, flunarizine,
lidoflazine, lomerizine, bencyclane, etafenone, and perhexiline)
trimetazidine, dipyridamole, etafenone, dilazep, trapidil,
nicorandil, enoxaparin, and aspirin. Examples of the diuretic
include thiazide diuretics (such as hydrochlorothiazide,
methyclothiazide, trichlormethiazide, benzylhydrochlorothiazide,
and penflutizide), loop diuretics (such as furosemide, etacrynic
acid, bumetanide, piretanide, azosemide, and torasemide), K.sup.+
sparing diuretics (spironolactone, triamterene, and potassium can
renoate), osmotic diuretics (such as isosorbide, D-mannitol, and
glycerin), nonthiazide diuretics (such as meticrane, tripamide,
chlorthalidone, and mefruside), and acetazolamide. Examples of the
cardiotonic include digitalis formulations (such as digitoxin,
digoxin, methyldigoxin, deslanoside, vesnarinone, lanatoside C, and
proscillaridin), xanthine formulations (such as aminophylline,
choline theophylline, diprophylline, and proxyphylline),
catecholamine formulations (such as dopamine, dobutamine, and
docarpamine), PDE III inhibitors (such as amrinone, olprinone, and
milrinone), denopamine, ubidecarenone, pimobendan, levosimendan,
aminoethylsulfonic acid, vesnarinone, carperitide, and colforsin
daropate. Examples of the antiarrhythmic drug include ajmaline,
pirmenol, procainamide, cibenzoline, disopyramide, quinidine,
aprindine, mexiletine, lidocaine, phenyloin, pilsicainide,
propafenone, flecainide, atenolol, acebutolol, sotalol,
propranolol, metoprolol, pindolol, amiodarone, nifekalant,
diltiazem, bepridil, and verapamil. Examples of the
antihyperlipidemic drug include atorvastatin, simvastatin,
pravastatin sodium, fluvastatin sodium, clinofibrate, clofibrate,
simfibrate, fenofibrate, bezafibrate, colestimide, and
colestyramine.
Formulations and Dosages of Pharmaceutical Compositions.
[0050] Typically, an effective amount of the compositions of the
present invention, sufficient for achieving a therapeutic or
prophylactic effect, range from about 0.000001 mg per kilogram body
weight per day to about 10,000 mg per kilogram body weight per day.
Suitably, the dosage ranges are from about 0.0001 mg per kilogram
body weight per day to about 10000 mg per kilogram body weight per
day. For administration of an agent, the dosage ranges may be from
about 0.0001 to 10000 mg/kg, and more usually 0.1 to 10000 mg/kg
every week, every two weeks or every three weeks, of the host body
weight. An exemplary treatment regime entails administration once
per every two weeks or once a month or once every 3 to 6 months.
The agent usually administered on multiple occasions. Intervals
between single dosages can be daily, weekly, monthly or yearly.
Alternatively, the agents can be administered as a sustained
release formulation, in which case less frequent administration is
required. Dosage and frequency vary depending on the half-life of
the agent in the subject. The dosage and frequency of
administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent
intervals over a long period of time. Some subjects continue to
receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated, and preferably until the subject shows
partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic
regime.
[0051] Toxicity. Suitably, an effective amount (e.g., dose) of an
agent described herein will provide therapeutic benefit without
causing substantial toxicity to the subject. Toxicity of the agent
described herein can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., by
determining the LD.sub.50 (the dose lethal to 50% of the
population) or the LD.sub.100 (the dose lethal to 100% of the
population). The dose ratio between toxic and therapeutic effect is
the therapeutic index. The data obtained from these cell culture
assays and animal studies can be used in formulating a dosage range
that is not toxic for use in human. The dosage of the agent
described herein lies preferably within a range of circulating
concentrations that include the effective dose with little or no
toxicity. The dosage can vary within this range depending upon the
dosage form employed and the route of administration utilized. The
exact formulation, route of administration and dosage can be chosen
by the individual physician in view of the subject's condition.
See, e.g., Fingl et al., In: The Pharmacological Basis of
Therapeutics, Ch. 1 (1975).
[0052] According to one embodiment, the agents can be incorporated
into pharmaceutical compositions suitable for administration. In
some embodiments, the pharmaceutical compositions may comprise
purified or substantially purified extracts of Geum japonicum and a
pharmaceutically-acceptable carrier in a form suitable for
administration to a subject. In other embodiments, the
pharmaceutical compositions may comprise
Pharmaceutically-acceptable carriers are determined in part by the
particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions for administering the compositions (see, e.g.,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa. 18.sup.th ed., 1990). The pharmaceutical compositions are
generally formulated as sterile, substantially isotonic and in full
compliance with all Good Manufacturing Practice (GMP) regulations
of the U.S. Food and Drug Administration.
[0053] The terms "pharmaceutically-acceptable,"
"physiologically-tolerable," and grammatical variations thereof, as
they refer to compositions, carriers, diluents and reagents, are
used interchangeably and represent that the materials are capable
of administration to or upon a subject without the production of
undesirable physiological effects to a degree that would prohibit
administration of the composition. For example,
"pharmaceutically-acceptable excipient" means an excipient that is
useful in preparing a pharmaceutical composition that is generally
safe, non-toxic, and desirable, and includes excipients that are
acceptable for veterinary use as well as for human pharmaceutical
use. Such excipients can be solid, liquid, semisolid, or, in the
case of an aerosol composition, gaseous.
"Pharmaceutically-acceptable salts and esters" means salts and
esters that are pharmaceutically-acceptable and have the desired
pharmacological properties. Such salts include salts that can be
formed where acidic protons present in the agent are capable of
reacting with inorganic or organic bases. Suitable inorganic salts
include those formed with the alkali metals, e.g., sodium and
potassium, magnesium, calcium, and aluminum. Suitable organic salts
include those formed with organic bases such as the amine bases,
e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine,
N-methylglucamine, and the like. Such salts also include acid
addition salts formed with inorganic acids (e.g., hydrochloric and
hydrobromic acids) and organic acids (e.g., acetic acid, citric
acid, maleic acid, and the alkane- and arene-sulfonic acids such as
methanesulfonic acid and benzenesulfonic acid).
Pharmaceutically-acceptable esters include esters formed from
carboxy, sulfonyloxy, and phosphonoxy groups present in the agent,
e.g., C.sub.1-6 alkyl esters. When there are two acidic groups
present, a pharmaceutically-acceptable salt or ester can be a
mono-acid-mono-salt or ester or a di-salt or ester; and similarly
where there are more than two acidic groups present, some or all of
such groups can be salified or esterified. The agent named in this
invention can be present in unsalified or unesterified form, or in
salified and/or esterified form, and the naming of such agent is
intended to include both the original (unsalified and unesterified)
compound and its pharmaceutically-acceptable salts and esters.
Also, certain agents named in this invention can be present in more
than one stereoisomeric form, and the naming of such agent is
intended to include all single stereoisomers and all mixtures
(whether racemic or otherwise) of such stereoisomers. A person of
ordinary skill in the art, would have no difficulty determining the
appropriate timing, sequence and dosages of administration for
particular drugs and compositions of the present invention.
[0054] Examples of such carriers or diluents include, but are not
limited to, water, saline, Ringer's solutions, dextrose solution,
and 5% human serum albumin. Liposomes and non-aqueous vehicles such
as fixed oils may also be used. The use of such media and compounds
for pharmaceutically active substances is well known in the art.
Except insofar as any conventional media or compound is
incompatible with the agent, use thereof in the compositions is
contemplated. Supplementary active compounds can also be
incorporated into the compositions.
[0055] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration. The
compositions of the present invention can be administered by
parenteral, topical, intravenous, oral, subcutaneous,
intraarterial, intradermal, transdermal, rectal, intracranial,
intraperitoneal, intranasal; intramuscular route or as inhalants.
The agent can optionally be administered in combination with other
agents that are at least partly effective in treating various
diseases.
[0056] Solutions or suspensions used for parenteral, intradermal,
or subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial compounds such as benzyl alcohol
or methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating compounds such as ethylenediaminetetraacetic
acid (EDTA); buffers such as acetates, citrates or phosphates, and
compounds for the adjustment of tonicity such as sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as
hydrochloric acid or sodium hydroxide. The parenteral preparation
can be enclosed in ampoules, disposable syringes or multiple dose
vials made of glass or plastic.
[0057] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, e.g.,
water, ethanol, polyol (e.g., glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), and suitable mixtures
thereof. The proper fluidity can be maintained, e.g., by the use of
a coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal compounds, e.g.,
parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the
like. In many cases, it will be preferable to include isotonic
compounds, e.g., sugars, polyalcohols such as manitol, sorbitol,
sodium chloride in the composition. Prolonged absorption of the
injectable compositions can be brought about by including in the
composition a compound which delays absorption, e.g., aluminum
monostearate and gelatin.
[0058] Sterile injectable solutions can be prepared by
incorporating the agents in the required amount in an appropriate
solvent with one or a combination of ingredients enumerated above,
as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the binding agent into a
sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, methods of preparation are vacuum drying and
freeze-drying that yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof. The agents of this invention can
be administered in the form of a depot injection or implant
preparation which can be formulated in such a manner as to permit a
sustained or pulsatile release of the active ingredient.
[0059] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the binding agent can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding compounds, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating compound such
as alginic acid, Primogel, or corn starch; a lubricant such as
magnesium,stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening compound such as sucrose or saccharin; or a
flavoring compound such as peppermint, methyl salicylate, or orange
flavoring.
[0060] In one embodiment, the agents are prepared with carriers
that will protect the agent against rapid elimination from the
body, such as a controlled release formulation, including implants
and microencapsulated delivery systems, or protect the drug from
degraded by the acid of the stomach. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically-acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, e.g., as described in U.S. Pat. No. 4,522,811.
EXAMPLES
[0061] The present technology is further illustrated by the
following examples, which should not be construed as limiting in
any way.
Example 1
Preparation of an Organic Extract of Geum japonicum
[0062] A bio-assay guided strategy was used for screening plant
constituents to identify a composition of compounds showing the
action on reducing peripheral resistance of small arteries through
stimulating the growth of arterioles and micro-vessels. Briefly, 10
kg dried Geum japonicum collected from Anhui Province was cut into
small pieces, which was percolated with 75% ethanol (10.times.
volume) at 40.degree. C. for 3 days. The extract was
electro-sprayed to yield a brown powder.
Example 2
OEGJ-Enhanced Proliferation and Differentiation of Vessel
Endothelial Cells
[0063] Vessel endothelial cell proliferation analysis was conducted
using an MTT assay following the modified ATCC protocol. Human
umbilical vein endothelial cells (HUVECs, 2.times.10.sup.3/well)
were seeded onto a 96-well culture plate with growth medium (F12K
medium with 15% FBS, 6 U/ml heparin and 30 .mu.g/ml endothelial
cell growth supplement). After cell attachment, the medium was
changed to Ham's F12K medium with 2% FBS for 12 hours. Cells were
then treated with OEGJ (prepared as described in Example 1) of
gradient concentrations 50, 100 and 200 .mu.g/ml, respectively, for
48 hours. The optical density (OD) was measured by Tecan Sunrise
plate reader (GmbH, Australia). For evaluation of the phenotype and
differentiation of the cultured endothelial cells, all wells with
different treatments were examined under an inverted microscope
before MTT measurement.
[0064] It was found that OEGJ not only promoted the proliferation
of the HUVECs at low concentration of OEGJ, but also enhanced the
differentiation of HUVECs forming thin, elongated and connected
tube-like structures in culture, indicating its potential in
promoting angiogenesis (FIG. 1).
Example 3
Therapeutic Effects of OEGJ in a Rat Models of Hypertension
[0065] Our preliminary studies suggested that permanent ligation of
bilateral carotid arteries (2VO) will cause mild hypertension due
to the increased peripheral resistance of small arteries in the
brain. After the 2VO, the cerebral blood supply should maximally
rely on bilateral vertebral arteries, which supplies about 40% of
the total blood volume to the brain normally. To compensate for the
reduced 60% of blood supply, which was normally supplied by the
bilateral carotid arteries, the heart needs to build up more
pressure, which has been confirmed by blood pressure measurements
during the experiment, to overcome the resistance of the small
arteries in the brain for delivery of more volume of blood to the
brain through the vertebral arteries.
[0066] To this end, male Sprague-Dawley (SD) rats, weighing 250-300
g were used. The study was conducted in accordance with the
National Regulations of Experimental Animal Administration, and all
animal experiments were approved by the Committee of Experimental
Animal Administration of Zhangjiang High-tech Park. For 2VO (n=12),
both of the common carotid arteries were exposed through a midline
cervical incision under anesthesia and then they were ligated by
6-0 nylon suture, and cut by microscissors. The wound was
thereafter closed with a suture. The rats (n=6) subjected to the
operation of midline cervical incision but without common carotid
artery ligation were taken as sham-operated controls. After
recovering from anesthesia, the experimental animals were allowed
free access to food and water.
[0067] The rats(n=6) of 2VO in the OEGJ treatment group were
intragastricly administered daily with an OEGJ suspension (480
mg/kg/day in water) for 4 weeks. The animals of 2VO in vehicle
treatment group (n=6) were administered with an equal volume of
water daily. Three of the rats in sham treated group were
intragastricly administered daily with an OEGJ suspension (480
mg/kg/day in water) for 4 weeks and the remaining other 3 animals
were administered with an equal volume of water daily. The blood
pressure and the blood flow to the brain were analyzed 4 weeks
after the treatment.
[0068] Measurement of blood pressure. Systolic blood pressure (SBP)
and mean blood pressure (MBP) of the experimental animals were
measured after four weeks by a computerized, automated system with
a tail-cuff (BP-98A, Softron) after a warming period in
unanesthetized rats. The room temperature was maintained constant
at 27.degree. C. It was found that the blood pressure was elevated
(154.25.+-.3.95 mmHg) in vehicle treated 2VO rats 4 weeks after
treatment (FIG. 2), which is significantly higher than that (around
125 mmHg) in sham operated animals. By contrast, the blood pressure
in OEGJ treated 2VO rats was 135.25.+-.2.14 mmHg (FIG. 2), which is
significantly lower than that in vehicle treated control rats, but
slightly higher than the level in sham-operated rats indicating
brain collateral vessel formation that reduced the peripheral
resistance of brain arteries or vessel dilation due to OEGJ
treatment.
[0069] To rule out the possibility of vessel dilation effect of
OEGJ, we measured the brain blood flow volume and blood pressure
again in the 2VO experimental animals two weeks after OEGJ
treatment. It was found that two weeks after termination of the
OEGJ treatment, the blood flow volume remained similar to the value
of last measurement in OEGJ-treated rats with blood pressure
approximately 128 mmHg. While in vehicle treated rats, both the
blood flow volume and blood pressure were about the same to the
values of last measurements two weeks ago. In conclusion, the
significantly decreased blood supply to the brain due to 2VO was
restored by OEGJ treatment. The effect is probably due to the
enhanced collateral vessel formation in the ischemic brain that
reduced the resistance of the arterioles of the brain, which was
confirmed by histological studies (blood vessel counting) of the
same brain samples.
[0070] Doppler ultrasound evaluation of brain blood flow. If OEGJ
treatment stimulated the growth of collateral vessels in the brain
that would reduce the peripheral resistance of small arteries in
the brain, then the total cerebral blood flow volume would be
increased under a certain blood pressure. To demonstrate this
hypothesis, we measured the blood pressure as stated above and
further evaluated the cerebral blood flow volume of the
experimental animals using a Toshiba Aplio XG ultrasound with
PLT-1202S linear array transducer by measuring extracranial basal
artery of the experimental animals. After permanent 2VO, the blood
supply to the rat brain depends dominantly on the bilateral common
vertebral arteries-basal artery, which normally supplies
approximately 40% of the total blood volume to the brain. Our
results showed that although the blood flow volume of the basal
artery in sham operated rats is around 12.4.+-.3.5 ml/min, it
reached 16.04.+-.6.4 ml/min in vehicle-treated control due to the
compensation mechanism to the 2VO (FIG. 3), which accounts for 52%
of the normal total blood volume to the brain. Interestingly, the
blood flow volume of the basal artery in OEGJ-treated animals
increased up to 25.9.+-.11.80 ml/min (FIG. 3), which is
approximately 83.5% of the normal level of the total cerebral blood
flow volume.
[0071] To determine whether the OEGJ-mediated reduction effect of
peripheral blood vessel resistance were resulted from vessel
dilation or growth of new collateral vessels in brain, we measured
the blood flow volume again in these experimental animals two weeks
after OEGJ treatment. It was found that two weeks after termination
of the OEGJ treatment, the blood flow volume remained similar
(21.1.+-.6.3 ml/min) to the value of last measurement in OEGJ
treated rats with blood pressure approximately 128 mmHg similar
level to the normal blood pressure. While in vehicle treated rats,
both the blood flow volume (14.6 ml/min) and blood pressure (148
mmHg) were about similar to the values of last measurements two
weeks ago.
[0072] Based on the formula: Blood pressure (BP)/blood flow volume
(BF)=peripheral vessel resistance (PR), for the normal rats: at the
time of first measurement, Rn0=125/12.4=10.08, and 2 weeks later,
Rn2=121/12=10.1; for the vehicle treated control rats:
Rc0=154.25/16.04=9.62, 2 weeks post termination of vehicle
treatment, Rc2=148/14.6=10.14; for the OEGJ treated rats:
Ro0=135.25/25.9=5.22, 2 weeks post termination of OEGJ treatment,
Ro2=128/21.1=6.06 (FIG. 3). It was derived from above calculations
that the peripheral vessel resistance between normal and vehicle
treated control rats showed almost no difference (.about.10).
However, the peripheral vessel resistance of OEGJ treated group is
about 43% lower, an indication of growth of new collateral arteries
that increased the sectional area of peripheral arteries in brain,
which was further confirmed by vessel counting in histological
analysis.
[0073] Confirmation of collateral neovascularization in the brain.
It was found that the average weight of the brain in OEGJ treated
rats is 9.1.+-.2.3% heavier than that in vehicle treated control
rats (P<0.01). Brains from the experimental animals sacrificed
after cerebral blood flow measurement were removed, fixed in
formalin and embedded in paraffin. Thin sections (5 .parallel.m)
were cut from each slide and stained with H&E staining. The
vascular densities were determined on the thin sections by counting
the numbers of vessels within the cortex of frontal lobe and around
hippocampus regions using a light microscope under a high power
field (HPF) (40.times.). Six random and non-overlapping HPFs within
the frontal lobe or hippocampus were inspected 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.
[0074] It was found that the numbers of vessels are about
61.7.+-.20.3/HPF in the regions of cortex in frontal lobe (FIG. 4)
and 56.4.+-.12.3/HPF around the regions of hippocampus in OEGJ
treated rats. By contrast, the numbers of vessels are about
38.5.+-.12.6/HPF in regions of cortex in frontal lobe (FIG. 4) and
30.7.+-.10.5/HPF around the regions of hippocampus in vehicle
treated rats. In summary, OEGJ treatment induced 37.4% more
collateral vessels formed in ischemic brains that resulted in 30.8%
more blood supply to the ischemic brain and 14.5% reduction of the
blood pressure in the OEGJ treated 2VO induced hypertension animals
(FIG. 5).
Example 4
Therapeutic Effects of OEGJ in Mild Hypertension Animal Models
[0075] The therapeutic effects of OEGJ in several mild hypertension
animal models were examined. All studies were conducted in
accordance with the National Regulations of Experimental Animal
Administration, and all animal experiments were approved by the
Committee of Experimental Animal Administration of Zhangjiang
High-tech Park. The animal models included a partial ligation of
uniilateral carotid arteries in rat, APP mice, stroke rat, and
senescence accelerated mouse induced mild hypertensionanimal
models, which are described below.
[0076] Induction of a milder high blood pressure rat model. To this
end, a partial ligation (60%) of right carotid artery (PLRCA) in SD
rats (n=12) was performed. The right common carotid artery was
exposed through a right midline cervical incision under anesthesia.
The exposed right carotid artery was then partially (60%) ligated
by a 6-0 nylon suture, and cut by microscissors. The wound was
thereafter closed with a suture. Six rats subjected to the
operation of right midline cervical incision but without common
carotid artery ligation were taken as sham-operation control. After
recovering from anesthesia, the experimental animals were allowed
free access to food and water. The rats were randomly divided into
the OEGJ treatment group (n=6) and vehicle treatment group
(n=6).
[0077] APP mice develop fibrillar amyloid plaques. As the APP mice
age, they exhibit impairments in spatial learning and memory with a
mildly elevated blood pressure. SAMP10 mice, developed by Takeda
and his colleagues (Takeda et al., 1991), show characteristics of
rapid aging. They develop early abnormalities in learning and
memory with mild high blood pressure when they age. In this study,
both APP (n=30) and SAMP10 mice (n=30) were used. Both the APP mice
and SAMP 10 mice were randomly divided into the OEGJ treatment
group (n=15) and vehicle treatment group (n=15) respectively.
[0078] Rat models of ischemic stroke induced high blood pressure.
Ischemic stroke was induced in SD rats by surgery according to the
methods used previously (Mayzel-Oreg et al., 2004). Briefly, the
common carotid artery (CCA), internal carotid artery (ICA), and
external carotid artery (ECA) around the carotid bifurcation were
exposed through a midline incision in the right side of the neck.
CCA was ligated proximal to the carotid bifurcation. Saline
solution (0.5 ml) containing .about.1000 microspheres (80-150
.mu.M) was injected by a syringe inserted into the ECA pointing
toward the carotid bifurcation. After ligation of the ECA distal to
the injection site and removal of CCA ligation, the injected
microspheres entered the ICA resulted in multi-infarct ischemic
stroke in the brain. Rats (n=28) were divided into OEGJ treatment
group (n=12) and vehicle treatment group (n=16) according to their
neurological gradings so that rats in each group had an overall
similar grade.
[0079] The rats and mice in OEGJ treatment groups of all above
animal models were intragastricly treated with an OEGJ suspension
(480 mg/kg/day in water) for 4 weeks respectively. The rats and
mice in the vehicle-treated groups were intragastricly administered
with an equal volume of water daily for the same period.
[0080] The results showed that the average blood pressure of the
vehicle treated rats (PLRCA) was increased to approximately 142
mmHg and the total blood flow volume was around 37 ml/min (FIG. 6).
By contrast, the elevated blood pressure due to the PLRCA was
reduced to a normal level (121 mmHg) in OEGJ treated rats, while
the total cerebral blood flow volume significantly increased up to
58 ml/min to maintain the blood supply to the ischemic brain (FIG.
6). In APP mice (6-7 months old), the average blood pressure of
mice in vehicle treated group was .about.124 mmHg (FIG. 7). By
contrast, the average blood pressure of mice in OEGJ treatment
group was .about.114 mmHg, which is about 10% lower than that in
the vehicle treated control mice (FIG. 7). It was also found that
the average weight of the brains in OEGJ treatment group is about
15% heavier than that in vehicle treatment group. The histological
studies also demonstrated that significantly more newly grown
vessels were found in the cortex and hippocampus of brains in OEGJ
treated APP mice. In conclusion, one month OEGJ treatment not only
induced growth of new collateral vessels in brain and other
tissues, but also decreased the systemic blood pressure (.about.114
mmHg) in APP mice. In contrast, the blood pressure in vehicle
treated APP mice was about 10% higher (124 mmHg) than that in OEGJ
treated (FIG. 7). Furthermore, it was also demonstrated that, after
one month OEGJ (480 mg/kg) treatment, the mildly increased blood
pressures (130-150 mmHg) in rat stroke model and SAMP10 mouse model
reduced by approximately 10%, similar to the normal blood pressure
level in these animals.
Example 5
The Therapeutic Effects of the OEGJ on Hypertension in Human
Subjects
[0081] Based on above rather promising results from the
OEGJ-enhanced proliferation and differentiation of vessel
endothelial cells in cell cuture systems and more importantly, OEGJ
mediated substantial treatments on 2VO, stroke, PLRCA and APP
induced hypertension in animal models, the potential as to whether
the OEGJ-induced substantial treatment effects observed in
hypertension animal model can be similarly translated in a clinical
setting was tested in hypertension patients on the basis of mercy
treatment. Our preliminary clinical mercy treatment was performed
on 10 patients with primary or secondary hypertension.
[0082] All patients (n=10) with primary or secondary hypertension
enrolled in the test reported having a long history of
hypertension. Their hypertension could not be satisfactorily
treated with currently commercial available medications in recent
years. Their high blood pressure did not respond well to the
traditional therapeutic strategy or combinations. At the time of
examination, their blood pressures were around 162.+-.20/106.+-.10
mmHg. Furthermore, apart from high blood pressure, they also
complained that they had been bothered with frequent headache,
dizziness, chest suppression, shortness of breath and blurred
vision.
[0083] They were treated on request and written consent with OEGJ
(2-3 grams/day, oral administration) for 4-8 weeks. After 1-2 weeks
oral administration of the OEGJ, all of these patients experienced
a smooth decrease of blood pressure. After four to eight weeks
treatment, the blood pressure of all these patients were back to
within normal range (119.+-.10/79.+-.6 mmHg), and they reported
relief from the accompaning symptoms of hypertension as mentioned
above (Table 1).
TABLE-US-00001 TABLE 1 Relief of hypertension related symptoms
after treatment Hypertension Related Symptoms Before Treatment
After Treatment Headache +++~++++ - Dizziness ++~+++ - Chest
Suppression +~++ - Shortness of Breath ++~+++ - Blurred Vision +~++
-
Example 6
Treatment of Hypertension Using MEGJ
[0084] In a preliminary clinical test with the methanol extract of
Geum japonicum Thunb. variant (MEGJ) (the composition of MEGJ is
similar to OEGJ) for its therapeutic effects on hypertension, it
was found that two weeks MEGJ treatment (orally, 3-4 grams/day)
could smoothly decrease by 10-20% the blood pressure of patients
who did not respond well to the conventional anti-high-blood
pressure treatment. Furthermore, the reduced blood pressure can be
maintained for several months without any further treatment,
indicating its significant potential for treating hypertension, and
especially for intractable hypertension. The mechanism underlying
the remarkable therapeutic effect of MEGJ on hypertension is
postulated to increase the total cross-sectional area of the
arteriole bed in the treated subjects.
[0085] One week of MEGJ treatment in 5 patients with borderline
hypertension (140-150/90-95 mm Hg) lowered blood pressure to
115-125/70-75 mm Hg for more than 3 months. Another 6 patients with
refractory hypertension (140-180/95-140) received MEGJ treatment
for one month after failing to respond to conventional
anti-hypertension treatment. In the first week of treatment with
MEGJ, blood pressure was not significantly lowered. However, after
two weeks of treatment with MEGJ, the blood pressures of these
patients were significantly and smoothly lowered to 135-145/85-100
mmHg and the symptoms of headache, dizziness, tinnitus, confusion,
papilloedema were significantly improved or disappeared (Table 2).
After four weeks of treatment with MEGJ, the blood pressures of all
6 patients were lowered and could be maintained for several months
without any further medication.
[0086] In conclusion, two to four week oral administration of MEGJ
can gradually and significantly lower the elevated blood pressures
of patients, who have borderline or refractory hypertension, to a
normal level, and the symptoms derived from high blood pressure
were also significantly improved or eliminated. The MEGJ-induced
blood pressure lowering effect seemed different from current
clinically available anti-high blood pressure drugs. Increasing the
total cross-sectional area of the arteriole bed in treated subjects
is considered to be one of the major mechanisms for smoothly
lowering blood pressure as an effective and possible cure for
hypertension.
TABLE-US-00002 TABLE 2 MEGJ treatment of hypertension. Symptoms
Before treatment After treatment Blood pressure 140-180/95-140
120-140/75-95 Headache ++ --- +++ - Tinnitus + --- ++ - --- +
Confusion - --- ++ - Papilloedema + --- +++ - --- + Dizziness ++
--- +++ - --- +
[0087] While certain embodiments have been illustrated and
described, it should be understood that changes and modifications
can be made therein in accordance with ordinary skill in the art
without departing from the technology in its broader aspects as
defined in the following claims.
[0088] The present disclosure is not to be limited in terms of the
particular embodiments described in this application. Many
modifications and variations can be made without departing from its
spirit and scope, as will be apparent to those skilled in the art.
Functionally equivalent methods and apparatuses within the scope of
the disclosure, in addition to those enumerated herein, will be
apparent to those skilled in the art from the foregoing
descriptions. Such modifications and variations are intended to
fall within the scope of the appended claims. The present
disclosure is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled. It is to be understood that this disclosure is
not limited to particular methods, reagents, compounds compositions
or biological systems, which can, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting.
[0089] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0090] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges disclosed herein also encompass any and all
possible subranges and combinations of subranges thereof. Any
listed range can be easily recognized as sufficiently describing
and enabling the same range being broken down into at least equal
halves, thirds, quarters, fifths, tenths, etc. As a non-limiting
example, each range discussed herein can be readily broken down
into a lower third, middle third and upper third, etc. As will also
be understood by one skilled in the art all language such as "up
to," "at least," "greater than," "less than," and the like include
the number recited and refer to ranges which can be subsequently
broken down into subranges as discussed above. Finally, as will be
understood by one skilled in the art, a range includes each
individual member. Thus, for example, a group having 1-3 units
refers to groups having 1, 2, or 3 units. Similarly, a group having
1-5 units refers to groups having 1, 2, 3, 4, or 5 units, and so
forth.
[0091] While various aspects and embodiments have been disclosed
herein, other aspects and embodiments will be apparent to those
skilled in the art. The various aspects and embodiments disclosed
herein are for purposes of illustration and are not intended to be
limiting, with the true scope and spirit being indicated by the
following claims.
REFERENCES
[0092] 1. Heart Disease and Stroke Statistics--2007 Update, Heart
Disease and Stroke Statistics--2007 Update, A Report From the
American Heart Association Statistics Committee and Stroke
Statistics Subcommittee. [0093] 2. British Heart Foundation.
European Cardiovascular Disease Statistics; 2000 Edition. [0094] 3.
Pierdomenico, S D, Lapenna, D, Bucci, A. et al., Cardiovascular
outcome in treated hypertensive patients with responder, masked,
false resistant, and true resistant hypertension. Am J Hypertens,
2005, 18:1422. [0095] 4. Muxfeldt, E S, Bloch, K V, Nogueira Ada,
R, Salles, G F. True resistant hypertension: is it possible to be
recognized in the office? Am J Hypertens, 2005, 18:1534. [0096] 5.
Calhoun, D A, Jones, D, Textor, S. et al., Resistant hypertension:
diagnosis, evaluation, and treatment. A scientific statement from
the American Heart Association Professional Education Committee of
the Council for High Blood Pressure Research. Hypertension, 2008,
51:1403. [0097] 6. Berlowitz, D R, Ash, A S, Hickey, E C. et al.,
Inadequate management of blood pressure in a hypertensive
population. N Engl J Med, 1998, 339:1957. [0098] 7. Cushman, W C,
Ford, C E, Cutler, J A. et al., Success and predictors of blood
pressure control in diverse North American settings: the
antihypertensive and lipid-lowering treatment to prevent heart
attack trial (ALLHAT). J Clin Hypertens (Greenwich), 2002, 4:393.
[0099] 8. Cheung W L, Cheng L, Liu H, Gu X, Li M., The dual actions
of angiogenesis and anti-apoptosis induced by an isolated fraction
from Geum japonicum repair muscle ischemia. Arch Biochem Biophys,
2007, 459:91-97. [0100] 9. MTT Cell Proliferation Assay
Instructions. ATCC. [0101] 10. Bunag R D, Validation in awake rats
of a tail-cuff method for measuring systolic pressure. J Appl
Physiol., 1973, 34:279-82. [0102] 11. Yen T T, Powell C E, Pearson
D V, An indirect method of measuring the blood pressure of rats
without heating. In Spontaneous Hypertension: its Pathogenesis and
Complications. DHEW Publication No. 77-1179. Bethesda, Md.: US
Public Health Service, 1977, p 486. [0103] 12. Medsger, Oliver
Perry (1972) Edible Wild Plants, London, Collier-Macmillan Limited.
[0104] 13. Takeda T, Hosokawa M & Higuchi K (1991)
Senescence-accelerated mouse (SAM): a novel murine model of
accelerated senescence. Journal of the American Geriatrics Society
39: 911-919. [0105] 14. O. Mayzel-Oreg, T. Omae, M. Kazemi, F. Li,
M. Fisher and Y. Cohen et al. Unilateral intracarotid injection of
holmium microspheres to induce bilateral MRI-validated cerebral
embolization in rats. J Neurosci Methods, 2004, 176: 152-156.
* * * * *