U.S. patent application number 15/707557 was filed with the patent office on 2018-06-14 for methods and compositions involving (s)-bucindolol.
The applicant listed for this patent is ARCA BIOPHARMA, INC.. Invention is credited to Michael R. Bristow, Jonathan D. Port.
Application Number | 20180161309 15/707557 |
Document ID | / |
Family ID | 40531457 |
Filed Date | 2018-06-14 |
United States Patent
Application |
20180161309 |
Kind Code |
A1 |
Bristow; Michael R. ; et
al. |
June 14, 2018 |
METHODS AND COMPOSITIONS INVOLVING (S)-BUCINDOLOL
Abstract
Disclosed is bucindolol substantially free of its
R-stereoisomer. Also disclosed are pharmaceutical compositions that
include bucindolol substantially free of its R-stereoisomer or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier. Also disclosed are methods of treating a
patient that involve administering to the patient a therapeutically
effective amount of a composition of the present invention.
Inventors: |
Bristow; Michael R.;
(Englewood, CO) ; Port; Jonathan D.; (Denver,
CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARCA BIOPHARMA, INC. |
Westminster |
CO |
US |
|
|
Family ID: |
40531457 |
Appl. No.: |
15/707557 |
Filed: |
September 18, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15268137 |
Sep 16, 2016 |
9763916 |
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15707557 |
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14590661 |
Jan 6, 2015 |
9446023 |
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15268137 |
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13056916 |
May 4, 2011 |
8946284 |
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PCT/US2009/032144 |
Jan 27, 2009 |
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14590661 |
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61085586 |
Aug 1, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61B 17/04 20130101;
A61L 29/16 20130101; A61M 25/0045 20130101; A61L 2300/204 20130101;
A61P 25/06 20180101; A61B 17/80 20130101; A61L 31/16 20130101; A61P
27/06 20180101; A61P 11/00 20180101; A61K 31/4045 20130101; A61B
17/72 20130101; A61B 17/1214 20130101; A61F 2/01 20130101; A61F
2/82 20130101; A61L 27/54 20130101; C07D 209/14 20130101; A61F 2/24
20130101; A61P 9/04 20180101; A61P 9/06 20180101; A61P 9/12
20180101; A61F 2/06 20130101; A61K 45/06 20130101; A61P 29/00
20180101; A61B 17/86 20130101; A61F 2250/0067 20130101; A61F 2/0063
20130101 |
International
Class: |
A61K 31/4045 20060101
A61K031/4045; A61L 29/16 20060101 A61L029/16; C07D 209/14 20060101
C07D209/14; A61B 17/04 20060101 A61B017/04; A61B 17/12 20060101
A61B017/12; A61B 17/72 20060101 A61B017/72; A61B 17/80 20060101
A61B017/80; A61B 17/86 20060101 A61B017/86; A61F 2/00 20060101
A61F002/00; A61F 2/01 20060101 A61F002/01; A61F 2/06 20060101
A61F002/06; A61F 2/24 20060101 A61F002/24; A61F 2/82 20060101
A61F002/82; A61K 45/06 20060101 A61K045/06; A61L 27/54 20060101
A61L027/54; A61M 25/00 20060101 A61M025/00; A61L 31/16 20060101
A61L031/16 |
Claims
1.-46. (canceled)
47. A method of increasing the capacity of a cell to generate NO,
comprising contacting said cell with a composition comprising a
pharmaceutically acceptable carrier and bucindolol substantially
free of its R-stereoisomer.
48. The method of claim 47, wherein the cell is an endothelial
cell, an epithelial cell, or a stem cell.
49. The method of claim 47, wherein the cell is an endothelial
cell.
50. The method of claim 47, wherein the cell is in a patient.
51. The method of claim 47, wherein the cell is in an animal.
52. The method of claim 51, wherein the animal is a mouse, a rat, a
rabbit, a cat, a dog, a horse, a sheep, a goat, a cow, or a
primate.
53.-62. (canceled)
Description
[0001] The present application is a divisional of U.S. patent
application Ser. No. 15/268,137, filed Sep. 16, 2016, which is a
continuation of U.S. patent application Ser. No. 14/590,661, filed
Jan. 6, 2015, now U.S. Pat. No. 9,446,023, which is a divisional of
U.S. patent application Ser. No. 13/056,916, filed May 4, 2011, now
U.S. Pat. No. 8,946,284, issued Feb. 3, 2015, which is a national
phase application under 35 U.S.C. .sctn. 371 of International
Application No. PCT/US2009/032144, filed Jan. 27, 2009, which
claims the benefit of priority to U.S. Provisional Patent
Application Ser. No. 61/085,586, filed Aug. 1, 2008, each of which
are hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The present invention relates generally to the fields of
pharmacology and clinical medicine. More specifically, the present
invention is directed to pharmaceutical compositions and methods
for treatment of disease in humans that concern S-bucindolol.
2. Description of Related Art
[0003] The human endothelium has an essential role in regulating
arterial blood flow and preserving normal vascular physiology.
Important activities of the endothelium are mediated by the
production of signaling molecules, especially nitric oxide (NO).
Endothelial dysfunction, on the other hand, is linked to
atherosclerosis and its clinical manifestations (coronary artery
disease, heart failure) (Harrison et al., 1987; Liao, 1998; Oemar
et al., 1998). Key risk factors for atherosclerosis, including
dyslipidemia, smoking and diabetes, can be specifically linked to
abnormalities in NO-mediated endothelial dilation (Harrison et al.,
1987; Liao, 1998; Oemar et al., 1998). In addition, a reduction in
NO bioavailability contributes to elevated vascular resistance and
loss of sensitivity to stimuli of vasodilation, hallmark features
of hypertension (Paniagua et al., 2001; Panza et al., 1990; Taddei
et al., 1993).
[0004] Beyond vasodilation, NO has well-characterized vascular
benefits, including inhibition of smooth muscle cell proliferation
and migration, adhesion of leukocytes to the endothelium, and
platelet aggregation (Harrison, 1997). In patients at higher risk
for cardiovascular disease and its clinical consequences (e.g.,
African Americans), there is also evidence for reduced NO-mediated
vasodilation associated with increased superoxide generation in
endothelial cells (Campia et al., 2002; Kalinowski et al., 2004;
Stein et al., 1997). Thus, agents that directly stimulate NO
release may have important therapeutic advantages in the prevention
and treatment of cardiovascular disease.
[0005] Hispanics are the largest and fastest-growing minority group
in the United States, and Mexican Americans are the largest
sub-group of Hispanics. Epidemiologic studies indicate that
Mexicans have higher rates of coronary heart disease (CHD) risk
equivalents, including type 2 diabetes mellitus, metabolic syndrome
and some primary forms of dyslipidemia (Stern et al., 1991;
Aguilar-Salinas et al., 2001; Aguilar-Salinas et al., 2003). By the
age of 50, epidemiologic studies indicate that 28% of men and 21%
of women in Mexico already exhibit some form of dyslipidemia
(Aguilar-Salinas et al.,2001). In the San Antonio Heart Study, it
was reported that after adjusting for age and gender, U.S.-born
Mexican Americans were 1.4 times as likely to die of cardiovascular
(CV) disease as non-Hispanic whites (Hunt et al., 2002). For
cardiovascular disease, U.S.-born Mexican Americans were 1.7 times
more likely to die from CVD and 1.9 times more likely to die from
CHD than non-Hispanic whites (Hunt et al., 2002). To understand the
basis for this enhanced risk, a recent study has identified genetic
variants that confer higher susceptibility to dyslipidemia, but
more studies are needed in this area (Aguilar-Salinas et al., 2003;
Huerta-Vazquez et al., 2005). There are also well known
environmental factors that contribute to higher risk in individual
Mexican Americans, including a high-fat and high-calorie diet,
tobacco use, alcohol consumption and sedentary lifestyle.
[0006] Hypertension is a risk factor that is less likely treated
and controlled among Hispanics, as compared to the overall U.S.
population. This was a key finding from the National Health and
Nutrition Examination Surveys (NHANES) for 1999-2002. This report
has identified racial/ethnic disparities in the awareness of,
treatment for, and control of hypertension. NHANES is a stratified,
multistage probability sample of the civilian,
non-institutionalized U.S. population. During 1999-2002, the
age-adjusted prevalence of hypertension in the study population was
28.6% (CI=26.8%-30.4%). The prevalence of hypertension increased
with age, as expected, and was higher among women than men. Among
adults with hypertension, the proportion aware of having this
condition was 70.3% among non-Hispanic blacks, 62.9% among
non-Hispanic whites, but only 49.8% among Mexican Americans. The
age-adjusted proportion that reported treatment was 55.4% among
non-Hispanic blacks, 48.6% among non-Hispanic whites, and only
34.9% among Mexican Americans. Only 29% of U.S. adults with
hypertension had controlled BP levels (<140/90 mmHg). The
proportion with controlled BP was similar among non-Hispanic blacks
(29.8%) and non-Hispanic whites (29.8%) but substantially lower
among Mexican Americans (17.3%). These findings indicate the
challenge of effectively treating and controlling hypertension in
the rapidly growing Hispanic and Mexican American population.
[0007] While epidemiologic studies indicate a higher risk of CV
disease and lower BP control among Mexican Americans, the
underlying pathophysiology is not well understood. Studies in other
high-risk populations, such as African Americans, indicate that the
higher risk is related to decreased responsiveness of conductance
vessels to both endogenous and exogenous stimulants of NO, as
compared with age-matched whites (Campia et al., 2002). To
understand the basis for this difference, it was reported that
there is lower bioavailability of NO from endothelium of black
Americans, despite much higher levels of endothelial-dependent NO
synthase (eNOS) (Kalinowski et al., 2004). The cellular basis for
this paradox was the finding that excessive O.sub.2.sup.-
generation by NAD(P)H-oxidase and uncoupled eNOS resulted in the
loss of functional NO due to its reactivity with O.sub.2.sup.-,
resulting in peroxynitrite (ONOO.sup.-) formation, a potent oxidant
with the capacity to produce adverse biological effects (Kalinowski
et al., 2004).
[0008] Thus, there is the need for improved therapy of hypertension
and CV disease, particularly among racial groups where there is a
high prevalence of these diseases.
SUMMARY OF THE INVENTION
[0009] The present invention relates to the finding that the
S-stereoisomer of bucindolol (S-bucindolol) has a greater capacity
of inducing cells to generate NO compared to bucindolol racemate
while simultaneously reducing ONOO.sup.- production. The present
invention is also related to the finding that S-bucindolol
demonstrates particularly favorable activity in racial groups
including African Americans, Mexican Americans, and non-Hispanic
whites.
[0010] Certain embodiments of the present invention concern methods
and compositions involving bucindolol
(2-(3-(1-(1H-indol-3-yl)-2-methylpropan-2-ylamino)-2-hydroxypropoxy)benzo-
nitrile), substantially free of its R-stereoisomer. A composition
is "substantially free" of R-bucindolol if it includes a mixture of
S-bucindolol and (optionally) R-bucindolol wherein the weight of
R-bucinolol, if present, is no more than about 20% of the total
weight of S-bucindolol and R-bucindolol in the composition. In some
embodiments, the composition may contain no more than about 20, 19,
18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4.9, 4.8, 4.7,
4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4,
3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1,
2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1.0% or any
range derivable therein by weight of R-bucindolol relative to the
total weight of S-bucindolol and R-bucindolol in the composition.
In some particular embodiments, the composition that is
substantially free of R-bucindolol contains no more than about 20%
by weight of R-bucindolol relative to the total weight of
S-bucindolol and R-bucindolol in the composition. In more
particular embodiments, the composition contains no more than about
10% by weight of R-bucindolol relative to the total weight of
S-bucindolol and R-bucindolol in the composition. In more
particular embodiments, the inventive composition contains no more
than about 10% of R-bucindolol relative to the total weight of
S-bucindolol and R-bucindolol in the composition. In even more
particular embodiments, the inventive composition contains no more
than about 1% of R-bucindolol relative to the total weight of
S-bucindolol and R-bucindolol in the composition.
[0011] In some embodiments of the invention, it is contemplated
that bucindolol includes pharmaceutically acceptable salts of
bucindolol. Thus, for example, a composition comprising
S-bucindolol may include a pharmaceutically acceptable salt of
S-bucindolol. In particular embodiments of the present invention,
embodiments include bucindolol or pharmaceutical compositions of
bucindolol that do not include any pharmaceutically acceptable
salts of bucindolol.
[0012] In certain embodiments of the present invention, the
bucindolol in the composition is substantially purified.
"Substantially purified" as set forth herein refers to a
composition comprising bucindolol wherein the composition includes
at least about 80% S-bucindolol. In some embodiments, the
composition includes at least about 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.2, 99.2, 99.3, 99.4,
99.5, 99.6, 99.7, 99.8, or 99.9% by weight of S-bucindolol relative
to total bucindolol in the composition.
[0013] The pharmaceutical composition may optionally include one or
more additional pharmaceutical agents. Any pharmaceutical agent is
contemplated for inclusion in the compositions. Examples of
specific agents are set forth in the specification below. In some
embodiments, the pharmaceutical agent is an agent that can be
applied in the treatment or prevention of a cardiovascular disease,
a neurological disease, an infectious disease, an inflammatory
disease, a neoplasm, a gastrointestinal disease, a genitourinary
disease, a pulmonary disease, or an immune disease. In some
embodiments the pharmaceutical agent is an additional
.beta.-adrenergic receptor blocker. Non-limiting examples of
beta-adrenergic receptor blockers include AC 623, acebutolol,
alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol,
bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol,
bufuralol, bunitrolol, bupranolol, butidrine hydrocholoride,
butofilolol, carazolol, carteolol, carvedilol, celiprolol,
cetamolol, cloranolol, dilevalol, esmolol, indenolol, labetalol,
landiolol, levobunolol, mepindolol, metipranolol, metoprolol,
moprolol, nadolol, nadoxolol, nebivolol, nifenalol, nipradilol,
oxprenolol, penbutolol, pindolol, practolol, pronethalol,
propranolol, sotalol, sulfinalol, talinolol, tertatolol, tilisolol,
timolol, toliprolol, and xibenolol.
[0014] In other embodiments, the composition may include a nitric
oxide (NO) enhancing agent. Examples of NO enhancing agents are
well known to those of ordinary skill in the art. Examples of such
agents include a RAS inhibitor, a statin, a PDE5 inhibitor, a
NO-conjugated drug, or a diazeniumdiolate. Non-limiting examples of
RAS inhibitors include captopril, cilazapril, enalapril,
fosinopril, lisinopril, quinapril, ramapril, zofenopril,
candesartan cilexetil, eprosartan, irbesartan, losartan,
tasosartan, tehnisartan, and valsartan, or a pharmaceutically
acceptable salt thereof. Non-limiting examples of statins include
atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin
calcium, and simvastatin. Non-limiting examples of NO-conjugated
drugs include S--NO-glutathione, NO-naproxen, NO-aspirin,
NO-ibuprofen, NO-Diclofenac, NO-Flurbiprofen, NO-Ketoprofen,
NO-releasing compound-7, NO-releasing compound-5, NO-releasing
compound-12, or NO-releasing compound-18. Other examples of NO
enhancing agents include L-arginine, arginine alpha-ketoglutarate,
GEA 3175, sodium nitroprusside, glyceryl trinitrate,
S-nitroso-N-acetyl-penicillamine, nitroglycerin, and diethylamine
NONOate. Information concerning NO generating compounds for
treating hypertension and atherosclerosis can be found in U.S. Pat.
Nos. 7,396,829, 7,348,319, 7,155,284, 7,052,695, 6,358,536, and
5,208,233, each of which is herein specifically incorporated by
reference. Information regarding nebivolol as an NO-enhancing agent
can be found in U.S. Pat. No. 7,138,430, herein specifically
incorporated by reference.
[0015] The invention also concerns methods of treating a patient
involving administering to a patient a therapeutically effective
amount of a composition comprising bucindolol wherein the
composition is substantially free of the R-stereoisomer of
bucindolol. The composition can be any of those compositions set
forth above.
[0016] In certain embodiments, the patient self-identifies as a
Caucasian. In more particular embodiments, the patient
self-identifies as a non-Hispanic white. In other embodiments, the
patient self-identifies as an individual of African descent. In
still further embodiments, the patient self-identifies as a
Hispanic. In more particular embodiments, the patient
self-identifies as a Mexican American. In some embodiments the
patient has a disease or condition such that the patient is in need
of a NO enhancing agent. For example, the patient may have a
headache, hypoxic respiratory failure, pulmonary hypertension,
right ventricular heart failure, congestive heart failure,
respiratory distress syndrome, impotence, hypertension, angina,
myocardial infarction, or cardiac arrhythmia.
[0017] In some embodiments, the patient is in need of a
beta-blocker. For example, the patient may be a patient in need of
treatment or prevention of hypertension, angina, myocardial
infarction, mitral valve prolapse, cardiac arrhythmia, congestive
heart failure, hypertrophic obstructive cardiomyopathy, acute
dissecting aortic aneurysm, portal hypertension, anxiety disorder,
glaucoma, migraine headache, migraine prophylaxis, tremor due to
anxiety, tremor due to hyperthyroidism, essential tremor,
pheochromocytoma, or hyperhidrosis.
[0018] The method may optionally involve administering to the
patient a secondary form of therapy. The secondary form of therapy
may be any type of therapy. For example, the secondary form of
therapy may be a pharmaceutical agent or a surgical procedure.
Non-limiting examples of surgical procedures include angioplasty,
valve replacement surgery, heart transplant, coronary artery bypass
grafting, and peripheral vascular surgery. The secondary therapy
may be administered prior to, concurrently with, or following
administration of the therapeutic compositions set forth herein. In
particular embodiments, the secondary therapy is a pharmaceutical
agent. The pharmaceutical agent may be administered separately from
the composition of the present invention, or may be included as a
component of a composition as set forth herein. Examples of such
pharmaceutical agents are set forth above and elsewhere in this
specification.
[0019] Administration of the pharmaceutical compositions set forth
herein may be by any method known to those of ordinary skill in the
art. Examples include, but are not limited to, oral, intravenous,
intramuscular, intra-arterial, intramedullary, intrathecal,
intraventricular, intradermal, intratracheal, intravesicular,
intraocular, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, or rectal administration.
Further details on techniques for formulation and administration
may be found in the specification below.
[0020] In some embodiments, the method further includes contacting
the patient with a medical device that includes S-bucindolol. For
example, the medical device may include a coating that includes
S-bucindolol, a matrix that includes S-bucindolol, or a reservoir
that includes a therapeutic composition as set forth above. The
device may be inserted into the patient temporarily or implanted in
the patient or placed on a body surface of the patient. Examples of
such body surfaces include skin surfaces or mucosal surfaces.
[0021] The medical device may be any medical device known to those
of ordinary skill in the art. Non-limiting examples of such medical
devices include a stent, a graft, a heart valve, a filter, a
catheter, a coil, a mesh repair material, a plate, a rod, a screw,
or a suture.
[0022] The present invention also concerns methods of increasing
the capacity of a cell to generate NO, involving contacting the
cell with a composition that includes bucindolol substantially free
of its R-stereoisomer or a pharmaceutically acceptable salt
thereof, and a pharmaceutically acceptable carrier. The cell may be
any type of cell. In particular embodiments the cell is an
endothelial cell, an epithelial cell, or a stem cell. In certain
embodiments, the cell is a cell that is in a patient or an animal.
Non-limiting examples of animals include a mouse, a rat, a rabbit,
a cat, a dog, a horse, a sheep, a goat, a cow, or a primate.
[0023] The invention also concerns medical devices that include a
coating, a matrix, or a chamber, wherein the coating, matrix, or
chamber includes bucindolol substantially free of the
R-stereoisomer. Non-limiting examples of such medical devices
include a stent, a graft, a heart valve, a filter, a catheter, a
coil, a mesh repair material, a plate, a rod, a screw, and a
suture. An example of a type of filter is an inferior vena caval
filter. An example of a type of catheter is a drug infusion
catheter. An example of a type of coil is an embolic coil.
[0024] It is specifically contemplated that any limitation
discussed with respect to one embodiment of the invention may apply
to any other embodiment of the invention. Furthermore, any
composition of the invention may be used in any method of the
invention, and any method of the invention may be used to produce
or to utilize any composition of the invention.
[0025] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternative are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0026] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device and/or method being employed to determine the value.
[0027] As used herein the specification, "a" or "an" may mean one
or more, unless clearly indicated otherwise. As used herein in the
claim(s), when used in conjunction with the word "comprising," the
words "a" or "an" may mean one or more than one. As used herein
"another" may mean at least a second or more.
[0028] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0029] The following figures form part of the present specification
and are included to further demonstrate certain aspects of the
present invention. The invention may be better understood by
reference to one or more of these drawings in combination with the
detailed description of specific embodiments presented herein.
[0030] FIG. 1. Schematic diagram of a NO nanosensor placed in close
proximity to the surface of a single endothelial cell. The
nanosensor measures the levels of NO, O.sub.2.sup.-, and ONOO.sup.-
from the intact endothelium in real time. The sensors are made by
depositing a sensing material on the tip of carbon fiber with a
diameter of about 0.5 .mu.m. The fibers are sealed with
nonconductive epoxy and electrically connected to wires (gold,
copper) with conductive silver epoxy.
[0031] FIG. 2. CaI-stimulated NO release in HUVECs isolated from
non-Hispanic white, Mexican American and African American donors.
Values are reported as mean.+-.S.D. (N=5). *p<0.01 versus
Non-Hispanic white controls (ANOVA Dunnett multiple comparisons
test; Overall ANOVA: p<0.0001; F=47.375).
[0032] FIG. 3. Effects of bucindolol, (S)-bucindolol,
(R)-bucindolol and atenolol on CaI-stimulated NO release in HUVECs
isolated from non-Hispanic white donors. Values are reported as
mean.+-.S.D. (N=4-5). *p<0.001 and .sup..dagger.p<0.05 versus
control; .sup.Bp<0.01 versus cognate bucindolol treatment;
.sup.Sp<0.001 versus cognate (S)-bucindolol treatment; and
.sup.Rp<0.001 versus cognate (R)-bucindolol treatment (ANOVA
Student-Newman-Keuls multiple comparisons test; Overall ANOVA:
p<0.0001; F=31.062). Abbreviations: (S)-Bucin=(S)-Bucindolol;
(R)-Bucin=(R)-Bucindolol.
[0033] FIG. 4 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol
and atenolol on CaI-stimulated NO release in HUVECs isolated from
Mexican-American donors. Values are reported as mean.+-.S.D.
(N=4-5). *p<0.001 versus control; .sup.Bp<0.001 versus
cognate bucindolol treatment; .sup.Sp<0.001 versus cognate
(S)-bucindolol treatment; and .sup.Rp<0.001 versus cognate
(R)-bucindolol treatment (ANOVA Student-Newman-Keuls multiple
comparisons test; Overall ANOVA: p<0.0001; F=19.458).
Abbreviations: (S)-Bucin=(S)-Bucindolol;
(R)-Bucin=(R)-Bucindolol.
[0034] FIG. 5 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol
and atenolol on CaI-stimulated NO release in HUVECs isolated from
African American donors. Values are reported as mean.+-.S.D.
(N=4-5). *p<0.001 and .sup..dagger.p<0.05 versus control;
.sup.Bp<0.05 versus cognate bucindolol treatment;
.sup.Sp<0.001 versus cognate (S)-bucindolol treatment; and
.sup.Rp<0.01 versus cognate (R)-bucindolol treatment (ANOVA
Student-Newman-Keuls multiple comparisons test; Overall ANOVA:
p<0.0001; F=21.419. Abbreviations: (S)-Bucin=(S)-Bucindolol;
(R)-Bucin=(R)-Bucindolol.
[0035] FIG. 6 CaI-stimulated ONOO.sup.- release in HUVECs isolated
from non-Hispanic white, Mexican American and African American
donors. Values are reported as mean.+-.S.D. (N=5). *p<0.01
versus control (ANOVA Dunnett multiple comparisons test; Overall
ANOVA: p<0.0001; F=55.340).
[0036] FIG. 7 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol
and atenolol on CaI-stimulated ONOO.sup.- release in HUVECs
isolated from non-Hispanic white donors. Values are reported as
mean.+-.S.D. (N=4-5). *p<0.05, .sup..dagger.p<0.01 and
.sub..sctn.p<0.001 versus control; .sup.Bp<0.05 versus
cognate bucindolol treatment; .sup.Sp<0.01 versus cognate
(S)-bucindolol treatment; and .sup.Rp<0.05 versus cognate
(R)-bucindolol treatment (ANOVA Student-Newman-Keuls multiple
comparisons test; Overall ANOVA: p<0.0001; F=7.575).
Abbreviations: (S)-Bucin=(S)-Bucindolol;
(R)-Bucin=(R)-Bucindolol.
[0037] FIG. 8 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol
and atenolol on CaI-stimulated ONOO.sup.- release in HUVECs
isolated from Mexican American donors. Values are reported as
mean.+-.S.D. (N=4-5). *p<0.05, .sup..dagger.p<0.01 and
.sup..sctn.p<0.001 versus control; .sup.Bp<0.05 versus
cognate bucindolol treatment; .sup.Sp<0.05 versus cognate
(S)-bucindolol treatment; and .sup.Rp<0.05 versus cognate
(R)-bucindolol treatment (ANOVA Student-Newman-Keuls multiple
comparisons test; Overall ANOVA: p<0.0001; F=15.481).
Abbreviations: (S)-Bucin=(S)-Bucindolol;
(R)-Bucin=(R)-Bucindolol.
[0038] FIG. 9 Effects of bucindolol, (S)-bucindolol, (R)-bucindolol
and atenolol on CaI-stimulated ONOO.sup.- release in HUVECs
isolated from African American donors. Values are reported as
mean.+-.S.D. (N=4-5). *p<0.05, .sup..dagger.p<0.01 and
.sup..sctn.p<0.001 versus control; .sup.Bp<0.001 versus
cognate bucindolol treatment; .sup.Sp<0.05 versus cognate
(S)-bucindolol treatment; and .sup.Rp<0.01 versus cognate
(R)-bucindolol treatment (ANOVA Student-Newman-Keuls multiple
comparisons test; Overall ANOVA: p<0.0001; F=14.628).
Abbreviations: (S)-Bucin=(S)-Bucindolol;
(R)-Bucin=(R)-Bucindolol.
[0039] FIG. 10 CaI-stimulated NO/ONOO.sup.- release ratio in HUVECs
isolated from non-Hispanic white, Mexican American, and African
American donors. Values are reported as mean.+-.S.D. (N=5).
*p<0.01 versus control (ANOVA Dunnett multiple comparisons test;
Overall ANOVA: p<0.0001; F=79.897).
[0040] FIG. 11 Effects of bucindolol, (S)-bucindolol,
(R)-bucindolol and atenolol on CaI-stimulated NO/ONOO.sup.- release
in HUVECs isolated from non-Hispanic white donors. Values are
reported as mean.+-.S.D. (N=4-5). *p<0.001, and
.sup..dagger.p<0.05 versus control; .sup.Bp<0.01 versus
cognate bucindolol treatment; .sup.Sp<0.01 versus cognate
(S)-bucindolol treatment; and .sup.Rp<0.01 versus cognate
(R)-bucindolol treatment (ANOVA Student-Newman-Keuls multiple
comparisons test; Overall ANOVA: p<0.0001; F=21.782).
Abbreviations: (S)-Bucin=(S)-Bucindolol;
(R)-Bucin=(R)-Bucindolol.
[0041] FIG. 12 Effects of bucindolol, (S)-bucindolol,
(R)-bucindolol and atenolol on CaI-stimulated NO/ONOO.sup.- release
in HUVECs isolated from Mexican American donors. Values are
reported as mean.+-.S.D. (N=4-5). *p<0.001, and
.sup..dagger.p<0.01 versus control; .sup.Bp<0.05 versus
cognate bucindolol treatment; .sup.Sp<0.05 versus cognate
(S)-bucindolol treatment; and .sup.Rp<0.05 versus cognate
(R)-bucindolol treatment (ANOVA Student-Newman-Keuls multiple
comparisons test; Overall ANOVA: p<0.0001; F=29.540).
Abbreviations: (S)-Bucin=(S)-Bucindolol;
(R)-Bucin=(R)-Bucindolol.
[0042] FIG. 13 Effects of bucindolol, (S)-bucindolol,
(R)-bucindolol and atenolol on CaI-stimulated NO/ONOO.sup.- release
in HUVECs isolated from African American donors. Values are
reported as mean.+-.S.D. (N=4-5). *p<0.001, and
.sup..dagger.p<0.01 versus control; .sup.Bp<0.01 versus
cognate bucindolol treatment; .sup.Sp<0.05 versus cognate
(S)-bucindolol treatment; and .sup.Rp<0.001 versus cognate
(R)-bucindolol treatment (ANOVA Student-Newman-Keuls multiple
comparisons test; Overall ANOVA: p<0.0001; F=30.266).
Abbreviations: (S)-Bucin=(S)-Bucindolol;
(R)-Bucin=(R)-Bucindolol.
[0043] FIG. 14A, 14B. 14A--Competition curve between S- or
R-bucindolol in human LV membranes that are 86% .beta..sub.1. AR
and genotypically 389 Arg/Arg. The respective K.sub.is are 0.49 nM
and 14.0 nM for the S- and R-isomers. 14B--Competition curve
between S- or R-bucindolol in human LV membranes that are 83%
.beta..sub.1. AR and genotypically 389 Gly/Gly. The respective
K.sub.is are 0.59 nM and 26.3 nM for the S- and R-isomers.
[0044] FIG. 15. .sup.1H NMR spectrum of (R)-bucindolol (sample in
DMSO-d.sub.6); 300 MHz.
[0045] FIG. 16. .sup.1H NMR spectrum of (S)-bucindolol (sample in
DMSO-d.sub.6); 300 MHz.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0046] Bucindolol is a nonselective .beta.-blocking agent with mild
vasodilatory properties. The present invention is in part based on
the finding that the mechanism of vasodilation with bucindolol
involves endothelial-dependent nitric oxide (NO) release, and that
it is effective in higher risk racial groups, such as African
Americans and Mexican Americans. In this regard, the inventors
examined the effects of bucindolol and its separate enantiomers on
endothelial-dependent NO and nitroxidative stress (peroxynitrite)
release in cells from healthy white, African American and Mexican
American donors. The effects of bucindolol and its enantiomers were
compared to another .beta..sub.1-selective antagonist, atenolol. It
was found that bucindolol had a dual effect on endothelial function
by increasing the capacity of cells to generate NO while
simultaneously reducing ONOO.sup.- production in a stereoselective
manner. The favorable activity of bucindolol on the NO/ONOO.sup.-
ratio was significant and highly dose-dependent in three different
racial groups, including African Americans, Mexican Americans and
non-Hispanic whites. The activity of (S)-bucindolol was superior to
bucindolol racemate but not reproduced by atenolol under identical
conditions.
A. BETA BLOCKERS AND BUCINDOLOL
[0047] 1. .beta.-Blockers
[0048] Treatment for heart failure has involved targeting
adrenergic receptors (AR).
[0049] There are at least nine sub-types of adrenergic receptors
(Dohlman et al., 1991; and Liggett et al., 1993), of which at least
three sub-types are .beta.-adrenergic receptors.
[0050] The .beta..sub.1 adrenergic receptor (.beta..sub.1AR) is the
principle subtype expressed on cardiac myocytes. The human heart
expresses both the .beta..sub.1AR and the .beta..sub.2AR subtypes
(Bristow et al, 1986; Bristow et al., 1988). Each receptor mediates
positive inotropic and chronotropic responses to endogenous
catecholamines and exogenously administered agonists (Bristow et
al., 1986; Brodde et al., 1986; Brodde et al., 1992).
[0051] The .beta..sub.1AR triggers the heart's contractile response
when activated, as it is by norepinephrine. In addition, the
.beta..sub.1 receptor has a central role in the progression of
cardiomyopathy and other disease pathways. Increased activation of
this receptor and its associated myopathic and arrhythmic pathways
plays a major role in the natural history of heart failure. Once
the cardiomyopathic process has begun, chronic activation of
.beta..sub.1-adrenergic receptors can accelerate disease
progression, as the failing heart tries to compensate for its
impaired functioning by releasing more norepinephrine and
increasing .beta..sub.1-receptor signaling. The theory of
.beta.-receptor blockade rests in part on counteracting this
cardiomyopathic pathway by blocking the .beta..sub.1-receptor and
reducing norepinephrine signaling.
[0052] The .beta..sub.1 adrenergic receptor has been cloned and
sequenced (Frielle et al., 1987). The gene has been localized to
chromosome q24-q26 of chromosome 10 (Yang-Feng et al., 1990). The
human .beta..sub.1AR has a deduced amino acid sequence of 477 amino
acids.
[0053] At coding nucleotide position 1165 of the .beta..sub.1AR
gene, either cytosine or guanine can be found in the human
population, which results in either Arg or Gly being encoded at
amino acid position 389 (Mason et al., 1999). This position is
within an intracellular domain of the receptor that is involved
with coupling to the stimulatory G-protein, Gs. In fibroblasts
transfected to express equal levels of the two receptors, the
.beta..sub.1-Arg389 receptor display substantially greater
stimulation of adenylyl cyclase compared to .beta..sub.1-Gly389
(Mason et al., 1999). A less common polymorphism of the
.beta..sub.1AR, Gly49, has also been identified but there are
discrepant reports as to its functional implications (Rathz et al.,
2002; Levin et al., 2002).
[0054] The .beta..sub.1-AR 389Arg/Arg polymorphism is actually the
most prevalent form of the .beta..sub.1 adrenergic receptor and is
present in about 50% of the U.S. population (slightly less in
African-Americans). The other variant of this receptor has a
glycine (Gly) at the 389 position and is considered the wild type
only because it was cloned first. Liu et al. (2003) report finding
that a greater response (in terms of changes in heart rate) to
metoprolol was associated with Arg389 compared to Gly389.
[0055] While .beta..sub.1 agonists such as dobutamine, are used for
treating acute deterioration of patients with failing ventricular
function, prolonged exposure of the heart from administered
agonists, or the elevated endogenous catecholamine agonists
produced by the body, leads to worsening heart failure. Indeed
.beta..sub.1AR and .beta..sub.2AR become desensitized in heart
failure, which is thought to be a mechanism of self-protection
against the high levels of catecholamines that exist in heart
failure. The administration of .beta. antagonists can improve
ventricular function and clinical outcomes, presumably by blocking
these deleterious effects of catecholamines. And indeed, cardiac
.beta.AR expression and function improve during .beta. blockade
treatment of heart failure. The vast majority of the deleterious
effects of catecholamines, and the success of .beta. blocker
therapy is due to variants of the .beta..sub.1AR subtype. (Zhu et
al., 2001; and Bristow et al., 2003).
[0056] .beta.-adrenergic receptor antagonists (also termed
.beta.-blockers) have emerged as a major treatment modality in
chronic heart failure. Initially these agents were thought to be
contraindicated in heart failure, since increased adrenergic drive
was thought to be critical for supporting the failing heart. In
fact, in early experience with the 1.sup.st generation compound
propranolol, administration of standard doses was frequently
associated with worsening of heart failure (Stephen, 1968).
However, using low starting doses and slow up-titration, 2.sup.nd
generation (selective .beta..sub.1-blockers) or 3.sup.rd generation
(nonselective .beta.-blocker-vasodilators) generation compounds
have been shown to reverse contractile dysfunction as well as
structural and molecular remodeling, and to improve heart failure
morbidity and mortality (Bristow, 2000); CIBIS-II Investigators and
Committees. The cardiac insufficiency bisoprolol study II: a
(CIBIS-II, 1999); MERIT-HF Study Group. Effect of metoprolol CR/XL
in chronic heart failure: Metoprolol CR/XL Randomized Intervention
Trial in Congestive Heart Failure (MERIT-HF, 1999). Packer et al.
(2001); BEST Trial Investigators, (2001); Lowes et al., 2002). In
part, these beneficial effects are thought to be due to a
protection of the failing heart, which has limited metabolic and
physiologic reserves, from persistent adverse biological effects
mediated by elevated norepinephrine levels found in the syndrome
(Bristow, 2000; Cohn et al., 1984; and Liggett, 2001). In addition,
.beta.-blockers have been shown to partially reverse the molecular
phenotype of heart failure (Lowes et al., 2002), so these agents
are capable of both preventing and reversing progressive myocardial
failure and remodeling Eichhorn and Bristow, Circulation 1996).
[0057] Bucindolol and metoprolol have some notable differences in
their pharmacologic properties (Bristow, 2000; and Bristow et al.,
1997). In particular, bucindolol lowers norepinephrine, dilates the
peripheral vasculature, and more potently blocks the human
.beta..sub.1-adrenergic receptor.
[0058] While a common pharmacologic property of all .beta.-blocking
agents that have been used to treat HF is that they block the
.beta..sub.1AR, which in the failing human heart has been estimated
to transduce up to approximately 90% of the pathologic adrenergic
stimulation (Zhu et al., 2001; and Bristow et al., 2003), the
available .beta.-blockers have a number of distinguishing
properties including .beta.AR-subtype selectivity, affinity for
.alpha..sub.1AR, partial agonist activity, sympatholysis (Bristow
et al., 2004) and vasodilation (Bristow, 2000; and Bristow et al.,
1997).
[0059] .beta.-blockers have significant structural differences.
Moreover, they have different pharmacological properties.
Carvedilol, for instance, is an efficient .beta..sub.1-AR and
.beta..sub.2-AR blocker, as well as an .alpha..sub.1-AR blocker. In
contrast, bucindolol is a weak .alpha..sub.1-AR blocker, and
metoprolol and bisoprolol do not block .alpha..sub.1-AR at all.
Significantly, bucindolol is unique among .beta.-blockers in its
sympatholytic properties, in contrast to carvedilol, metoprolol,
and bisoprolol, which have no such properties. Compared to other
.beta.-blocking agents bucindolol uniquely lowers systemic
norepinephrine levels (Lowes et al., 2000; Bristow et al., 1997;
BEST NEJM, Bristow, 2004), and is a full agonist for the
.beta..sub.3-adrenergic receptor (Strosberg, 1997).
[0060] 2. Bucindolol
[0061] Bucindolol is a 3rd generation, .beta.-blocker-vasodilator
with the chemical name and structure of
(2-{2-hydroxy-3{{2-(3-indolyl)-1,1-dimethylethyl}amino}propoxy}-benzonitr-
ile hydrochloride). It was first developed for treatment of
hypertension, and subsequently for the treatment of heart failure.
Because of its low inverse-agonist and vasodilator properties the
nonselective .beta.-blockade of bucindolol is relatively well
tolerated by heart failure patients, and in part for this reason in
1994 bucindolol was selected by the NIH and VA Cooperative Clinical
Trials Group to test the hypothesis that a .beta.-blocker could
reduce mortality in advanced heart failure. The test of this
hypothesis was the BEST Trial, which was conducted between May 31,
1995 and Jul. 29, 1999.
[0062] The Beta-blocker Evaluation of Survival Trial ("BEST") was
stopped prematurely on recommendation of the Data and Safety
Monitoring Committee, at a time when the hazard ratio for the
primary endpoint of all-cause mortality was apparently 0.90 (C.I.s
0.78-1.02) (BEST Investigators, 2001; Domanski et al., 2003).
However, the results for the entire BEST cohort were positive for
the high order secondary endpoint of mortality or heart failure
hospitalization, which was reduced by bucindolol by 19% with a
p-value of <0.0001 (Domanski et al., 2003). This endpoint is in
fact increasingly viewed as the preferred primary endpoint for HF
pivotal trials.
[0063] The reasons why BEST was stopped were 1) confirmation by
BEST Trial data generated in Class III, non-Black patients of the
then recently published information from CIBIS-II (CIBIS
Investigators, 1999) and MERIT-HF (MERIT-HF Study Group, 1999)
trials that these types of heart failure patients have a
substantial survival benefit from .beta.-blockade, 2) increasing
loss of equipoise among investigators, who believed that the
efficacy of .beta.-blockade in heart failure had been demonstrated,
and 3) inefficacy and trends toward adverse events in subgroups
(Class IV and Blacks) that had not been previously investigated in
.beta.-blocker heart failure trials. Further development of
bucindolol was then abandoned because it was not clear bucindolol
could be successfully marketed, even if approved.
[0064] Therefore, in this large survival trial in which the end
point evaluation was overall survival, the BEST clinical trial was
terminated early because of confirmation of benefit that had
recently been shown in other trials, and the inability to extend
the efficacy of bucindolol to patient subgroups that had not been
previously evaluated in large scale clinical trials (BEST
Investigators, 2001). At that time, there was no significant
difference in mortality observed between those treated with
bucindolol or with a placebo. In distinct contrast to the results
of BEST, similar studies with the .beta.-adrenergic antagonists
bisoprolol (termed "CIBIS-II" trial), metoprolol (termed "MERIT-HF"
trial), and carvedilol (termed "COPERNICUS" trial) reported very
favorable differences (34-35% reductions in mortality) between
those treated with the antagonists and those treated with a
placebo. The BEST investigators speculated that one possible
explanation for the difference in the results "may derive from the
unique pharmacological properties of bucindolol."
[0065] In the CIBIS-II trial, the study was also stopped early, but
because the mortality rates were significantly less in those
treated with bisoprolol. CIBIS-II Investigators, 1999. Similarly,
in the MERIT-HF study with metoprolol, the study was ended
prematurely because the predefined criterion had been met and
exceeded. MERIT-HF Study Group, 1999. The COPERNICUS study
involving carvedilol was also halted early because of the
significant benefits observed with treatment. Packer et al., 2001.
The BEST investigators noted that their results raised questions
about the equivalency of .beta.-blockers.
[0066] Therefore, there are therapeutic differences between
bucindolol and other .beta.-blockers, and there was a significant
question regarding the therapeutic efficacy of bucindolol overall.
Consequently, any relationship between bucindolol and particular
genetic variants was not evident.
[0067] The benefit of retrospective analysis based on the genetic
data disclosed herein highlights the unique pharmacologic features
of bucindolol that contribute to its effectiveness in treating
heart failure patients. Two of these features are also instrumental
in the interaction of the drug with the adrenergic receptor gene
variants.
[0068] The first of these features is sympatholysis, or the ability
of a drug to lower adrenergic drive directly (lower norepinephrine
levels in blood and tissue). As noted above, among .beta.-blockers
that have been used to treat heart failure, bucindolol is unique in
this regard (BEST Trial Investigators, 2001; Lowes et al., 2000;
Bristow et al., 2004). The sympatholytic effects of bucindolol are
likely due to .beta..sub.2-receptor blockade coupled with not
enough .alpha..sub.1-blockade to activate adrenergic drive. Other
properties of bucindolol that could contribute to sympatholysis are
nitric oxide generation and .beta..sub.3-receptor agonism
(Strosberg, 1997). These latter two properties, plus or minus weak
.alpha..sub.1-receptor blockade, likely account for the mild
vasodilator properties of bucindolol (Gilbert et al., 1990) which,
unlike carvedilol, are not sufficiently powerful to trigger reflex
adrenergic activation.
[0069] When present in modest amounts, (smaller reductions in
norepinephrine) sympatholysis is a favorable property, contributing
to the therapeutic anti-adrenergic effect of bucindolol. This is a
potentially superior mechanism of action to simple .beta.-blockade,
as excess norepinephrine is removed from the system. Norepinephrine
is toxic to heart muscle and in excess amounts triggers various
cardiac disease pathways. However, when exaggerated, sympatholysis
can be harmful, and can increase mortality (Bristow et al. 2004).
As discussed below, genetic targeting of bucindolol allows this
property to function only in a favorable manner.
[0070] The second pharmacologic property of bucindolol that
interacts with a pharmacogenetic target is high affinity
.beta..sub.1-receptor blockade (Hershberger et al., 1990; Asano et
al., 2001). Bucindolol has high affinity for human
.beta..sub.1-receptors, as well as for .beta..sub.2-receptors
(Hershberger et al., 1990). In addition, through a non-agonist
effect on either translation or protein turnover, bucindolol lowers
.beta..sub.1-receptor density (Asano et al., 2001). Because it is
so well tolerated, bucindolol can be administered at very high
.beta.-blocking doses, and in addition bucindolol uniquely
(compared to carvedilol or metoprolol) inactivates constitutively
active .beta..sub.1 389Arg/Arg receptors (Liggett et al., 2006;
Walsh et al., 2008) and each of these properties contributes to its
salutary effects on the high functioning human
.beta..sub.1-receptor 389Arg/Arg gene variant (Examples, Mason et
al., 1999; Liggett et al. 2006). Although bucindolol has intrinsic
symapthomimetic activity (ISA) in rat myocardium in functioning
human cardiac tissue bucindolol is devoid of ISA (Bristow et al.,
1994; Sederberg et al., 2000; Bristow et al., 1998, Example 7).
This can clearly be seen in FIG. 13, panels A and B, where no
significant increase in force development occurs in isolated
failing human right ventricular trabeculae, even in the presence of
signal transduction augmentation with the diterpene compound
forskolin, in either the .beta..sub.1AR Arg/Arg or Gly carrier
genotypes. In contrast, as shown in FIG. 13 panel C, xamoterol as a
positive control ISA compound exhibits an increase in force in both
low and high signal transduction activation in the .beta..sub.1AR
Arg/Arg genotype, but only in the high activation state rendered by
forskolin pretreatment in Gly carriers. Finally, as shown in FIG.
13, in preparations of isolated human heart, bucindolol has unique
effects on .beta..sub.1AR Arg/Arg vs Gly carrier receptors. Under
conditions of low levels of signal transduction (low receptor
activation) in the failing heart (Panel A), bucindolol functions as
a neutral antagonist (no agonist or inverse agonist activity) at
the human myocardial .beta..sub.1Arg/Arg receptor, but when signal
transduction is high as when adenylyl cyclase is directly activated
by forskolin (Panel B), bucindolol functions as an inverse agonist,
inactivating the receptor as indicated by a statistically
significant slope factor up to the highest concentration achievable
in plasma by therapeutic doses, 10-6 M. No such effect occurs in
Gly carrier receptors, where bucindolol functions as an inverse
agonist in low activation states, and a neutral antagonist in the
presence of forskolin. These data suggest that bucindolol is
uniquely effective in antagonizing high activation states of the
.beta..sub.1389Arg/Arg receptor, the form of the receptor that
would be expected to be the most cardiomyopathic.
[0071] These properties are likely reasons for the surprising and
unexpected results that were observed with the Arg389 genetic
variant in the .beta..sub.1AR and the Del322-325 genetic variant in
.alpha..sub.2cAR in the context of bucindolol treatment.
[0072] 3. Stereoisomers of Bucindolol
[0073] Racemic mixtures of bucindolol or its stereoisomers can be
obtained from commercial sources or can be produced by methods
well-known to those of ordinary skill in the art. Commercial
sources of bucindolol and its enantiomers include Knoll AG and
Bristol-Myers Squib Co. Information regarding synthesis of
bucindolol can be obtained from any of a variety of sources known
to those of ordinary skill in the art, such as U.S. Pat. No.
6,927,036 and WO 1987003584, each of which is herein specifically
incorporated by reference.
[0074] S-bucindolol can also be prepared by the resolution of
racemic materials, using conventional means such as fractional
crystallization, simple crystallization and chromatography on a
chiral substrate, extraction, distillation, column chromatography,
high performance liquid chromatography, and the like. Additional
information regarding preparation of S-bucindolol and separation of
S-bucindolol from a racemic mixture is discussed in the
specification below.
B. PREVENTION AND THERAPY
[0075] 1. Diseases to be Treated or Prevented
[0076] Some embodiments of the present invention concern methods of
treating a patient. The patient may have any disease or condition
for which treatment of S-bucindolol is indicated. For example, the
disease or condition may be one for which treatment with a
NO-enhancer is indicated. The disease or condition may be one for
which treatment with a beta-blocker is indicated. Examples of such
diseases and conditions are discussed elsewhere in this
specification.
[0077] "Treatment" and "treating" as used herein refer to
administration or application of a therapeutic agent to a subject
or performance of a procedure or modality on a subject for the
purpose of obtaining a therapeutic benefit of a disease or
health-related condition. For example, a pharmaceutical composition
that includes S-bucindolol may be administered to a subject to
reduce the symptoms of congestive heart failure.
[0078] The term "therapeutic benefit" or "therapeutically
effective" as used throughout this application refers to anything
that promotes or enhances the well-being of the subject with
respect to the medical treatment of this condition. This includes,
but is not limited to, a reduction in the frequency or severity of
the signs or symptoms of a disease. For example, reducing the
symptoms of congestive heart failure may include reducing
peripheral edema or increasing exercise tolerance.
[0079] Other embodiments of the present invention concern methods
of preventing a disease in a patient. "Prevention" and "preventing"
are used according to their ordinary and plain meaning to mean
"acting before" or such an act. In the context of a particular
disease or health-related condition, those terms refer to
administration or application of an agent, drug, or remedy to a
subject or performance of a procedure or modality on a subject for
the purpose of blocking the onset of a disease or health-related
condition. For example, a composition comprising S-bucindolol may
be administered to a patient to prevent onset of a myocardial
infarction or to prevent the development of symptoms associated
with congestive heart failure.
[0080] 2. Routes of Administration
[0081] Administration of the pharmaceutical compositions comprising
S-bucindolol set forth herein may be by any number of routes
including, but not limited to oral, intravenous, intramuscular,
intra-arterial, intramedullary, intrathecal, intraventricular,
intradermal, intratracheal, intravesicle, intraocular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.). In certain embodiments bucindolol is formulated for
oral administration.
[0082] 3. Formulations
[0083] Where clinical applications are contemplated, pharmaceutical
compositions will be prepared in a form appropriate for the
intended application. Generally, this will entail preparing
compositions that are essentially free of pyrogens, as well as
other impurities that could be harmful to humans or animals.
[0084] One will generally desire to employ appropriate salts and
buffers to render delivery vectors stable and allow for uptake by
target cells. Buffers also will be employed when recombinant cells
are introduced into a patient. Aqueous compositions of the present
invention comprise an effective amount of the vector or cells,
dissolved or dispersed in a pharmaceutically acceptable carrier or
aqueous medium. The phrase "pharmaceutically" or "pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce adverse, allergic, or other untoward reactions when
administered to an animal or a human. As used herein,
"pharmaceutically acceptable carrier" includes solvents, buffers,
solutions, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like
acceptable for use in formulating pharmaceuticals, such as
pharmaceuticals suitable for administration to humans. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active ingredients of the present
invention, its use in therapeutic compositions is contemplated.
Supplementary active ingredients also can be incorporated into the
compositions, provided they do not inactivate the vectors or cells
of the compositions.
[0085] The active compositions of the present invention may include
classic pharmaceutical preparations. Administration of these
compositions according to the present invention may be via any
common route so long as the target tissue is available via that
route. This includes oral, nasal, or buccal. Alternatively,
administration may be by intradermal, subcutaneous, intramuscular,
intraperitoneal or intravenous injection, or by direct injection
into cardiac tissue. Such compositions would normally be
administered as pharmaceutically acceptable compositions, as
described supra.
[0086] The active compounds may also be administered parenterally
or intraperitoneally. By way of illustration, solutions of the
active compounds as free-base or pharmacologically acceptable salts
can be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations generally contain a preservative to prevent the growth
of microorganisms.
[0087] The pharmaceutical forms suitable for injectable use
include, for example, sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. Generally, these preparations
are sterile and fluid to the extent that easy injectability exists.
Preparations should be stable under the conditions of manufacture
and storage and should be preserved against the contaminating
action of microorganisms, such as bacteria and fungi. Appropriate
solvents or dispersion media may contain, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetable oils. The proper fluidity can be maintained,
for example, 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. The prevention of the action of
microorganisms can be brought about by various antibacterial an
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0088] For oral administration the polypeptides of the present
invention generally may be incorporated with excipients and used in
the form of non-ingestible mouthwashes and dentifrices. A mouthwash
may be prepared incorporating the active ingredient in the required
amount in an appropriate solvent, such as a sodium borate solution
(Dobell's Solution). Alternatively, the active ingredient may be
incorporated into an antiseptic wash containing sodium borate,
glycerin and potassium bicarbonate. The active ingredient may also
be dispersed in dentifrices, including: gels, pastes, powders and
slurries. The active ingredient may be added in a therapeutically
effective amount to a paste dentifrice that may include water,
binders, abrasives, flavoring agents, foaming agents, and
humectants.
[0089] The compositions of the present invention generally may be
formulated in a neutral or salt form. Pharmaceutically-acceptable
salts include, for example, acid addition salts (formed with the
free amino groups of the protein) derived from inorganic acids
(e.g., hydrochloric or phosphoric acids, or from organic acids
(e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the free carboxyl groups of the protein can also be
derived from inorganic bases (e.g., sodium, potassium, ammonium,
calcium, or ferric hydroxides) or from organic bases (e.g.,
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0090] Upon formulation, solutions are preferably administered in a
manner compatible with the dosage formulation and in such amount as
is therapeutically effective. The formulations may easily be
administered in a variety of dosage forms such as injectable
solutions, drug release capsules and the like. For parenteral
administration in an aqueous solution, for example, the solution
generally is suitably buffered and the liquid diluent first
rendered isotonic for example with sufficient saline or glucose.
Such aqueous solutions may be used, for example, for intravenous,
intramuscular, subcutaneous and intraperitoneal administration.
Preferably, sterile aqueous media are employed as is known to those
of skill in the art, particularly in light of the present
disclosure. By way of illustration, a single dose may be dissolved
in 1 ml of isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject. Moreover, for human administration, preparations should
meet sterility, pyrogenicity, general safety and purity standards
as required by FDA Office of Biologics standards.
[0091] 4. Controlled/Extended/Sustained/Prolonged Release
Administration
[0092] Another aspect of this invention provides methods of
treating patients by delivering the pharmaceutical compositions set
forth herein as a controlled release formulation. As used herein,
the terms "controlled," "extended," "sustained," or "prolonged"
release of the composition of the present invention will
collectively be referred to herein as "controlled release," and
includes continuous or discontinuous, and linear or non-linear
release of the composition of the present invention. There are many
advantages for a controlled release formulation of
.beta.-blockers.
[0093] a. Tablets
[0094] A controlled release tablet suitable for purposes of this
invention is disclosed in U.S. Pat. No. 5,126,145, which is
incorporated by reference herein. This tablet comprises, in
admixture, about 5-30% high viscosity hydroxypropyl methyl
cellulose, about 2-15% of a water-soluble pharmaceutical binder,
about 2-20% of a hydrophobic component such as a waxy material,
e.g., a fatty acid, and about 30-90% active ingredient.
[0095] b. Films
[0096] This invention further provides a prophylaxis for or method
of treating a patient following an invasive cardiac procedure
comprising administering biodegradable, biocompatible polymeric
film comprising S-bucindolol, to a patient. The polymeric films are
thin compared to their length and breadth. The films typically have
a uniform selected thickness between about 60 micrometers and about
5 mm. Films of between about 600 micrometers and 1 mm and between
about 1 mm and about 5 mm thick, as well as films between about 60
micrometers and about 1000 micrometers, and between about 60 and
about 300 micrometers are useful in the manufacture of therapeutic
implants for insertion into a patient's body. The films can be
administered to the patient in a manner similar to methods used in
adhesion surgeries. For example, a .beta.-blocker, such as
bucindolol, film formulation can be sprayed or dropped onto a
cardiac tissue site or artery during surgery, or a formed film can
be placed over the selected tissue site. In an alternative
embodiment, the film can be used as controlled release coating on a
medical device such as a stent, as is discussed in further detail
below.
[0097] Either biodegradable or nonbiodegradable polymers may be
used to fabricate implants in which the .beta.-blocker is uniformly
distributed throughout the polymer matrix. A number of suitable
biodegradable polymers for use in making the biodegradable films of
this invention are known to the art, including polyanhydrides and
aliphatic polyesters, preferably polylactic acid (PLA),
polyglycolic acid (PGA) and mixtures and copolymers thereof, more
preferably 50:50 copolymers of PLA:PGA and most preferably 75:25
copolymers of PLA:PGA. Single enantiomers of PLA may also be used,
preferably L-PLA, either alone or in combination with PGA.
Polycarbonates, polyfumarates and caprolactones may also be used to
make the implants of this invention.
[0098] The amount of the S-bucindolol to be incorporated into the
polymeric films of this invention is an amount effective to show a
measurable effect in treating diseases having similar
pathophysiological states, such as but not limited to, heart
failure, pheochromocytoma, migraines, cardiac arrhythmias,
hypertension, aschemia, cardiomyopathy, and various anxiety
disorders. The composition of the present invention can be
incorporated into the film by various techniques such as by
solution methods, suspension methods, or melt pressing.
[0099] c. Transdermal Patch Device
[0100] Transdermal delivery involves delivery of a therapeutic
agent through the skin for distribution within the body by
circulation of the blood. Transdermal delivery can be compared to
continuous, controlled intravenous delivery of a drug using the
skin as a port of entry instead of an intravenous needle. The
therapeutic agent passes through the outer layers of the skin,
diffuses into the capillaries or tiny blood vessels in the skin and
then is transported into the main circulatory system.
[0101] Transdermal patch devices that provide a controlled,
continuous administration of a therapeutic agent through the skin
are also well known in the art. Such devices, for example, are
disclosed in U.S. Pat. Nos. 4,627,429; 4,784,857; 5,662,925;
5,788,983; and 6,113,940, which are all incorporated herein by
reference. Characteristically, these devices contain a drug
impermeable backing layer which defines the outer surface of the
device and a permeable skin attaching membrane, such as an adhesive
layer, sealed to the barrier layer in such a way as to create a
reservoir between them in which the therapeutic agent is placed. In
one embodiment of the present invention a formulation of the
.beta.-blocker is introduced into the reservoir of a transdermal
patch and used by a patient who is homozygous Arg389 at the
.beta..sub.1AR genes.
[0102] 5. Medical Devices
[0103] Another embodiment contemplates the incorporation of
S-bucindolol or a composition comprising S-bucindolol as set forth
herein into a medical device that is then positioned to a desired
target location within the body, whereupon the S-bucindolol elutes
from the medical device. As used herein, "medical device" refers to
a device that is introduced temporarily or permanently into a
mammal for the prophylaxis or therapy of a medical condition. These
devices include any that are introduced subcutaneously,
percutaneously or surgically to rest within an organ, tissue or
lumen. Medical devices include, but are not limited to, stents,
synthetic grafts, artificial heart valves, artificial hearts and
fixtures to connect the prosthetic organ to the vascular
circulation, venous valves, abdominal aortic aneurysm (AAA) grafts,
inferior venal caval filters, catheters including permanent drug
infusion catheters, embolic coils, embolic materials used in
vascular embolization (e.g., PVA foams), mesh repair materials, a
Dracon vascular particle orthopedic metallic plates, rods and
screws and vascular sutures.
[0104] In one embodiment, the medical device such as a stent or
graft is coated with a matrix. The matrix used to coat the stent or
graft according to this invention may be prepared from a variety of
materials. A primary requirement for the matrix is that it be
sufficiently elastic and flexible to remain unruptured on the
exposed surfaces of the stent or synthetic graft.
[0105] 6. Dosages
[0106] The amount of S-bucindolol or composition comprising
S-bucindolol that is administered or prescribed to the patient can
be about, at least about, or at most about 0.1, 0.5, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,
300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,
430, 440, 441, 450, 460, 470, 480, 490, 500 mg of total bucindolol
or S-bucindolol, or any range derivable therein. Alternatively, the
amount administered or prescribed may be about, at least about, or
at most about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007,
0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,
1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9,
4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg/kg of
total bucindolol or S-bucindolol, or any range derivable therein,
with respect to the weight of the patient.
[0107] When provided in a discrete amount, each intake of
S-bucindolol or composition comprising S-bucindolol can be
considered a "dose." A medical practitioner may prescribe or
administer multiple doses over a particular time course (treatment
regimen) or indefinitely.
[0108] The therapeutic composition may be administered 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40,
50, 60, 70, 80, or more times or any range derivable therein. It is
further contemplated that the drug may be taken for an indefinite
period of time or for as long as the patient exhibits symptoms of
the medical condition for which the therapeutic agent was
prescribed. Also, the drug may be administered every 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24 hours, or 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5
weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or
any range derivable therein. Alternatively, it may be administered
systemically over any such period of time and be extended beyond
more than a year.
C. OTHER THERAPEUTIC OPTIONS
[0109] In certain embodiments of the invention, methods may involve
administering a beta-blocker that is not bucindolol for the
treatment of a disease or disorder in a subject. These agents may
be prescribed or administered instead of or in addition to
bucindolol.
[0110] As a second therapeutic regimen, the agent may be
administered or taken at the same time as S-bucindolol, or either
before or after S-bucindolol. The treatment may improve one or more
symptoms of disease such as providing increased exercise capacity,
increased cardiac ejection volume, decreased left ventricular end
diastolic pressure, decreased pulmonary capillary wedge pressure,
increased cardiac output or cardiac index, lowered pulmonary artery
pressures, decreased left ventricular end systolic and diastolic
dimensions, decreased left and right ventricular wall stress,
decreased wall tension and wall thickness, increased quality of
life, and decreased disease-related morbidity and mortality.
[0111] In another embodiment, it is envisioned to use S-bucindolol
in combination with other therapeutic modalities. Thus, in addition
to the therapies described above, one may also provide to the
patient more "standard" pharmaceutical cardiac therapies. Examples
of other therapies include, without limitation, other beta
blockers, anti-hypertensives, cardiotonics, anti-thrombotics,
vasodilators, hormone antagonists, iontropes, diuretics, endothelin
antagonists, calcium channel blockers, phosphodiesterase
inhibitors, ACE inhibitors, angiotensin type 2 antagonists and
cytokine blockers/inhibitors, and HDAC inhibitors.
[0112] Combinations may be achieved by contacting cardiac cells
with a single composition or pharmacological formulation that
includes both agents, or by contacting the cell with two distinct
compositions or formulations, at the same time, wherein one
composition includes the expression construct and the other
includes the agent. Alternatively, the therapy using S-bucindolol
may precede or follow administration of the other agent(s) by
intervals ranging from minutes to weeks. In embodiments where the
other agent and expression construct are applied separately to the
cell, one would generally ensure that a significant period of time
did not expire between the time of each delivery, such that the
agent and expression construct would still be able to exert an
advantageously combined effect on the cell. In such instances, it
is contemplated that one would typically contact the cell with both
modalities within about 12-24 hours of each other and, more
preferably, within about 6-12 hours of each other, with a delay
time of only about 12 hours being most preferred. In some
situations, it may be desirable to extend the time period for
treatment significantly, however, where several days (2, 3, 4, 5, 6
or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administrations.
[0113] It also is conceivable that more than one administration of
either S-bucindolol, or the other agent will be desired. In this
regard, various combinations may be employed. By way of
illustration, where the S-bucindolol is "A" and the other agent is
"B", the following permutations based on 3 and 4 total
administrations are exemplary:
TABLE-US-00001 A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B
B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B
Other combinations are likewise contemplated.
[0114] 1. Pharmacological Therapeutic Agents
[0115] Pharmacological therapeutic agents and methods of
administration, dosages, etc., are well known to those of skill in
the art (see for example, the "Physicians Desk Reference",
Klaassen's "The Pharmacological Basis of Therapeutics",
"Remington's Pharmaceutical Sciences", and "The Merck Index,
Eleventh Edition", incorporated herein by reference in relevant
parts), and may be combined with the invention in light of the
disclosures herein. Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject, and such individual
determinations are within the skill of those of ordinary skill in
the art.
[0116] Non-limiting examples of a pharmacological therapeutic agent
that may be used in the present invention include an
antihyperlipoproteinemic agent, an antiarteriosclerotic agent, an
antithrombotic/fibrinolytic agent, a blood coagulant, an
antiarrhythmic agent, an antihypertensive agent, a vasopressor, a
treatment agent for congestive heart failure, an antianginal agent,
an antibacterial agent or a combination thereof.
[0117] In addition, it should be noted that any of the following
may be used to develop new sets of cardiac therapy target genes as
.beta.-blockers were used in the present examples (see below).
While it is expected that many of these genes may overlap, new gene
targets likely can be developed.
[0118] In certain embodiments, administration of an agent that
lowers the concentration of one of more blood lipids and/or
lipoproteins, known herein as an "antihyperlipoproteinemic," may be
combined with a cardiovascular therapy according to the present
invention, particularly in treatment of athersclerosis and
thickenings or blockages of vascular tissues. In certain aspects,
an antihyperlipoproteinemic agent may comprise an
aryloxyalkanoic/fibric acid derivative, a resin/bile acid
sequesterant, a HMG CoA reductase inhibitor, a nicotinic acid
derivative, a thyroid hormone or thyroid hormone analog, a
miscellaneous agent or a combination thereof.
[0119] Non-limiting examples of aryloxyalkanoic/fibric acid
derivatives include beclobrate, enzafibrate, binifibrate,
ciprofibrate, clinofibrate, clofibrate (atromide-S), clofibric
acid, etofibrate, fenofibrate, gemfibrozil (lobid), nicofibrate,
pirifibrate, ronifibrate, simfibrate and theofibrate.
[0120] Non-limiting examples of resins/bile acid sequesterants
include cholestyramine (cholybar, questran), colestipol (colestid)
and polidexide.
[0121] Non-limiting examples of HMG CoA reductase inhibitors
include lovastatin (mevacor), pravastatin (pravochol) or
simvastatin (zocor).
[0122] Non-limiting examples of nicotinic acid derivatives include
nicotinate, acepimox, niceritrol, nicoclonate, nicomol and
oxiniacic acid.
[0123] Non-limiting examples of thyroid hormones and analogs
thereof include etoroxate, thyropropic acid and thyroxine.
[0124] Non-limiting examples of miscellaneous
antihyperlipoproteinemics include acifran, azacosterol, benfluorex,
.beta.-benzalbutyramide, carnitine, chondroitin sulfate,
clomestrone, detaxtran, dextran sulfate sodium,
5,8,11,14,17-eicosapentaenoic acid, eritadenine, furazabol,
meglutol, melinamide, mytatrienediol, ornithine, .gamma.-oryzanol,
pantethine, pentaerythritol tetraacetate, .alpha.-phenylbutyramide,
pirozadil, probucol (lorelco), .beta.-sitosterol, sultosilic
acid-piperazine salt, tiadenol, triparanol and xenbucin.
[0125] Non-limiting examples of an antiarteriosclerotic include
pyridinol carbamate.
[0126] In certain embodiments, administration of an agent that aids
in the removal or prevention of blood clots may be combined with
administration of a modulator, particularly in treatment of
athersclerosis and vasculature (e.g., arterial) blockages.
Non-limiting examples of antithrombotic and/or fibrinolytic agents
include anticoagulants, anticoagulant antagonists, antiplatelet
agents, thrombolytic agents, thrombolytic agent antagonists or
combinations thereof.
[0127] In certain aspects, antithrombotic agents that can be
administered orally, such as, for example, aspirin and wafarin
(coumadin), are preferred.
[0128] A non-limiting example of an anticoagulant include
acenocoumarol, ancrod, anisindione, bromindione, clorindione,
coumetarol, cyclocumarol, dextran sulfate sodium, dicumarol,
diphenadione, ethyl biscoumacetate, ethylidene dicoumarol,
fluindione, heparin, hirudin, lyapolate sodium, oxazidione,
pentosan polysulfate, phenindione, phenprocoumon, phosvitin,
picotamide, tioclomarol and warfarin.
[0129] Non-limiting examples of antiplatelet agents include
aspirin, a dextran, dipyridamole (persantin), heparin,
sulfinpyranone (anturane) and ticlopidine (ticlid).
[0130] Non-limiting examples of thrombolytic agents include tissue
plaminogen activator (activase), plasmin, pro-urokinase, urokinase
(abbokinase) streptokinase (streptase), anistreplase/APSAC
(eminase).
[0131] In certain embodiments wherein a patient is suffering from a
hemmorage or an increased likelyhood of hemmoraging, an agent that
may enhance blood coagulation may be used. Non-limiting examples of
a blood coagulation promoting agent include thrombolytic agent
antagonists and anticoagulant antagonists.
[0132] Non-limiting examples of anticoagulant antagonists include
protamine and vitamine K1.
[0133] Non-limiting examples of thrombolytic agent antagonists
include amiocaproic acid (amicar) and tranexamic acid (amstat).
Non-limiting examples of antithrombotics include anagrelide,
argatroban, cilstazol, daltroban, defibrotide, enoxaparin,
fraxiparine, indobufen, lamoparan, ozagrel, picotamide, plafibride,
tedelparin, ticlopidine and triflusal.
[0134] Non-limiting examples of antiarrhythmic agents include Class
I antiarrythmic agents (sodium channel blockers), Class II
antiarrythmic agents (beta-adrenergic blockers), Class II
antiarrythmic agents (repolarization prolonging drugs), Class IV
antiarrhythmic agents (calcium channel blockers) and miscellaneous
antiarrythmic agents.
[0135] Non-limiting examples of sodium channel blockers include
Class IA, Class IB and Class IC antiarrhythmic agents. Non-limiting
examples of Class IA antiarrhythmic agents include disppyramide
(norpace), procainamide (pronestyl) and quinidine (quinidex).
Non-limiting examples of Class IB antiarrhythmic agents include
lidocaine (xylocaine), tocainide (tonocard) and mexiletine
(mexitil). Non-limiting examples of Class IC antiarrhythmic agents
include encainide (enkaid) and flecainide (tambocor).
[0136] Non-limiting examples of a beta blocker, otherwise known as
a .beta.-adrenergic blocker, a .beta.-adrenergic antagonist or a
Class II antiarrhythmic agent, include acebutolol (sectral),
alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol,
bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol,
bufuralol, bunitrolol, bupranolol, butidrine hydrochloride,
butofilolol, carazolol, carteolol, carvedilol, celiprolol,
cetamolol, cloranolol, dilevalol, epanolol, esmolol (brevibloc),
indenolol, labetalol, levobunolol, mepindolol, metipranolol,
metoprolol, moprolol, nadolol, nadoxolol, nifenalol, nipradilol,
oxprenolol, penbutolol, pindolol, practolol, pronethalol,
propanolol (inderal), sotalol (betapace), sulfinalol, talinolol,
tertatolol, timolol, toliprolol and xibinolol. In certain aspects,
the beta blocker comprises an aryloxypropanolamine derivative.
Non-limiting examples of aryloxypropanolamine derivatives include
acebutolol, alprenolol, arotinolol, atenolol, betaxolol,
bevantolol, bisoprolol, bopindolol, bunitrolol, butofilolol,
carazolol, carteolol, carvedilol, celiprolol, cetamolol, epanolol,
indenolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol,
nipradilol, oxprenolol, penbutolol, pindolol, propanolol,
talinolol, tertatolol, timolol and toliprolol.
[0137] Non-limiting examples of an agent that prolong
repolarization, also known as a Class III antiarrhythmic agent,
include amiodarone (cordarone) and sotalol (betap ace).
[0138] Non-limiting examples of a calcium channel blocker,
otherwise known as a Class IV antiarrythmic agent, include an
arylalkylamine (e.g., bepridile, diltiazem, fendiline, gallopamil,
prenylamine, terodiline, verapamil), a dihydropyridine derivative
(felodipine, isradipine, nicardipine, nifedipine, nimodipine,
nisoldipine, nitrendipine) a piperazinde derivative (e.g.,
cinnarizine, flunarizine, lidoflazine) or a micellaneous calcium
channel blocker such as bencyclane, etafenone, magnesium,
mibefradil or perhexiline. In certain embodiments a calcium channel
blocker comprises a long-acting dihydropyridine (nifedipine-type)
calcium antagonist.
[0139] Non-limiting examples of miscellaneous antiarrhymic agents
include adenosine (adenocard), digoxin (lanoxin), acecainide,
ajmaline, amoproxan, aprindine, bretylium tosylate, bunaftine,
butobendine, capobenic acid, cifenline, disopyranide,
hydroquinidine, indecainide, ipatropium bromide, lidocaine,
lorajmine, lorcainide, meobentine, moricizine, pirmenol,
prajmaline, propafenone, pyrinoline, quinidine polygalacturonate,
quinidine sulfate and viquidil.
[0140] Non-limiting examples of antihypertensive agents include
sympatholytic, alpha/beta blockers, alpha blockers,
anti-angiotensin II agents, beta blockers, calcium channel
blockers, vasodilators and miscellaneous antihypertensives.
[0141] Non-limiting examples of an alpha blocker, also known as an
.alpha.-adrenergic blocker or an .alpha.-adrenergic antagonist,
include amosulalol, arotinolol, dapiprazole, doxazosin, ergoloid
mesylates, fenspiride, indoramin, labetalol, nicergoline, prazosin,
terazosin, tolazoline, trimazosin and yohimbine. In certain
embodiments, an alpha blocker may comprise a quinazoline
derivative. Non-limiting examples of quinazoline derivatives
include alfuzosin, bunazosin, doxazosin, prazosin, terazosin and
trimazosin.
[0142] In certain embodiments, an antihypertensive agent is both an
alpha and beta adrenergic antagonist. Non-limiting examples of an
alpha/beta blocker comprise labetalol (normodyne, trandate).
[0143] Non-limiting examples of anti-angiotension II agents include
include angiotensin converting enzyme inhibitors and angiotension
II receptor antagonists. Non-limiting examples of angiotension
converting enzyme inhibitors (ACE inhibitors) include alacepril,
enalapril (vasotec), captopril, cilazapril, delapril, enalaprilat,
fosinopril, lisinopril, moveltopril, perindopril, quinapril and
ramipril. Non-limiting examples of an angiotensin II receptor
blocker, also known as an angiotension II receptor antagonist, an
ANG receptor blocker or an ANG-II type-1 receptor blocker (ARBS),
include angiocandesartan, eprosartan, irbesartan, losartan and
valsartan.
[0144] Non-limiting examples of a sympatholytic include a centrally
acting sympatholytic or a peripherially acting sympatholytic.
Non-limiting examples of a centrally acting sympatholytic, also
known as an central nervous system (CNS) sympatholytic, include
clonidine (catapres), guanabenz (wytensin) guanfacine (tenex) and
methyldopa (aldomet). Non-limiting examples of a peripherally
acting sympatholytic include a ganglion blocking agent, an
adrenergic neuron blocking agent, a .beta.-adrenergic blocking
agent or a alpha1-adrenergic blocking agent. Non-limiting examples
of a ganglion blocking agent include mecamylamine (inversine) and
trimethaphan (arfonad). Non-limiting of an adrenergic neuron
blocking agent include guanethidine (ismelin) and reserpine
(serpasil). Non-limiting examples of a .beta.-adrenergic blocker
include acenitolol (sectral), atenolol (tenormin), betaxolol
(kerlone), carteolol (cartrol), labetalol (normodyne, trandate),
metoprolol (lopressor), nadanol (corgard), penbutolol (levatol),
pindolol (visken), propranolol (inderal) and timolol (blocadren).
Non-limiting examples of alpha1-adrenergic blocker include prazosin
(minipress), doxazocin (cardura) and terazosin (hytrin).
[0145] In certain embodiments a cardiovasculator therapeutic agent
may comprise a vasodilator (e.g., a cerebral vasodilator, a
coronary vasodilator or a peripheral vasodilator). In certain
preferred embodiments, a vasodilator comprises a coronary
vasodilator. Non-limiting examples of a coronary vasodilator
include amotriphene, bendazol, benfurodil hemisuccinate,
benziodarone, chloracizine, chromonar, clobenfurol, clonitrate,
dilazep, dipyridamole, droprenilamine, efloxate, erythrityl
tetranitrane, etafenone, fendiline, floredil, ganglefene, herestrol
bis(.beta.-diethylaminoethyl ether), hexobendine, itramin to
sylate, khellin, lidoflanine, mannitol hexanitrane, medibazine,
nicorglycerin, pentaerythritol tetranitrate, pentrinitrol,
perhexiline, pimefylline, trapidil, tricromyl, trimetazidine,
trolnitrate phosphate and visnadine.
[0146] In certain aspects, a vasodilator may comprise a chronic
therapy vasodilator or a hypertensive emergency vasodilator.
Non-limiting examples of a chronic therapy vasodilator include
hydralazine (apresoline) and minoxidil (loniten). Non-limiting
examples of a hypertensive emergency vasodilator include
nitroprusside (nipride), diazoxide (hyperstat IV), hydralazine
(apresoline), minoxidil (loniten) and verapamil.
[0147] Non-limiting examples of miscellaneous antihypertensives
include ajmaline, .gamma.-aminobutyric acid, bufeniode,
cicletainine, ciclosidomine, a cryptenamine tannate, fenoldopam,
flosequinan, ketanserin, mebutamate, mecamylamine, methyldopa,
methyl 4-pyridyl ketone thiosemicarbazone, muzolimine, pargyline,
pempidine, pinacidil, piperoxan, primaperone, a protoveratrine,
raubasine, rescimetol, rilmenidene, saralasin, sodium nitrorus
side, ticrynafen, trimethaphan camsylate, tyrosinase and
urapidil.
[0148] In certain aspects, an antihypertensive may comprise an
arylethanolamine derivative, a benzothiadiazine derivative, a
N-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine
derivative, a guanidine derivative, a hydrazines/phthalazine, an
imidazole derivative, a quanternary ammonium compound, a reserpine
derivative or a suflonamide derivative.
[0149] Non-limiting examples of arylethanolamine derivatives
include amosulalol, bufuralol, dilevalol, labetalol, pronethalol,
sotalol and sulfinalol.
[0150] Non-limiting examples of benzothiadiazine derivatives
include althizide, bendroflumethiazide, benzthiazide,
benzylhydrochlorothiazide, buthiazide, chlorothiazide,
chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide,
epithiazide, ethiazide, fenquizone, hydrochlorothizide,
hydroflumethizide, methyclothiazide, meticrane, metolazone,
paraflutizide, polythizide, tetrachlormethiazide and
trichlormethiazide.
[0151] Non-limiting examples of N-carboxyalkyl(peptide/lactam)
derivatives include alacepril, captopril, cilazapril, delapril,
enalapril, enalaprilat, fosinopril, lisinopril, moveltipril,
perindopril, quinapril and ramipril.
[0152] Non-limiting examples of dihydropyridine derivatives include
amlodipine, felodipine, isradipine, nicardipine, nifedipine,
nilvadipine, nisoldipine and nitrendipine.
[0153] Non-limiting examples of guanidine derivatives include
bethanidine, debrisoquin, guanabenz, guanacline, guanadrel, gu
anazodine, guanethidine, guanfacine, guanochlor, guanoxabenz and
guanoxan.
[0154] Non-limiting examples of hydrazines/phthalazines include
budralazine, cadralazine, dihydralazine, endralazine,
hydracarbazine, hydralazine, pheniprazine, pildralazine and
todralazine.
[0155] Non-limiting examples of imidazole derivatives include
clonidine, lofexidine, phentolamine, tiamenidine and
tolonidine.
[0156] Non-limiting examples of quanternary ammonium compounds
include azamethonium bromide, chlorisondamine chloride,
hexamethonium, pentacynium bis(methylsulfate), pentamethonium
bromide, pentolinium tartrate, phenactropinium chloride and
trimethidinium methosulfate.
[0157] Non-limiting examples of reserpine derivatives include
bietaserpine, deserpidine, rescinnamine, reserpine and
syrosingopine.
[0158] Non-limiting examples of sulfonamide derivatives include
ambuside, clopamide, furosemide, indapamide, quinethazone,
tripamide and xipamide.
[0159] Vasopressors generally are used to increase blood pressure
during shock, which may occur during a surgical procedure.
Non-limiting examples of a vasopressor, also known as an
antihypotensive, include amezinium methyl sulfate, angiotensin
amide, dimetofrine, dopamine, etifelmin, etilefrin, gepefrine,
metaraminol, midodrine, norepinephrine, pholedrine and
synephrine.
[0160] Non-limiting examples of agents for the treatment of
congestive heart failure include anti-angiotension II agents,
afterload-preload reduction treatment, diuretics and inotropic
agents.
[0161] In certain embodiments, an animal patient that cannot
tolerate an angiotension antagonist may be treated with a
combination therapy. Such therapy may combine adminstration of
hydralazine (apresoline) and isosorbide dinitrate (isordil,
sorbitrate).
[0162] Non-limiting examples of a diuretic include a thiazide or
benzothiadiazine derivative (e.g., althiazide, bendroflumethazide,
benzthiazide, benzylhydrochlorothiazide, buthiazide,
chlorothiazide, chlorothiazide, chlorthalidone, cyclopenthiazide,
epithiazide, ethiazide, ethiazide, fenquizone, hydrochlorothiazide,
hydroflumethiazide, methyclothiazide, meticrane, metolazone,
paraflutizide, polythizide, tetrachloromethiazide,
trichlormethiazide), an organomercurial (e.g., chlormerodrin,
meralluride, mercamphamide, mercaptomerin sodium, mercumallylic
acid, mercumatilin dodium, mercurous chloride, mersalyl), a
pteridine (e.g., furterene, triamterene), purines (e.g.,
acefylline, 7-morpholinomethyltheophylline, pamobrom,
protheobromine, theobromine), steroids including aldosterone
antagonists (e.g., canrenone, oleandrin, spironolactone), a
sulfonamide derivative (e.g., acetazolamide, ambuside, azosemide,
bumetanide, butazolamide, chloraminophenamide, clofenamide,
clopamide, clorexolone, diphenylmethane-4,4'-disulfonamide,
disulfamide, ethoxzolamide, furosemide, indapamide, mefruside,
methazolamide, piretanide, quinethazone, torasemide, tripamide,
xipamide), a uracil (e.g., aminometradine, amisometradine), a
potassium sparing antagonist (e.g., amiloride, triamterene) or a
miscellaneous diuretic such as aminozine, arbutin, chlorazanil,
ethacrynic acid, etozolin, hydracarbazine, isosorbide, mannitol,
metochalcone, muzolimine, perhexiline, ticrnafen and urea.
[0163] Non-limiting examples of a positive inotropic agent, also
known as a cardiotonic, include acefylline, an acetyldigitoxin,
2-amino-4-picoline, amrinone, benfurodil hemisuccinate,
bucladesine, cerberosine, camphotamide, convallatoxin, cymarin,
denopamine, deslanoside, digitalin, digitalis, digitoxin, digoxin,
dobutamine, dopamine, dopexamine, enoximone, erythrophleine,
fenalcomine, gitalin, gitoxin, glycocyamine, heptaminol,
hydrastinine, ibopamine, a lanatoside, metamivam, milrinone,
nerifolin, oleandrin, ouabain, oxyfedrine, prenalterol,
proscillaridine, resibufogenin, scillaren, scillarenin,
strphanthin, sulmazole, theobromine and xamoterol.
[0164] In particular aspects, an intropic agent is a cardiac
glycoside, a beta-adrenergic agonist or a phosphodiesterase
inhibitor. Non-limiting examples of a cardiac glycoside includes
digoxin (lanoxin) and digitoxin (crystodigin). Non-limiting
examples of a .beta.-adrenergic agonist include albuterol,
bambuterol, bitolterol, carbuterol, clenbuterol, clorprenaline,
denopamine, dioxethedrine, dobutamine (dobutrex), dopamine
(intropin), dopexamine, ephedrine, etafedrine, ethylnorepinephrine,
fenoterol, formoterol, hexoprenaline, ibopamine, isoetharine,
isoproterenol, mabuterol, metaproterenol, methoxyphenamine,
oxyfedrine, pirbuterol, procaterol, protokylol, reproterol,
rimiterol, ritodrine, soterenol, terbutaline, tretoquinol,
tulobuterol and xamoterol. Non-limiting examples of a
phosphodiesterase inhibitor include amrinone (inocor).
[0165] Antianginal agents may comprise organonitrates, calcium
channel blockers, beta blockers and combinations thereof.
[0166] Non-limiting examples of organonitrates, also known as
nitrovasodilators, include nitroglycerin (nitro-bid, nitrostat),
isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate
(aspirol, vaporole).
[0167] 2. Surgical Therapeutic Agents
[0168] In certain aspects, the secondary therapeutic agent may
comprise a surgery of some type, which includes, for example,
preventative, diagnostic or staging, curative and palliative
surgery. Surgery, and in particular a curative surgery, may be used
in conjunction with other therapies, such as the present invention
and one or more other agents.
[0169] Such surgical therapeutic agents for vascular and
cardiovascular diseases and disorders are well known to those of
skill in the art, and may comprise, but are not limited to,
performing surgery on an organism, providing a cardiovascular
mechanical prostheses, angioplasty, coronary artery reperfusion,
catheter ablation, providing an implantable cardioverter
defibrillator to the subject, mechanical circulatory support or a
combination thereof. Non-limiting examples of a mechanical
circulatory support that may be used in the present invention
comprise an intra-aortic balloon counterpulsation, left ventricular
assist device or combination thereof.
D. EXAMPLES
[0170] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Effects of Bucindolol and its Enantiomers on Nitric Oxide and
Peroxynitrite Release from White, African American and Mexican
American Endothelial Cells: Comparison to Atenolol
Methods
[0171] Donors and Cell Cultures. Human umbilical vein endothelial
cells were isolated into primary cultures from healthy female
donors by Clonetics (San Diego, Calif.) and purchased as
proliferating cells. All cell culture donors were healthy, with no
pregnancy or prenatal complications. None of the donors took any
drugs regularly and all were nonsmokers and consumed regular
caloric/content diet. The cultured cells were incubated in 95%
air/5% CO.sub.2 at 37.degree. C. and passage by an enzymatic
(trypsin) procedure. The confluent cells (4 to 5.times.10.sup.5
cells per 35-mm dish) were placed with minimum essential medium
containing 3 mM L-arginine and 0.1 mM
(6R)-5,6,7,8-tetrahydrobiopterin (BH.sub.4). Before the
experiments, the cells (from second or third passage) were rinsed
twice with Tyrode-HEPES buffer with 1.8 mM CaCl2. Bucindolol and
its enanatiomers were obtained from Arca Discovery (Denver,
Colo.).
[0172] Measurement of NO and ONOO.sup.- Levels. Measurement of NO
was carried out with electrochemical nanosensors (FIG. 1). Their
design was based on previously developed and well-characterized
chemically modified carbon-fiber technology (Lvovich and Scheeline,
1997; Malinski and Taha, 1992). Each of the nanosensors was
constructed by depositing a sensing material on the tip of a carbon
fiber (length 4-5 .mu.m, diameter 0.2-0.5 .mu.m). The fibers were
sealed with nonconductive epoxy and electrically connected to
copper wires with conductive silver epoxy. The inventors used a
conductive film of polymeric nickel (II) tetrakis
(3-methoxy-4-hydroxyphenyl) porphyrin for the NO-sensor.
[0173] The NO nanosensors (diameter 1-2 .mu.m) with a platinum wire
(0.1 mm) counter electrode and saturated calomel reference
electrode (SCE) were applied. Differential pulse voltammetry (DPV)
and amperometry were performed with a computer-based Gamry VFP600
multichannel potentiostat. DPV was used to measure the basal NO
concentrations, and amperometry was used to measure changes in NO
concentrations from its basal level with time. The DPV current at
the peak potential characteristic for NO oxidation (0.65 V)
reduction was directly proportional to the local concentrations of
these compounds in the immediate vicinity of the sensor. Linear
calibration curves (current vs. concentration) were constructed for
each sensor from 10 nM to 3 .mu.M before and after measurements
with aliquots of NO standard solutions, respectively. The detection
limit of the sensors was 1.0 nM.
[0174] The quantification of each analyte (concentration in nmol/L)
was performed using a maximum current from amperograms and standard
calibration curves. The reproducibility of measurements with
nanosensors is relatively high, as previously described (Lvovich
and Scheeline, 1997; Malinski and Taha, 1992). The NO nanosensor
modules were lowered with the help of a computer-controlled
micromanipulator until it reached the surface of the cell membrane
(a small piezoelectric signal, 0.1-0.2 pA, of 1-3 milliseconds
duration was observed at this point). The sensors were slowly
raised 5.+-.2 .mu.m from the surface of a single endothelial
cell.
[0175] The HUVEC preparation is stable over the course of these
experiments with the cells remaining viable and active responses to
NO stimulation in culture for >24 hours. For robust statistical
analysis, randomly selected cells were used for each concentration
and type of drug used in these analyses.
Results
[0176] This study demonstrated reduced NO bioavailability in tissue
from patients in high-risk populations for hypertension, such as
African Americans and Mexican Americans. As shown in FIG. 2,
endothelial-dependent NO release from Mexican American and African
American donors was 25% (305.+-.28 nM, mean.+-.S.D.) and 39%
(251.+-.27 nM) lower, respectively, than in non-Hispanic whites
(409.+-.23 nM), following stimulation with a receptor-independent
stimulus (1.0 .mu.M CaI).
[0177] Treatment with the .beta..sub.1-selective antagonist
bucindolol, and especially its active enantiomer (S)-bucindolol,
caused a dose-dependent increase in the capacity of the endothelium
to generate NO (FIGS. 3-5). Pretreatment (6 hr) of the cells with
bucindolol racemate (1.0 .mu.M) increased NO release and enhanced
endothelial activity in white donors (16% to 475.+-.27 nM), Mexican
American donors (5% to 321.+-.28 nM), and African American donors
(22% to 306.+-.25 nM).
[0178] In addition to reduced NO production, cells from these
racial groups showed evidence of increased nitroxidative stress.
The release of ONOO.sup.- from Mexican American and African
American donors was higher by 57% (493.+-.26 nM) and 37% (428.+-.26
nM), respectively, than in non-Hispanic white donors (313.+-.30
nM), following stimulation with CaI (FIG. 6). Treatment with the
bucindolol (1.0 M), and especially its active enantiomer
(S)-bucindolol, caused a pronounced reduction in nitroxidative
stress. Bucindolol reduced ONOO.sup.- levels by 19% (313.+-.30 nM
to 244.+-.29 nM) in cells from non-Hispanic white donors, by 14%
(493.+-.26 nM to 422.+-.27 nM) in cells from Mexican Americans and
by 12% in cell from African Americans (428.+-.26 nM to 378.+-.30
nM) as shown in FIGS. 7-9. The favorable effects of bucindolol were
much more apparent at 10.0 .mu.M.
[0179] As compared to bucindolol racemate, a favorable effect on
endothelial function was more pronounced with the active
1-selective enantiomer, (S)-bucindolol, while less activity was
seen with (R)-bucindolol. The differences in these enanatiomers
were especially evident in cells from non-Hispanic white donors.
Pretreatment (6 hr) of the cells with (S)-bucindolol (1.0 .mu.M)
increased NO release and enhanced endothelial activity in white
donors (35% to 553.+-.35 nM), Mexican American donors (17% to
358.+-.32 nM), and African American donors (27% to 318.+-.16 nM).
The (S)-bucindolol also reduced ONOO.sup.- levels by 23% to
240.+-.24 nM in cells from non-Hispanic white donors as well as 15%
to 419.+-.34 nM in cells from Mexican Americans and 18% in cell
from African Americans to 352.+-.26 nM (FIGS. 7-9).
[0180] The most comprehensive measurement of endothelial function
is the ratio of NO to ONOO.sup.- following treatment with these
agents. This study demonstrated large differences in endothelial
function in tissue from higher risk populations for hypertension,
such as African Americans and Mexican Americans (FIG. 10). The
basis for these differences is not understood but may be due to
genetic variability in the eNOS protein. Bucindolol racemate (1.0
.mu.M) increased the NO/ONOO.sup.- ratio in white donors (49%),
Mexican American donors (23%), and African American donors (38%) as
shown in FIGS. 11-13. An even greater effect was observed with
(S)-bucindolol. Again, the favorable effects of bucindolol and its
enanatiomers were much more apparent at the higher dose (10.0
.mu.M) by at least two fold. The effect at the high concentration
was especially evident in higher risks populations (African
Americans, Mexican Americans). In contrast, atenolol failed to
produce an effect even at the higher dose.
[0181] In summary, bucindolol had a dual effect on endothelial
function by increasing the capacity of cells to generate NO while
simultaneously reducing ONOO.sup.- production. The effect of
bucindolol on endothelial function was dose-dependent and
stereoselective; greater NO bioavailability was associated with its
active enantiomer, (S)-bucindolol. The basis for the benefit with
bucindolol may be due to interactions with novel receptor sites,
such as the beta3 adrenergic receptor, along its property of
inhibition of oxidative stress mechanisms (e.g., NADPH oxidase).
The favorable activity of bucindolol on endothelial function was
observed in three different racial groups, including African
Americans, Mexican Americans and non-Hispanic whites. The activity
of (S)-bucindolol was superior to bucindolol racemate and the
1-selective blocker atenolol.
Example 2
Stereospecificity of Bucindolol Binding to Human
.beta..sub.1-Adrenergic Receptors
[0182] FIG. 14 gives the binding of S- and R-bucindolol in left
ventricular membranes prepared from one patient who had the
.beta..sub.1 389 Arg/Arg receptor (FIG. 14A), and another with the
Gly/Gly (FIG. 14B) genotype. As can be observed, the S-isomer has
much higher affinity, in both genotypes. In addition, the K.sub.is
are similar for both the S- (0.5-0.6 nM) and R- (around 14-26 nM)
isomers in the Arg/Arg and Gly/Gly preparations. Similar results
were obtained in the presence of Gpp(NH)p.
Example 3
Separation of the R- and S-Enantiomers of Bucindolol
Hydrochloride
[0183] Chemistry. The usual procedure for the resolution of racemic
amine derivatives involves fractional recrystallization of a
mixture of the diastereomeric salts formed by combination of the
base with an optically active acid (e.g. d-tartaric). With
bucindolol, however, this process proved too tedious and time
consuming to be practical for medium scale work (5-25 g), and an
alternate method was sought.
[0184] Racemic alcohols have been resolved by separation of the
corresponding diastereomeric carbamates produced by reaction with
optically active arylalkyl isocyanates (Pirkle and Hoekstra, 1974;
Pirkle and Hauske, 1977a; Pirkle and Hauske, 1977b). Bucindolol,
with a secondary hydroxyl group in the side chain, would seem
ideally suited for reaction with this type of reagent. The reaction
of bucindolol with (R)-(-)-(1-naphthyl)ethyl isocyanate, however,
does not provide the anticipated carbamates but gives a pair of
diastereomeric urea (Kolomiets et al., 1980) derivatives
incorporating the basic nitrogen (Scheme 1). The greater reactivity
of the basic nitrogen is not overcome by steric hindrance from the
bulky N-substituent. Resolution is achieved via these ureas.
[0185] As the benzene solubility of one of the two isomeric ureas
is significantly less than the other, the initial separation
becomes relatively trivial and the less soluble isomer is obtained
in excellent purity. The residual material affords, by gravity
column chromatography, the companion isomer in very satisfactory
yield.
[0186] Generation of the individual optical antipodes is
accomplished easily by stirring each diastereomer with hydrazine
hydrate in ethanol solution. Treatment with pyruvic acid in this
last step effectively removes the
N-[(1-naphthyl)ethyl]hydrazinecarboxamide by-product and excess
hydrazine.
[0187] Thus the resolution of bucindolol into its respective
enantiomers is achieved via a facile new method that should be of
general use for new .beta.-blocker molecules in the future.
[0188] Assignment of absolute configuration to the enantiomers of
bucindolol (MJ 13105) is tentative, and based on the usual
assignment of S-configuration to the .beta.-adrenergic
aryloxypropanolamine enantiomer with negative rotation (Danilewicz
and Kemp, 1973).
[0189] Experimental. Melting points were determined using a
Thomas-Hoover capillary melting point apparatus and are
uncorrected. Analytical values of carbon, hydrogen, and nitrogen
are within 0.4% of theory and NMR, IR, and MS spectra are
consistent with the assigned structures. Optical rotation
measurements were obtained on a Bendix-NPL 1169 automatic
polarimeter with digital readout.
[0190] Silica gel 60 (EM Reagents) was used for column
chromatography.
[0191] The (g)-(-)-(1-naphthyl)ethyl isocyanate was purchased from
Aldrich Chemical Co.
[0192]
2-[2-Hydroxy-3-[[2-(1H-indol-3-yl)-1,1-dimethylethyl]amino]propoxy.-
about.benzo-nitrile (MJ 13105 free base). A hot solution of
bucindolol hydrochloride salt (100 g, 0.28 mol) and 2.5 l of
H.sub.2O was basified with a 10% solution of NaOH. After cooling,
the aqueous layer was decanted, and the residual gum rinsed with
H.sub.2O and crystallized from i-PrOH (500 mL) to provide 81 g of
MJ 13105 free base: mp 126-128.degree. C.
[0193] The aqueous layer was allowed to stand overnight at
25.degree. C., and the precipitate was collected by filtration,
washed with H.sub.2O, and air dried overnight to give a 3.5 g
second crop of MJ 13105 free base: mp 125-127.degree. C.
[0194] (S), (R) and (R),
(R)--N-[3-(2-Cyanophenoxy)-2-hydroxypropyl]-N-1,1-dimethyl-2-(1H-indol-3--
yl)ethyl]-N'-[1-(1-naphthyl)ethyl]urea. A mixture of
(R,S)-bucindolol (MJ 13105) free base (1.8 g, 0.0051 mol),
(R)-(-)-1-(1-naphthyl)-ethyl isocyanate (1.0 g, 0.0051 mol), and
benzene (100 mL) was stirred at 25.degree. C. for 6 h. The white
solid was removed by filtration and air dried to give 1.24 g of
(S),
(R)--N-[3-(2-cyanophenoxy)-2-hydroxypropyl]-N-[1,1-dimethyl-2-(1H-indol-3-
-yl)ethyl]-N'-[1-(1-naphthyl)ethyl]urea: mp 167-168.degree. C., one
spot on TLC (silica gel;
CH 2 Cl 2 / EtOAc , 9 : 1 ) , [ .alpha. ] 25 D - 14 .degree. ( C
0.5 % , CH 3 OH ) . ##EQU00001##
[0195] Anal. Calcd. for C.sub.35H.sub.36N.sub.4O.sub.3: C, 74.98;
H, 6.48; N, 10.00. Found: C, 74.89; H, 6.46; N, 9.74.
[0196] The filtrate was concentrated to dryness and the residue
chromatographed on silica gel with CH.sub.2Cl.sub.2EtOAc (9:1) to
give 0.70 g of
(R),(R)--N-[3-(2-cyanophenoxy)-2-hydroxypropyl]-N-[1,1-dimethyl-
-2-(1H-indol-3-yl)ethyl]-N'-[1-(1-naphthyl)ethyl urea as a
foam:
[ .alpha. ] 25 D - 119 .degree. ( C 0.5 % , CH 3 OH ) .
##EQU00002##
[0197] Anal. Calcd for C.sub.35H.sub.36N.sub.4O.sub.3-1/2 EtOAc:
C,73.49; H, 6.67; N, 9.27. Found: C, 73.29; H, 6.60; N, 9.18.
[0198] (S)-(-) and
(R)-(+)-2-[2-Hydroxy-3-[[2-(1H-indol-3-yl)-1,1-dimethylethyl]-amino]propo-
xy]benzonitrile cyclamate (MJ 13105-163-997 and MJ 13105-163-998).
The respective diastereomer of the urea derivative was heated at
reflux for 0.5 h in absolute EtOH with five equivalents of 99%
hydrazine hydrate. After evaporation of the solvent at reduced
pressure, the residue was dissolved in CH.sub.3CN, and five
equivalents of pyruvic acid were added. The solution was stirred at
25.degree. C. overnight and concentrated at reduced pressure to
give a residue that was dissolved in EtOAc. The EtOAc solution was
washed with three portions each of 1 N NaOH and H.sub.2O, dried
(anhyd. MgSO.sub.4), filtered, and concentrated. One equivalent of
cyclohexanesulfamic acid was added to a solution of the weighed
residue and absolute EtOH. After the mixture had cooled, the
precipitated salt was collected by filtration. Recrystallization
from EtOH-(i-Pr).sub.2O-(Darco G-60) gave the analytically pure
samples of each isomer.
( s ) - ( - ) - isomer , mp 18 O - 181 .degree. C . , [ .alpha. ]
25 D - 15.0 .degree. ( C 1 , CH 3 OH ) . ##EQU00003##
[0199] Anal. Calcd for C.sub.22H.sub.25N.sub.3O.sub.2
C.sub.6H.sub.13NO.sub.3S: C, 61.98; H, 7.06; N, 10.33. Found: C,
62.12; H, 7.08; N, 10.31.
( R ) - ( + ) - isomer , - mp 179 - 18 O .degree. C . [ .alpha. ]
25 D + 15.5 .degree. ( C 1 , CH 3 OH ) . ##EQU00004##
[0200] Anal. Calcd for C.sub.22H.sub.25N.sub.3O.sub.2
C.sub.6H.sub.13NO.sub.3S: C, 61.98; H, 7.06; N, 10.33. Found: C,
62-.07; H, 7.14; N, 10.11.
TABLE-US-00002 SCHEME 1 ##STR00001## ##STR00002##
(R)-(+)-bucindolol ##STR00003## diastereomer (R), (R) and
(S)-(-)-bucindolol ##STR00004## diastereomer (S), (R)
Example 4
Determination of Percent (R)- and (S)-Bucindolol Present in
Bucindolol Drug Substance and Drug Products by HPLC
Summary
[0201] Chiral separation of (R)- and (S)-bucindolol is achieved by
HPLC for the purpose of determining the percent ratios present in
drug substance or drug products (tablets or capsules).
Equipment (as Stated Below, or Equivalent)
[0202] HPLC system (equipped with a UV detector capable of
detection at 220 nm)
[0203] (Accompanying data systems are acceptable for
quantitation)
[0204] Chiral Technologies, Inc. Chiralpak AD (4.6 mm.times.25 cm)
column
[0205] 0.2 .mu.m filter, Acrodisc CR PTFE 0.2 .mu.m Product No.
4225 25 mm
Reagents
[0206] Reagent Alcohol (Ethanol), HPLC grade
[0207] Diethylamine, reagent grade
Reference Standards
[0208] (R)-Bucindolol HCl Reference Standard and (S)-Bucindolol HCl
Reference Standard or racemic Bucindolol HCl Reference Standard
Preparation of Solutions
[0209] Note: Prepare solutions in sufficient quantities to meet the
needs of the analysis use appropriate proportions of solutions to
maintain the ratio of the final solution.
[0210] Mobile Phase
[0211] Add 1 mL of diethylamine (DEA) to every 500 mL of ethanol.
Mix, filter, and degas under vacuum with sonication.
[0212] System Suitability Solution
[0213] Accurately weight 25.+-.1 mg of (R)-bucindolol and 25.+-.1
mg of (S)-bucindolol into the same 100 mL volumetric flask and
dissolve in mobile phase. Transfer 3 mL to a 10 mL volumetric flask
and dilute to volume with mobile phase.
[0214] Alternatively, weigh approximately 50 mg of racemic
bucindolol HCl reference standard into a 100 mL volumetric flask
and prepare as above.
Preparation of Samples
[0215] Prepare these solutions in duplicate.
[0216] Preparation of Drug Substance Samples
[0217] Accurately weigh 25.+-.1 mg of the drug substance sample
into a 50 mL volumetric flask and dissolve in mobile phase.
Transfer 3 mL to a 10 mL volumetric flask and dilute to volume with
mobile phase. Transfer the 10 mL aliquot to a disposable syringe
that is fitted with an 0.2 pm filter. Spend the first 2 mL to waste
and collect the remaining portion for analysis.
[0218] Preparation of Tablet Samples
[0219] Accurately weigh 20 tablets individually and determine the
average tablet weight. Grind the tablets as a composite. From the
composite, accurately weigh the equivalent of one tablet into an
appropriate volumetric flask (V1 in Table 1). Dilute to volume with
mobile phase and sonicate for 5 minutes. Transfer (T1) mL to an
appropriate volumetric flask (V2) and dilute to volume with mobile
phase (no further dilution is required for some doses, as indicated
by NA in the table). Transfer 10 mL of the final solution to a
disposable syringe that is fitted with an 0.2 .mu.m filter. Spend
the first 2 mL to waste and collect the remaining portion for
analysis.
TABLE-US-00003 TABLE 1 Dose Initial Volume Transfer Volume Final
Volume (mg) (V1) (mL) (T1) (mL) (V2) (mL) 3.0 25 NA NA 6.25 50 NA
NA 12.5 100 NA NA 25.0 50 6 25 50.0 100 6 25 100 250 3 10
[0220] Preparation of Capsule Samples
[0221] Empty the contents of 20 capsules into a vial and mix to
obtain a composite. From the composite, accurately weigh 500 mg
into an appropriate volumetric flask (V1 in Table 2). Dilute to
volume with mobile phase and sonicate for 5 minutes. Transfer (T1)
mL to an appropriate volumetric flask (V2) and dilute to volume
with mobile phase (no further dilution is required for some doses,
as indicated by NA in the table). Transfer 10 mL of the final
solution to a disposable syringe that is fitted with an 0.2 .mu.m
filter. Spend the first 2 mL to waste and collect the remaining
portion for analysis.
TABLE-US-00004 TABLE 2 Dose Initial Volume Transfer Volume Final
Volume (mg) (V1) (mL) (T1) (mL) (V2) (mL) 3.0 25 NA NA 6.25 50 NA
NA 12.5 100 NA NA 25.0 50 6 25 50.0 100 6 25 100 250 3 10
Chromatographic Conditions
TABLE-US-00005 [0222] Column Chiralpak AD (4.6 mm id .times. 25 cm)
Column temperature ambient Autosampler Tray Temp. ambient Mobile
Phase See section Mobile Phase Flow rate 0.5 mL/min Needle Wash
Mobile Phase section Injection Volume 10 .mu.L Injections per vial
2 Wavelength 220 nm Run time 15 min (may be adjusted as
appropriate
System Suitability
[0223] System Interferences
[0224] Perform duplicate injections of mobile phase as a blank. No
interfering peaks or artifacts should be present in the blank
chromatograms.
[0225] Relative Retention Times
[0226] Perform duplication injections of the system suitability
solution. Using the second injection, report the relative retention
times of each bucindolol peak. The relative retention times of (R)-
and (S)-bucindolol should be approximately 1.00 and 1.23,
respectively.
[0227] Peak Tailing
[0228] Using the same injection as for Relative Retention Times
above, calculate the tailing factors for both bucindolol peaks as
follows:
T = W 2 .times. F ##EQU00005## [0229] where: T=tailing factor
[0230] W=peak width at 5% of peak height [0231] F=width of line
from peak start to the retention time at 5% of peak height [0232]
The tailing factors for both peaks should be .ltoreq.1.5
System Suitability
[0233] Perform duplicate injections of each sample.
Calculations
[0234] For all substance and product samples, calculate the percent
(R)- and percent (S)-bucindolol as follows:
% (R)-bucindolol=(peak area (R).times.100%/(peak area (R)+peak area
(S))
% (S)-bucindolol=(peak area (S).times.100%/(peak area (R)+peak area
(S))
Example 5
Preparations of (S)-Bucindolol
##STR00005##
TABLE-US-00006 [0235] Reagents MW Density Amount Units mmol Eq.
Source 221.62 -- 14 g 631.71 1 Ald/02807CE (S)Glycidol 74.08 1.116
4.19 ml 631.71 1 Ald/2007DE Et.sub.3N 101.19 0.720 8.878 ml 6.3171
1 Toluene 150 ml
Procedure:
[0236] To a round-bottom flask 4.19 ml (S)-glycidol, 8.878 ml of
Et.sub.3N and 150 ml of Toluene was added. The reaction mixture was
stirred in a N.sub.2 atm. The reaction mixture was cooled to
-10.degree. C. Then Nitrosulfaryl chloride was added in 3 lots. The
reaction mixture was stirred for two hours. After the completion of
the reaction, water was added to the reaction mixture. The compound
was taken up in EtOAc. The EtOAc layer was washed with brine. The
EtOAc layer was dried and concentrated. The compound was purified
by column chromatography.
[0237] Theoretical Yield: 16.376 g
[0238] % Yield: 93%
[0239] Yield obtained: 15.140 g
[0240] .sup.1H NMR: verified
##STR00006##
TABLE-US-00007 Reagents MW Density Amount Units mmol Eq.
2-Cyanophenol 119.12 -- 1 g 8.394 1 Nolylate 259.24 -- 2.17 g 8.394
1 K.sub.2CO.sub.3 138.21 3.48 g 25.182 3 acetone 50 ml (HPLC
grade)
Procedure:
[0241] 2-Cyanophenol was taken up in acetone. To that
K.sub.2CO.sub.3 was added. The reaction mixture was refluxed for 30
min. Then the reaction mixture was cooled to room temperature.
Nolylate was added and again refluxed. The reaction was monitored
by HPLC. After the completion of the reaction, K.sub.2CO.sub.3 was
then filtered/removed using scintered glass crucible. The filtrate
was concentrated and purified by column chromatography (30% EtOAc
in hexanes).
[0242] Theoretical Yield: 1.470 g
[0243] Yield Obtained: 1.4 g
[0244] .sup.1H NMR: verified
##STR00007##
TABLE-US-00008 Reagents MW Density Amount Units mmol Eq. Epoxide
175.18 -- 7 g 39.958 1 Amine 188.269 -- 7.52 g 39.958 1 Ethanol 125
ml
Procedure:
[0245] Epoxide and amine dissolved in EtOH and refluxed. After the
completion of the reaction, the ethanol was removed. The residue
was purified by column chromatography.
[0246] Theoretical Yield: 14.52 g
[0247] Yield Obtained: 5.5 g, HPLC purity 100% (1.sup.st reaction)
[0248] 6.5 g HPLC purity 98% (2.sup.nd reaction)
[0249] .sup.1H NMR: verified for both
##STR00008##
TABLE-US-00009 Reagents MW Density Amount Units mmol Eq. SM 363.45
-- 5.5 g 15.132 1 HCl in 1.5M 10.08 ml 15.132 1 ether Diethyl 50 ml
ether
Procedure:
[0250] The starting material was dissolved in diethyl ether and to
that 1.5 M HCl was added and allowed to stir for 2 hours. After
that diethyl ether was removed. The resulting solid was dried.
[0251] Theoretical Yield: 6.06 g
[0252] Yield Obtained: 5.750 g
[0253] % Yield: 95%
[0254] Chiral HPLC purity: 100%
[0255] HPLC purity: 99%
[0256] .sup.1H NMR: verified
##STR00009##
TABLE-US-00010 Reagents MW Density Amount Units mmol Eq. Source
221.62 -- 15 g 67.683 1 AW/12007DE R-glycidol 74.08 1.116 4.49 ml
67.683 1 Ald/02020BH Et.sub.3N 101.19 0.720 9.512 ml 67.683 1
Toluene
Procedure: As in IN-SPL-C-11
[0257] To R-glycidol, toluene and Et.sub.3N were added. Then
Nitrosulfonyl chloride was added in lots at -10.degree. C. and
stirred for 2 hours. Following EtOAc/water work up, the residue was
purified by column chromatography.
[0258] Theoretical Yield: 17.54 g
[0259] Yield Obtained: 15.5 g
[0260] % Yield: 88%
[0261] .sup.1H NMR: verified
##STR00010##
TABLE-US-00011 Reagents MW Density Amount Units mmol Eq. Source
2-Cyanophenol 119.12 -- 6.89 g 57.86 1 AM R1/290328 Nolylate 259.24
-- 15.0 g 57.86 1 K.sub.2CO.sub.3 138.21 -- 23.9 g 173.58 3 Acetone
200 ml
Procedure:
[0262] 2-Cyanophenol in acetone and K.sub.2CO.sub.3 were heated at
reflux for 30 min. Then cooled Nolylate was added and heated to
reflux. As in Reaction 2, the reaction was monitored by HPLC. After
the completion of the reaction K.sub.2CO.sub.3 filtered/removed.
The filtrate was concentrated and purified by column
chromatography.
[0263] Theoretical Yield: 10.13 g
[0264] Yield Obtained: 7.3 g
[0265] % Yield: 72%
[0266] .sup.1H NMR: verified
##STR00011##
TABLE-US-00012 Reagents MW Density Amount Units mmol Eq. Epoxide
175.18 -- 7 g 39.958 1 Amine 188.269 -- 7.52 g 39.958 1 Ethanol 150
ml
Procedure:
[0267] The Epoxide and amine, each in Ethanol, were heated to
reflux. Then Ethanol was removed after the completion of reaction
and purified by column chromatography.
[0268] Theoretical Yield: 14.523 g
[0269] Yield Obtained: 10.5 g
[0270] % Yield: 72%
##STR00012##
TABLE-US-00013 Reagents MW Density Amount Units mmol Eq. Starting
363.453 -- 10 g 27.51 1 material HCl in 1.5M 18.34 ml 27.51 1 Ether
Diethyl 200 ml ether
Procedure:
[0271] Starting material was dissolved in diethyl ether. To that
1.5 M HCl was added and allowed to stir for 2 hours. Then ether was
removed. The resulting solid was dried.
[0272] Theoretical Yield: 11.030 g
[0273] Yield Obtained: 10.5 g
[0274] % Yield: 95%
[0275] Chiral HPLC purity: 100%
[0276] HPLC Purity: 99%
[0277] .sup.1H NMR: verified
Example 6
Characterization Data of (R)- and (S)-Bucindolol
[0278] See FIGS. 15 and 16 for .sup.1H NMR spectra of (R)- and
(S)-bucindolol.
(R)-Bucindolol:
TABLE-US-00014 [0279] TEST RESULT REFERENCE Appearance Light green
solid 300 MHz .sup.1H NMR Consistent-Attached Spectrum
(DMSO-d.sub.6) Mass Spectrum ESI, m/z 364 [M + H].sup.+, Attached
Chiral HPLC Analysis >99% (area %), >99% ee, CHIRALPAK AD
Column, Detector @ 280 nm, Attached Optical Rotation
[.alpha.].sup.22.9 .sub.D + 15.5.degree. (c 1.00, Methanol)
(S)-Bucindolol
TABLE-US-00015 [0280] TEST RESULT/REFERENCE Appearance White solid
300 MHz .sup.1H NMR Consistent-Attached Spectrum (DMSO-d.sub.6)
Mass Spectrum ESI, m/z 364 [M + H].sup.+, Attached Chiral HPLC
Analysis >99% (area %), >99% ee, CIHRALPAK AD Column,
Detector @ 280 nm, Attached Optical Rotation [.alpha.].sup.22.7
.sub.D - 15.9.degree. (c 1.00, Methanol)
[0281] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of some embodiments,
it will be apparent to those of skill in the art that variations
may be applied to the compositions and methods and in the steps or
in the sequence of steps of the method described herein without
departing from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents which are
both chemically and physiologically related may be substituted for
the agents described herein while the same or similar results would
be achieved. All such similar substitutes and modifications
apparent to those skilled in the art are deemed to be within the
spirit, scope and concept of the invention as defined by the
appended claims.
REFERENCES
[0282] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
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* * * * *