U.S. patent application number 16/655004 was filed with the patent office on 2020-02-13 for sialylation-increasing therapies for diseases associated with oxidative stress.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Servic. The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Servic, The United States of America, as represented by the Secretary, Department of Health and Human Servic. Invention is credited to Nuria Carrillo, Marjan Huizing, May C. Malicdan.
Application Number | 20200046747 16/655004 |
Document ID | / |
Family ID | 55661534 |
Filed Date | 2020-02-13 |
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United States Patent
Application |
20200046747 |
Kind Code |
A1 |
Huizing; Marjan ; et
al. |
February 13, 2020 |
SIALYLATION-INCREASING THERAPIES FOR DISEASES ASSOCIATED WITH
OXIDATIVE STRESS
Abstract
Methods are disclosed for treating a subject with a vascular or
cardiac disorder associated with oxidative stress. Methods are
disclosed for treating a subject with GNE myopathy that has
impaired cardiac function. These methods include administering to
the subject a therapeutically effective amount of a sialic acid
precursor, sialic acid, or one or more sialylated compounds,
mannosamine, N-acetyl mannosamine or a derivative thereof. In other
embodiments, methods are disclosed for detecting a disorder
associated with oxidative stress.
Inventors: |
Huizing; Marjan; (Santa
Cruz, CA) ; Malicdan; May C.; (Rockville, MD)
; Carrillo; Nuria; (Bethesda, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Servic |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Servic
Bethesda
MD
|
Family ID: |
55661534 |
Appl. No.: |
16/655004 |
Filed: |
October 16, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15553561 |
Aug 24, 2017 |
10493087 |
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PCT/US2016/019084 |
Feb 23, 2016 |
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16655004 |
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62120742 |
Feb 25, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0053 20130101;
A61P 9/00 20180101; A61K 31/702 20130101; A61K 47/20 20130101; A61K
31/7012 20130101; A61K 31/7088 20130101; A61K 31/7008 20130101;
A61K 9/127 20130101; A61K 45/06 20130101; A61K 9/50 20130101 |
International
Class: |
A61K 31/7008 20060101
A61K031/7008; A61K 47/20 20060101 A61K047/20; A61K 9/50 20060101
A61K009/50; A61K 9/127 20060101 A61K009/127; A61K 9/00 20060101
A61K009/00; A61P 9/00 20060101 A61P009/00; A61K 45/06 20060101
A61K045/06; A61K 31/7012 20060101 A61K031/7012; A61K 31/702
20060101 A61K031/702; A61K 31/7088 20060101 A61K031/7088 |
Goverment Interests
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with Government support under
project number ZIA HG000215 16 by the National Institutes of
Health, National Human Genome Research Institute. The Government
has certain rights in the invention.
Claims
1. A method for treating a subject with a cardiovascular disorder
associated with oxidative stress, comprising selecting a subject
that has the cardiovascular disorder associated with oxidative
stress and does not have a GNE myopathy; and administering to the
subject a therapeutically effective amount of a mannosamine,
N-acetyl mannosamine or a derivative thereof, wherein the
derivative is: ##STR00004## wherein: R.sub.1, R.sub.3, R.sub.4, or
R.sub.5 is hydrogen, lower alkanoyl, carboxylate or lower alkyl;
and R.sub.2 is lower alkyl, lower alkanoylalkyl, or lower alkyl
alkanoyloxy, thereby treating the cardiovascular disorder in the
subject.
2. The method of claim 1, wherein the subject has heart failure,
atherosclerotic cardiovascular disease, cardiomyopathy, a cardiac
arrhythmia, myocardial infarction, ischemic heart disease, stroke,
or peripheral arterial disease.
3. The method of claim 1, wherein the mannosamine, N-acetyl
mannosamine or the derivative thereof, is microencapsulated.
4. The method of claim 1, wherein the mannosamine, N-acetyl
mannosamine or the derivative thereof, is formulated in a coating,
envelope or protective matrix made from a liposome.
5. The method of claim 1, wherein the mannosamine, N-acetyl
mannosamine or the derivative thereof, is orally administered to
the mammal.
6. The method of claim 5, wherein the mannosamine, N-acetyl
mannosamine or the derivative thereof, is orally administered in
the form of a food product.
7. The method of claim 2, wherein the subject has the heart
failure, and wherein the method further comprises administering to
the subject a therapeutically effective amount of an
angiotensin-converting enzyme (ACE) inhibitor, a beta blocker, an
aldosterone antagonist, a diuretic, an angiotensin receptor blocker
(ARB), or a vasodilator.
8. The method of claim 2, wherein the subject has the myocardial
infarction, and wherein the method further comprises administering
to the subject a therapeutically effective amount of an
antiplatelet agent, an anticoagulation agent, or a lipid or blood
pressure regulating agent.
9. The method of claim 8, wherein the lipid regulating agent is a
statin, niacin, PCSK9-targeting drug, bile acid binding resin, or
HDL-cholesterol targeting drug.
10. The method of claim 1, wherein the subject has the
atherosclerotic cardiovascular disease and wherein the method
further comprises administering to the subject a therapeutically
effective amount of a statin, niacin, a fibrate, a bile acid
binding resin, a cholesterol absorption inhibitor, a
PCSK9-targeting drug, an LDL-targeting drug or an HDL-targeting
drug.
11. The method of claim 1, further comprising administering to the
subject an anti-oxidant.
12. The method of claim 11, wherein the anti-oxidant is
N-acetylcysteine.
13. The method of claim 1, wherein the cardiovascular disorder
associated with oxidative stress is also associated with
hyposialylation.
14. The method of claim 1, wherein the subject has heart damage
from administration of a chemotherapeutic agent.
15. The method of claim 14, wherein the chemotherapeutic agent is
doxorubicin.
16. A method for treating impaired cardiac function in a subject
that does not have a GNE myopathy, comprising selecting a subject
that has the impaired cardiac function and does not have the GNE
myopathy; and administering to the subject a therapeutically
effective amount of mannosamine, N-acetyl mannosamine or a
derivative thereof, wherein the derivative is: ##STR00005##
wherein: R.sub.1, R.sub.3, R.sub.4, or R.sub.5 is hydrogen, lower
alkanoyl, carboxylate or lower alkyl; and R.sub.2 is lower alkyl,
lower alkanoylalkyl, or lower alkyl alkanoyloxy, thereby improving
cardiac function in the subject.
17. The method of claim 16, further comprising performing a
diagnostic test to determine the cardiac function of the
subject.
18. The method of claim 17, wherein diagnostic test is a CT scan,
cardiac catherization, coronary CT angiogram, echocardiography,
ejection fraction testing, electrocardiogram, electrophysiology,
exercise stress test, magnetic resonance imaging, tilt-table
testing, transesphogeal echocardiogram, or an ultrasound.
19. The method of claim 16, wherein the mannosamine, N-acetyl
mannosamine or the derivative thereof, is microencapsulated.
20. The method of claim 16, wherein the, mannosamine, N-acetyl
mannosamine or the derivative thereof, is formulated in a coating,
envelope or protective matrix made from a liposome.
21. The method of claim 16, wherein the mannosamine, N-acetyl
mannosamine or the derivative thereof, is orally administered to
the mammal.
22. The method of claim 21, wherein the mannosamine, N-acetyl
mannosamine or the derivative thereof, is orally administered in
the form of a food product.
23. The method of claim 16, wherein the N-acetyl mannosamine or the
derivative thereof is administered at a dose of about 0.02 g/day to
about 25 g/day.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/553,561, filed Aug. 24, 2017, which is the U.S.
National Stage of International Application No. PCT/US2016/019084,
filed Feb. 23, 2016, which was published in English under PCT
Article 21(2), which claims the benefit of U.S. Provisional
Application No. 62/120,742, filed Feb. 25, 2015. The prior
applications are incorporated herein by reference in their
entirety.
FIELD
[0003] This application relates to the use of sialylation
increasing therapies, such as N-acetyl-mannosamine and derivatives
thereof, for the treatment of disorders associated with oxidative
stress, such as cardiovascular disorders associated with oxidative
stress, for example heart failure, myocardial infarction, and
atherosclerotic vascular disease.
BACKGROUND
[0004] Upon reaction with electrons, oxygen is transformed into
reactive oxygen species (ROS). All ROS types, including superoxide
anions and hydrogen peroxide, have unpaired valence electrons or
unstable bonds. ROS is known to destroy bacteria and destroy human
cells. In addition, exposure to high ROS concentrations can lead to
damage to proteins, lipids, and nucleic acids. Low to intermediate
ROS concentrations are believed to exert their effects rather
through regulation of cell signaling cascades.
[0005] A variety of diseases are believed to be caused by a surplus
of ROS, including cardiovascular diseases such as myocardial
infarction and atherosclerosis. A need remains for therapeutic
agents to treat subject with disorders associated with increased
production of reactive oxygen species.
SUMMARY
[0006] It is disclosed herein that reactive oxygen species (ROS)
generation is increased in GNE myopathy. In addition, it was
determined that cardiac muscles are involved in GNE myopathy.
Hyposialylation of cardiac muscles leads to impaired cardiac muscle
contractility, and can be improved with sialylation-increasing
therapies. The results show that sialylation increasing therapies
can be of use in disorders associated with reactive oxygen species,
specifically cardiovascular disorders.
[0007] Methods are disclosed for detecting a disorder associated
with oxidative stress. In some embodiments, methods are disclosed
for treating a subject with a cardiovascular disorder associated
with oxidative stress, comprising administering to the subject a
therapeutically effective amount of a sialic acid precursor, sialic
acid, or one or more sialylated compounds, mannosamine, N-acetyl
mannosamine or a derivative thereof.
[0008] In additional embodiments, methods are disclosed for
treating a subject with GNE myopathy that has impaired cardiac
function. The method include selecting a subject with GNE myopathy
that has impaired cardiac function; and administering to the
subject a therapeutically effective amount of a sialic acid
precursor, sialic acid, or one or more sialylated compounds,
mannosamine, N-acetyl mannosamine or a derivative thereof. The
method can include the use of these agents for the treatment of
heart failure, myocardial infarction, cardiovascular disorders, and
atherosclerotic vascular disease.
[0009] In additional embodiments, the method includes selecting a
subject with GNE myopathy who has not been identified as having
impaired cardiac function and testing the subject's cardiac
function. The method can also include administering to the subject
at therapeutically effective amount of an anti-oxidant accompanied
or unaccompanied by a therapeutically effective amount N-acetyl
mannosamine or a derivative thereof, mannosamine, a sialic acid
precursor, sialic acid, or one or more sialylated compounds. The
method can improve cardiac function in the subject. The method can
be used to treat previously unidentified of heart failure,
myocardial infarction, cardiovascular disorders, and/or
atherosclerotic vascular disease, or to prevent these
conditions.
[0010] The foregoing and other features and advantages of the
invention will become more apparent from the following detailed
description of several embodiments which proceeds with reference to
the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1A-1E. Atrogenes and oxidative stress related genes
are upregulated in GNE Myopathy (GM) muscles. (A) Expression of
atrogin-1/FBxo32, MuRF1/Trim63, Srxn1, MTs, and LC3b in untreated
(white bars, n=17), N-acetylcysteine (NAC) treated (gray bars, n=6
per group), and littermate controls (black bars, n=6). Gne.sup.-/-
hGNED176V-Tg muscles were measured by quantitative RT-PCR and
expressed as fold changes of littermates controls. Data represent
mean.+-.SEM of each group. (B-E) Microarray analysis followed by
gene ontology profiling. Each dot represents average expression
values for the same gene from Gne.sup.-/-hGNED176V-Tg (vertical
axis, n=9) and littermates (horizontal axis, n=3) muscles. Inverted
triangles show recovered expression values with NAC treatment
(vertical axis, n=6) for significantly upregulated genes. (B)
Muscle atrophy related genes. (C) Oxidative stress and redox
homeostasis related genes. (D) Autophagy related genes. (E)
Collagen organization and biosynthesis related genes. Detailed
genes are listed Tables 1-4 (FIG. 6).
[0012] FIGS. 2A-2D. ROS production in GNE Myopathy muscle was
measured in vivo using microdialysis. (A-B) Experimental setup and
representative HPLC chromatograms. Perfusion medium containing 5 mM
salicylate (SA) was pumped through a microdialysis probe and
dihydroxybenzoic acid (DHBA) in dialysate was detected by HPLC-ECD
system. A, baseline; B, post-stimulus. Electrical stimulus (ES)
consisted of 50V, 40 Hz, 3 ms pulses for 300 trains. (C-D) 2,
5-DHBA increments after contraction was significantly greater in
GNE Myopathy mice (n=9) than those in littermates (n=5). Pre-ES,
before ES; ES1, 1.sup.st 20 min after ES; ES2, 2.sup.nd 20 min
after ES. (C) Data represent mean.+-.SEM. (D) Each circle
represents a 2,5-DHBA level from an individual mouse.
[0013] FIGS. 3A-3F. Antioxidant capacity is impaired in
hyposialylated GNE Myopathy myotubes. (A) Intracellular ROS
generation was imaged by fluorescence microscopy with green DCF
staining. (B) DCF fluorescence was quantified by using a multi-well
fluorescence plate reader. Data represent mean.+-.SEM of each group
(n=14). (C-F) ROS levels and cells viability were analyzed with the
addition of increasing concentration of H.sub.2O.sub.2 (0.5, 1.0,
2.0, and 4.0 mM) or menadione (5, 10, 20, and 40 .mu.M) to culture
media. Control myotubes of each group were cultured in the same
condition without adding H.sub.2O.sub.2 or menadione. Each point is
mean.+-.SEM of four determinations. (C-D) Increased ROS levels with
the addition of pro-oxidants were determined by subtracting mean
fluorescence of control cells. (E-F) Viable cells were counted
using propidium iodide/Hoechst co-staining. Relative viability (%)
was calculated in comparison to control cells of each group.
[0014] FIGS. 4A-4F. Oral N-acetyl cysteine (NAC) administration
improved muscle force generation and motor performance in GNE
Myopathy mice. Gne(-/) hGNED176V-Tg mice (circles) were treated
with low dose (LD; light gray fill color; n=13) and high dose (HD;
dark grayfill color; n=13) NAC and compared to untreated group (NT;
white fill color; n=17). Littermate controls (reverse triangles)
were treated in the same conditions (LD; n=7, HD; n=7, and NT;
n=6). (A) Treadmill performance test evaluating the distance that
the mice could run. (B) Treadmill endurance test evaluating the
number of beam breaks during a constant loading. (C-F) Contractile
forces of gastrocnemius muscles. (C) Peak isometric twitch force
(Pt). (D) Maximum tetanic force (Po). (E) Specific isometric force
(Pt normalized by CSA). (F) Specific tetanic force (Po normalized
by CSA). Values from each mouse are shown with mean.+-.SEM
(*P<0.05, **P<0.01).
[0015] FIGS. 5A-5C. Skeletal muscle atrophy in GNE Myopathy mice
was ameliorated by NAC treatment. (A) Representative sarcolemmal
staining (caveolin) images from gastrocnemius muscles. (B) Muscle
fiber diameters in low (light gray; n=13) and high dose (dark gray;
n=13) NAC treated GNE Myopathy mice were compared to those in
untreated controls (white; n=17). Data presented with mean.+-.SEM
(*P<0.05, **P<0.01). (C) Fiber diameter histogram from a
mouse in each group was compared.
[0016] FIGS. 6A-6O. Tables 1-4. Collagen organization and
biosynthesis related genes. Detailed genes lists.
[0017] FIGS. 7A-7B. (A) Paraffin-embedded heart sections from GNE
myopathy mutant mice [Gne(-/-)hGNED176V-Tg mice (GNE (-/-) and
unaffected littermates (GNE +/+)] were stained with three lectins
informative for sialylation status and co-stained with the nuclear
dye DAPI (blue). Left ventricular cardiac muscle tissue was imaged
and showed selective hyposialylation in GNE myopathy compared with
control muscle, demonstrated by apparent normal staining of WGA
(binding to most sialic acid groups), but decreased staining of SNA
(predominantly binding terminal .alpha.(2,6)-linked sialic acid on
all glycans). In addition, staining of VVA (predominantly binding
terminal GalNAc, without sialic acid attached, O-linked to serine
or threonine residues of glycoproteins) was not significantly
increased in GNE myopathy heart specimen. (B) Paraffin-embedded
muscle sections (gastrocnemius) from GNE myopathy mutant mice (-/-)
and unaffected control mice (control) were stained with the lectins
SNA and VVA (green) and co-stained with the nuclear dye DAPI (blue)
[adapted from Niethamer et al., 2012]. GNE myopathy muscle
specimens show hyposialylation, as demonstrated by decreased
staining of SNA compared with control skeletal muscle. In addition,
staining of VVA showed an increased signal, indicating significant
hyposialylation of O-linked glycoproteins. Oral supplementation of
ManNAc (1 g/kg/day for 12 weeks) restored the sialylation status
back to the normal range, demonstrated by increased SNA, and
decreased/absent VVA signal intensities similar to control
specimens.
[0018] FIGS. 8A-8F: Echocardiogram findings in GNE myopathy mice.
(A) GNE myopathy mutant mice [Gne(-/-)hGNED176V-Tg] showed slightly
decreased ejection fractions, suggesting possibly decreased left
ventricle pumping capacity. (B) GNE myopathy mutant mice showed
decreased fractional shortening (the ratio between the diameter of
the left ventricle when it is relaxed and its diameter when it has
contracted) compared to control lieetermate mice. (C, D) GNE
myopathy mutant mice displayed borderline increased left ventricle
mass, implying increased wall or septal thickness. (E, F) GNE
myopathy mutant mice showed increased systolic and diastolic left
ventricle volumes.
[0019] FIGS. 9A-9E. Magnetic Resonance Imaging (MRI) findings in
GNE myopathy mice. (A) Still images of representative gradient echo
cine scans from GNE myopathy mutant [Gne(-/-)hGNED176V-Tg] and
control littermate mice hearts; both end of systole (ES) and end of
diastole (ED) images are displayed. Left images: a long axis
4-chamber cine scan of the whole heart; Right images: 2D spin echo
covering the chest and abdomen. (B) Mean ejection fractions
calculated from MRI data showed that GNE myopathy mutant mice have
ejection fraction and size within the normal range. (C) Cardiac
output, calculated from MRI data was markedly decreased in GNE
myopathy mutant mice compared to control. (D, E) MRI data showed
slightly increased ED and ES volumes in GNE myopathy mutant mice
hearts compared to control hearts.
[0020] FIG. 10. Electrocardiography (ECG) findings in GNE myopathy
mice. Both 3 and 6 lead ECG were performed on GNE myopathy mutant
[Gne(-/-) hGNED176V-Tg mice] and control littermate mice. ECG
findings show that GNE myopathy mutant mice had an increased PR
interval of 40.475 (.+-.2.11) ms; the normal range of PR intervals
is 31.7-36.5 ms. GNE myopathy mutant mice had QRS intervals within
the normal range.
[0021] FIG. 11. Paraffin-embedded heart sections from GNE myopathy
mutant mice [Gne(-/-)hGNED176V-Tg] mice (GNE (-/-) and unaffected
littermates (GNE +/+) were stained with the SNA lectin informative
for sialylation status (see also FIGS. 7A,B). As shown in FIG. 7A,
GNE -/- mice showed decreased staining of SNA (predominantly
binding terminal .alpha.(2,6)-linked sialic acid on all glycans) of
heart tissue compared to control littermates (GNE +/+). Heart
tissues collected from .about.1 year old GNE -/- mice treated since
age 10 weeks with either 2 g/kg/day ManNAc or 2 g/kg/day Neu5Ac
(sialic acid) (Malicdan et al. Nat Med 2009; 15: 690-695) showed
significantly increased sialylation of glycans compared to
untreated GNE -/- mice, indicating resialylation of glycans in
heart tissue after these sialylation-increasing therapies.
DETAILED DESCRIPTION
[0022] It is disclosed herein that disorders associated with
oxidative stress, such as cardiovascular disorders associated with
oxidative stress can be treated using an agent that increases
sialylation. It was determined that generation of reactive oxygen
species is increased in both in vitro and in vivo models of a
hyposialylation disorder. Methods for treating disorders associated
with oxidative stress, and methods for diagnosing these disorders
are disclosed.
Terms
[0023] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8). In order to facilitate review of the various
embodiments of this disclosure, the following explanations of
specific terms are provided:
[0024] Administration: Providing a compound to a subject by another
person or self-administration by the subject.
[0025] Animal: Living multi-cellular vertebrate organisms, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals. Similarly, the
term "subject" includes both human and non-human subjects,
including birds and non-human mammals, such as non-human primates,
companion animals (such as dogs and cats), livestock (such as pigs,
sheep, cows), as well as non-domesticated animals, such as big
cats, zebras, etc. The term subject applies regardless of the stage
in the organism's life-cycle.
[0026] Alteration: A statistically significant change in a
parameter as compared to a control. In one example, an "increase"
is a statistically significant elevation in a parameter, such as
the presence of a biological marker, or the ratio of two biological
markers, such as the T/ST ratio. The alternation can be measured as
compared to a control. Suitable statistical analyses are well known
in the art, and include, but are not limited to, Student's T test
and ANOVA assays. In one example, a "decrease" or "reduction" is a
statistically significant decline in a parameter, such as the
presence of a biological marker, such as the T/ST ratio as compared
to a control. In another example, an "increase" is a statistically
significant higher level of a parameter, such as the presence of a
biological marker, such as the T/ST ratio as compared to a control.
Suitable statistical analyses are well known in the art, and
include, but are not limited to, Student's T test and ANOVA
assays.
[0027] Atherosclerosis: The progressive narrowing and hardening of
a blood vessel over time. Atherosclerosis is a common form of
arteriosclerosis in which deposits of yellowish plaques (atheromas)
containing cholesterol, lipoid material and lipophages are formed
within the intima and inner media of large and medium-sized
arteries. Treatment of atherosclerosis includes reversing or
slowing the progression of atherosclerosis, for example as measured
by the presence of atherosclerotic lesions and/or functional signs
of the disease, such as improvement in cardiovascular function as
measured by signs (such as peripheral capillary refill), symptoms
(such as chest pain and intermittent claudication), or laboratory
evidence (such as that obtained by EKG, angiography, or other
imaging techniques). "Assessing atherosclerosis" indicates
determining if a subject of interest has atherosclerosis,
determining the prognosis of the subject of interest, and/or
determining if a therapeutic regimen administered to the subject is
effective in treating the subject.
[0028] Arrhythmia: A heart condition wherein the electrical
activity of the heart is irregular, or faster or slower than
normal. Tachycardia is general more than 100 beats per minute for a
human adult, bradycardia is generally below 60 beats were minute
for a human adult. The arrhythmia can be an atrial, ventricular, or
at the atrioventricular junction. Atrial arrhythmias include sinus
bradycardia, premature atrical contractions, wander atrial
pacemaker, atrial tachycardia, multifocal atrial tachycardia,
atrial flutter, and atrial fibrillation. Junction arrhythmias
include AVnodal reentrant tachycardia, junctional rhythm,
junctional tachycardia and premature junctional contraction.
Ventricular arrhythmias include premature ventricular contractions,
accelerated idioventricular rhythm, monomorphic ventricular
tachycardia, polymorphic ventricular tachycardia, and ventricular
fibrillation.
[0029] Beta blocker: A type of drug that targets the beta receptor,
which are found on the cells of heart muscles. Beta blockers
interfere with binding of epinephrine and other stress hormones to
the beta receptor. These drugs are often used for the management of
cardiac arrhythmias, prevention of heart attacks and heart failure,
and for treating hypertension.
[0030] Bile acid binding resins: Agents that lower LDL cholesterol.
Bile acids are the breakdown products of cholesterol and are
excreted by the liver via the bile. Bile acids are 90% reabsorbed
from the intestine and used to re-manufacture cholesterol in the
liver. Bile acid binding resins (also referred to as bind acid
sequestrants) interfere with this intestinal reabsorption by
binding bile acids in the gut and promoting their excretion from
the body.
[0031] Cardiomyopathy: Measurable deterioration of the ability of
the myocardium to contract, usually leading to heart failure.
Cardiomyopathy includes hypertrophic cardiomyopathy, arrhythmogenic
right ventricular cardiomyopathy, isolated ventricular
non-compaction, mitochondrial myopathy, dilated cardiomyopathy,
restrictive cardiomyopathy, peripartum cardiomyopathy, Takotsubo
cardiomyopathy, and Loeffler endocarditis.
[0032] Cardiovascular: Pertaining to the heart and/or blood
vessels.
[0033] Cardiovascular disease (CVD): Disorders of the heart and
blood vessels, such as atherosclerosis (ASCVD), coronary heart
disease, cerebrovascular disease, and peripheral vascular disease.
Cardiovascular diseases also include, for example, myocardial
infarction, stroke, angina pectoris, transient ischemic attacks,
and congestive heart failure. Atherosclerosis usually results from
the accumulation of fatty material, inflammatory cells,
extracellular matrices and plaque. Clinical symptoms and signs
indicating the presence of CVD may include one or more of the
following: chest pain and other forms of angina, shortness of
breath, sweatiness, Q waves or inverted T waves on an EKG, a high
calcium score by CT scan, at least one stenotic lesion on coronary
angiography, and heart attack. Subclinical ASCVD can be identified
by imaging tests (such as CT measures of coronary calcification, or
MRI measures of coronary or aortic plaque, and/or ultrasound
evidence of carotid plaque or thickening).
[0034] Cholesterol absorption inhibitor: A class of cholesterol
lowering drugs that block absorption of cholesterol at the brush
border of the intestine without affecting absorption of
tri-glycerides or fat soluble vitamins. These drugs are not
systemically absorbed and can lower cholesterol on their own (i.e.
without the use of additional drugs). An exemplary cholesterol
absorption inhibitor is ezetimibe (Ezetrol).
[0035] Cholesterol lowering agent: An agent that lowers the level
of cholesterol in a subject, such as a pharmaceutical, vitamin, or
small molecule. One of skill in the art can readily identify
assays, such as blood screening, to determine the effect of
cholesterol. Agents include, but are not limited to, niacin, the
statins (e.g., ZOCOR.TM., LIPITOR.TM., PRAVACOL.TM., LESCOR.TM.,
MEVACOR.TM.), bile acid binding resins (e.g., QUESTRAN.TM.), and
fibrates (e.g. LOPID.TM., LIPIDIL MICRO.TM.).
[0036] Control: A "control" refers to a sample or standard used for
comparison with an experimental sample. In some embodiments, the
control is a sample obtained from a healthy patient or a
non-diseased tissue sample obtained from a patient diagnosed with
the disorder of interest, such as a cardiovascular disorder
associated with oxidative stress, for example HF or ASCVD. In some
embodiments, the control is a historical control or standard
reference value or range of values (such as a previously tested
control sample, such as a group of patients with the disorder, or
group of samples that represent baseline or normal values, such as
the level of specific genes in non-diseased tissue).
[0037] Determining or Measuring: Identifying the presence of a
target molecule in a sample. There terms refer to measuring a
quantity or quantitating a target molecule in the sample, either
absolutely or relatively. For example, T and ST can be analyzed in
a sample from a subject of interest, such as a subject suspected of
having a hyposialylation disorder. The sample can be any biological
sample of interest, such as, but not limited to, a plasma sample,
serum sample, or tissue extract. Generally, detecting, measuring or
determining a biological molecule requires performing an assay,
such as mass spectrometry, and not simple observation.
[0038] Diagnosing or diagnosis of a disorder associated with
oxidative stress: Detecting the disorder by measuring specific
parameters. For example, a disorder can be detected by determining
the T/ST ratio in a biological sample. Diagnosis can encompass
laboratory confirmation of a pre-existing clinical condition or a
specific disease.
[0039] Diuretic: A drug that promotes the production of urine.
Diuretics are often used to treat heart failure, hypertension and
other diseases.
[0040] Fibrates: Agents that lower tri-glyceride levels and raise
HDL levels. Fibrates, also known as fibric acid derivatives, are
particularly useful in diabetic patients whose characteristic lipid
abnormality is high tri-glycerides and low HDL. In some patients
who have combined lipid abnormalities, fibrates are combined with
statins to lower both tri-glycerides and LDL and to raise HDL.
Exemplary fibrates include gemfibrozil (LOPID.TM.), fenofibrate
(Lipidil micro, Lipidil Supra, Lipidil EZ), and bezafibrate
(Bezalip).
[0041] Framingham Risk Score: A risk factor score that is used for
predicting future risk of coronary artery disease in individuals
free of disease, based on the measurement of Framingham risk
factors which include age, gender, systolic blood pressure (and use
of antihypertensive treatment), cigarette smoking, diabetes, as
well as total cholesterol (or low density lipoprotein cholesterol
(LDL cholesterol) and high density lipoprotein cholesterol (HDL
cholesterol) levels (Wilson et al., Circulation 1998; 97:
1837-47).
[0042] Glycoprotein: Proteins that contain oligosaccharide chains
(glycans) covalently attached to polypeptide side-chains. The
carbohydrate is attached to the protein in a cotranslational or
posttranslational modification process known as glycosylation.
There are two main types of glycosylation, N-glycosylation,
O-glycosylation. In N-glycosylation, the addition of the sugar
occurs on an amide nitrogen, such as in the side chain of
asparagine. In O-glycosylation, the addition of the sugar occurs on
a hydroxyl oxygen, such as on the side chain of hydroxylysine,
hydroxyproline, serine or threonine. The sugars commonly found in
eukaryotic glycoproteins include, but are not limited to,
.beta.-D-glucose, .beta.-D-galactose, .beta.-D-mannose,
.alpha.-L-fucose, N-Acetylgalactosamine, N-Acetylglucosamine,
N-Acetylneuraminic acid, and xylose.
[0043] Heart failure (HF): The physiological state in which cardiac
output is insufficient in meeting the needs of the body and lungs.
This condition is also called "congestive heart failure," and is
most commonly caused when cardiac output is low and the lungs
become congested with fluid due to an inability of heart output to
properly match venous return. Heart failure can also occur in
situations of high output, where the ventricular systolic function
is normal but the heart can't process the augmentation of blood
volume. This can occur in overload situation (blood or serum
infusions), renal diseases, chronic severe anemia, beriberi
(vitamin B 1/thiamine deficiency), thyrotoxicosis, Paget's disease,
arteriovenous fistulae, or arteriovenous malformations. Heart
failure includes left sided failure and right sided failure,
wherein the left and right ventricles are affected, respectively,
and biventricular failure. Ischemic heart disease (including
myocardial infarction), cigarette smoking, hypertension, obesity,
diabetes, and valvular heart disease are associated with increased
risk of heart failure. Viral myocarditis, human immunodeficiency
virus infections, connective tissue disease (such as systemic lupus
erythematous), drug (cocaine) abuse, and some chemotherapeutic
agents can cause heart failure.
[0044] Hyposialylation: Reduced or absent addition of sialic acid
(N-acetyl neuraminic acid (Neu5Ac) and its derivatives) to
galactose (Gal) or other underlying monosaccharides (such as, but
not limited to N-acetylgalactose (GalNAc)), Mannose (Man),
N-acetylglucosamine (GlcNAc), N-acetlylneuraminic acid (Neu5Ac) or
of sialic acid chains in polysialylation (PSA), such as on
PSA-NCAM.
[0045] Hyposialylation disorders are conditions with
hyposialylation of glycoproteins and glycolipids in affected
tissues. Hyposialylation of affected tissues can be detected, for
example, using histochemistry staining of fixed tissue slides with
specific lectins. A demonstration of a significant reduction (or
absence) of sialic acid, either by a statistically reduced
staining/binding of sialic acid recognizing lectins (such as, but
not limited to wheat germ agglutinin (WGA), Sambucus nigra
agglutinin (SNA), and Limax flavus agglutinin (LFA) or by presence
of staining of free monosaccharides underlying sialic acid on the
glycan chain, including galactose or GalNAc, by the lectins (such
as, but not limited to, helix pomatia agglutinin (HPA), Vicia
villosa agglutinin (VVA), jackfruit agglutinin (Jacalin), and
peanut agglutinin (PNA) can be used to identify hyposialylation
disorders, such as certain cases with myopathy (including the
adult-onset, progressive, autosomal recessive muscular disorder,
GNE myopathy, also called distal myopathy with rimmed vacuoles
(DMRV)/hereditary inclusion body myopathy (HIBM)), renal disorders
(including, but not limited to minimal change nephrosis, lupus
nephritis, IgA nephropathy, diabetic nephropathy), sleep disorders
(including those with reduced REM sleep), neurodegenerative
disorders (including those with amyloid depositions), cancers and
liver disorders. Western blotting or 2D gel electrophoresis
followed by lectin labeling or immunolabeling with a specific
antibody to a sialoglycan can also be used to detect
hyposialylation disorders. Methods for detecting are disclosed, for
example, in Kakani et al. Am J Pathol 2012: 180: 1431-1440 and
Niethamer et al. Mol Genet Metab 2012: 107:748-755.
[0046] Inhibiting or treating a disease: Inhibiting the full
development of a disease or condition, or decreasing intensity for
example, in a subject who has a cardiovascular disorder associated
with oxidative stress. "Treatment" refers to a therapeutic
intervention that ameliorates a sign or symptom of a disease or
pathological condition after it has begun to develop. The term
"ameliorating," with reference to a disease or pathological
condition, refers to any observable beneficial effect of the
treatment. The beneficial effect can be evidenced, for example, by
a delayed onset of clinical symptoms of the disease in a
susceptible subject, a reduction in severity of some or all
clinical symptoms of the disease, a slower progression of the
disease, an improvement in the overall health or well-being of the
subject, reports of reduced intensity of pain, or by other
parameters well known in the art that are specific to the
particular disease. A "prophylactic" treatment is a treatment
administered to a subject who does not exhibit signs of a disease
or exhibits only early signs for the purpose of decreasing the risk
of developing pathology.
[0047] Ion Exchange Chromatography: A chromatographic process that
allows the separation of ions and polar molecules based on their
charge. Ion-exchange chromatography retains analyte molecules on
the column based on coulombic (ionic) interactions. The stationary
phase surface displays ionic functional groups (R-X) that interact
with analyte ions of opposite charge. This type of chromatography
is further subdivided into cation exchange chromatography and anion
exchange chromatography. The ionic compound consisting of the
cationic species M+ and the anionic species B- can be retained by
the stationary phase.
[0048] Generally, a sample is introduced, either manually or with
an autosampler, into a sample loop of known volume. A buffered
aqueous solution (often called the "mobile phase") carries the
sample from the loop onto a column that contains a stationary phase
material that is typically a resin or gel matrix consisting of
agarose or cellulose beads with covalently bonded charged
functional groups. The target analytes (either anions or cations)
are retained on the stationary phase, but can be eluted by
increasing the concentration of a similarly charged species that
will displace the analyte ions from the stationary phase. For
example, in cation exchange chromatography, the positively charged
analyte can be displaced by the addition of positively charged
sodium ions. The analytes of interest are detected, such as by
conductivity or an ultraviolet (UV)/Visible light absorbance.
Generally, a chromatography data system (CDS) is used to control
the chromotography system.
[0049] Intravenous Immunoglobulin (IVIG): A blood product that
includes pooled polyvalent IgG extract from the plasma of a number
of blood donors. It is used as treatment for immune deficiencies
such as X-linked agammaglobulinemia, autoimmune diseases, such as
immune thrombocytopenia and Kawaski disease, and acute
infections.
[0050] Level of Expression: An amount, such as of a protein or an
mRNA, that can be measured in a biological sample.
[0051] Lipoprotein: A biochemical assembly that contains both
proteins and lipids, bound to the proteins, which allow fats to
move through the water inside and outside cells. There are five
major groups of lipoprotein particles, which, in order of molecular
size, largest to smallest, are chylomicrons, very low-density
lipoprotein (VLDL), intermediate-density lipoprotein (IDL),
low-density lipoprotein (LDL), and HDL. HDL contains the highest
proportion of protein to cholesterol; its most abundant
apolipoproteins are apo A-I and apo A-II. LDL contains
apolipoprotein B, and has a core consisting of linoleate and
includes esterified and non-esterified cholesterol molecules. LDL
particles are approximately 22 nm in diameter and have a mass of
about 3 million daltons. Lipoprotein a, (Lp(a)) is a lipoprotein
subclass; lipoprotein a consists of an LDL-like particle and the
specific apolipoprotein(a) [apo(a)], which is covalently bound to
the apolipoprotein B of the LDL like particle.
[0052] Mammal: This term includes both human and non-human mammals.
Examples of mammals include, but are not limited to: humans, pigs,
cows, goats, cats, dogs, rabbits, rats, and mice.
[0053] Mass Spectrometry: A process used to separate and identify
molecules based on their mass. Mass spectrometry ionizes chemical
compounds to generate charged molecules or molecule fragments and
measures their mass-to-charge ratios. In a typical MS procedure, as
sample is ionized. The ions are separated according to their
mass-to-charge ratio, and the ions are dynamically detected by some
mechanism capable of detecting energetic charged particles. The
signal is processed into the spectra of the masses of the particles
of that sample. The elements or molecules are identified by
correlating known masses by the identified masses.
[0054] "Time-of-flight mass spectrometry" (TOFMS) is a method of
mass spectrometry in which an ion's mass-to-charge ratio is
determined via a time measurement. Ions are accelerated by an
electric field of known strength. This acceleration results in an
ion having the same kinetic energy as any other ion that has the
same charge. The velocity of the ion depends on the mass-to-charge
ratio. The time that it subsequently takes for the particle to
reach a detector at a known distance is measured. This time will
depend on the mass-to-charge ratio of the particle (heavier
particles reach lower speeds). From this time and the known
experimental parameters one can find the mass-to-charge ratio of
the ion. "Liquid chromatography-mass spectrometry" or "LC-MS" is a
chemistry technique that combines the physical separation
capabilities of liquid chromatography (or HPLC) with the mass
analysis capabilities of mass spectrometry. Liquid chromatography
mass spectrometry (LC-MS) separates compounds chromatographically
before they are introduced to the ion source and mass spectrometer.
It differs from gas chromatography (GC-MS) in that the mobile phase
is liquid, usually a mixture of water and organic solvents, instead
of gas and the ions fragments. Most commonly, an electrospray
ionization source is used in LC-MS.
[0055] Mean and Standard Deviation: The arithmetic mean is the
"standard" average, often simply called the "mean".
x _ = 1 n i = 1 n x i ##EQU00001##
The mean is the arithmetic average of a set of values.
[0056] The standard deviation (represented by the symbol sigma,
.sigma.) shows how much variation or "dispersion" exists from the
mean. The standard deviation of a random variable, statistical
population, data set, or probability distribution is the square
root of its variance. The standard deviation is commonly used to
measure confidence in statistical conclusions. Generally, twice the
standard deviation is about the radius of a 95 percent confidence
interval. Effects that fall far outside the range of standard
deviation are generally considered statistically significant. One
of skill in the art can readily calculate the mean and the standard
deviation from a population of values.
[0057] Myocardial Infarction (MI): An event that occurs when blood
stops flowing properly to part of the heart and the heart muscle is
injured due to inadequate oxygen delivery. Acute myocardial
infarction refers to two subtypes of acute coronary syndrome,
namely non-ST-elevated myocardial infarction and ST-elevated
myocardial infarction, which are most frequently (but not always) a
manifestation of coronary artery disease. The most common
triggering event is the disruption of an atherosclerotic plaque in
an epicardial coronary artery, which leads to a clotting cascade,
sometimes resulting in total occlusion of the artery. If impaired
blood flow to the heart lasts long enough, it triggers a process
called the ischemic cascade; the heart cells in the territory of
the occluded coronary artery die, chiefly through necrosis. A
collagen scar forms in the heart in place of the damaged cells.
[0058] Niacin: A B-vitamin that is used as a medication for
patients with elevated levels of tri-glycerides and cholesterol. A
long-acting preparation of niacin is available as NIASPAN.RTM..
[0059] Prognosis: A prediction of the future course of a disease,
such as ASCVD or HF. The prediction can include determining the
likelihood of a subject to develop complications of ASCVD or HF, or
to survive a particular amount of time (e.g., determine the
likelihood that a subject will survive 1, 2, 3 or 5 years), to
respond to a particular therapy (e.g., lipid lowering therapy), or
combinations thereof.
[0060] Reactive Oxygen Species and Oxidative Stress: Reactive
oxygen species are oxygen radicals and hydrogen peroxide
(H.sub.2O.sub.2), singlet oxygen, lipid peroxides, O.sub.2--,
pro-oxidants and refers to molecules or ions formed by the
incomplete one-electron reduction of oxygen. These reactive oxygen
intermediates include singlet oxygen, superoxides; peroxides;
hydroxyl radical; and hypochlorous acid. They contribute to the
microbicidal activity of phagocytes, regulation of signal
transduction and gene expression, and the oxidative damage to
nucleic acids; proteins; and lipids.
[0061] Oxidative stress is an imbalance between the systemic
manifestation of reactive oxygen species and a biological system's
ability to readily detoxify the reactive intermediates or to repair
the resulting damage. Disturbances in the normal redox state of
cells can cause toxic effects through the production of peroxides
and free radicals that damage all components of the cell, including
proteins, lipids, and DNA. Chemically, oxidative stress is
associated with increased production of oxidizing species or a
significant decrease in the effectiveness of antioxidant defenses,
such as glutathione.
[0062] Renal hyposialylation disorder: A disease of the kidneys
characterized by decreased sialylation. In some subjects, the
glomeruli are hyposialylated. These disorders include some forms of
podocytopathies, minimal change nephrosis, focal and segmental
glomerulosclerosis, membranous glomerulonephritis, and other forms
of unexplained idiopathic nephrotic syndrome, as well as glomerular
basement membrane diseases such as Alport disease and thin membrane
disease. Such kidney disorders and conditions are sometimes
characterized by segmental splitting of the glomerular basement
membrane and/or podocytopathy due to disturbed polyanions on
podocyte membranes, or to changes in the amount or charge
(sialylation) of glomerular basement membrane components.
[0063] Sample (or biological sample): A biological specimen
containing genomic DNA, RNA (including mRNA), protein, or
combinations thereof, obtained from a subject. Examples include,
but are not limited to, peripheral blood, serum, plasma, urine,
fine needle aspirate, tissue biopsy, surgical specimen, and autopsy
material.
[0064] Sialic acid: A negative charged sugar that is a terminal
sugar on glycans. The most common sialic acid is
5-N-acetylneuraminic acid, a monosaccharide with a nine-carbon
backbone. Other less common sialic acids are N- or O-substituted
derivatives of 5-N-neuraminic acid. Sialic acids are found widely
distributed in animal tissues and to a lesser extent in other
species, ranging from plants and fungi to yeasts and bacteria,
mostly in glycoproteins and gangliosides. The amino group generally
bears either an acetyl or glycolyl group. The hydroxyl substituents
include acetyl, lactyl, methyl, sulfate, and phosphate groups.
Sialic acid is transferred to an oligosaccharide by a
sialyltransferase.
[0065] In renal functions, sialic acid residues are important for
maintenance of glomerular integrity, facilitating glomerular
filtration, and their deficiency is implicated in proteinuria
and/or hematuria. It has also been reported that glomerular
podocyte and podocyte foot process morphologies are maintained by
the anionic charge of sialic acid residues on podocyte
glycoproteins and glycolipids, and that a barrier to protein
permeability is controlled by functional endothelial glycocalyx,
rich in sialic acid.
[0066] Statin: Any of a class of lipid-lowering drugs that reduce
serum cholesterol levels by inhibiting a key enzyme involved in the
biosynthesis of cholesterol. Example statins include atorvastatin
(LIPITOR.RTM.), fluvastatin (LESCOL.RTM.), lovastatin
(MEVACOR.RTM., ALTOCOR.RTM., not marketed in the UK), pravastatin
(PRAVACHOL.RTM., SELEKTINE.RTM., LIPOSTAT.RTM.), rosuvastatin
(CRESTOR.RTM.), simvastatin (ZOCOR.RTM.). There are two groups of
statins: (1) Fermentation-derived: lovastatin, simvastatin and
pravastatin, and (2) Synthetic statins: fluvastatin, atorvastatin,
cerivastatin and rosuvastatin. Generally, statins act by
competitively inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG
CoA) reductase, an enzyme of the HMG-CoA reductase pathway, the
body's metabolic pathway for the synthesis of cholesterol.
[0067] The structure of one exemplary statin, lovastatin, is shown
below.
##STR00001##
[0068] Standard: A substance or solution of a substance of known
amount, purity or concentration that is useful as a control. A
standard can also be a known value or concentration of a particular
substance. A standard can be compared (such as by spectrometric,
chromatographic, spectrophotometric, or statistical analysis) to an
unknown sample (of the same or similar substance) to determine the
presence of the substance in the sample and/or determine the
amount, purity or concentration of the unknown sample. In one
embodiment, a standard is a particular T/ST ratio. In another
embodiment, a standard is a known ratio of T/ST that is found in a
sample from a subject that does not have a cardiac disorder
associated with oxidative stress.
[0069] Subject: Living organisms susceptible to hyposialylation
disorders; a category that includes both human and non-human
mammals, such as non-human primates.
[0070] Therapeutically effective amount: An amount of a
pharmaceutical preparation that alone, or together with a
pharmaceutically acceptable carrier or one or more additional
therapeutic agents, induces the desired response. A therapeutic
agent, such as MaNAc or any other sialylation increasing therapy,
is administered in therapeutically effective amounts.
[0071] Effective amounts a therapeutic agent can be determined in
many different ways, such as assaying for a reduction in
atherosclerotic disease or improvement of physiological condition
of a subject having vascular disease. Effective amounts also can be
determined through various in vitro, in vivo or in situ assays.
[0072] Therapeutic agents can be administered in a single dose, or
in several doses, for example daily, during a course of treatment.
However, the effective amount of can be dependent on the source
applied, the subject being treated, the severity and type of the
condition being treated, and the manner of administration.
[0073] In one example, it is an amount sufficient to partially or
completely alleviate symptoms of vascular disease within a subject.
Treatment can involve only slowing the progression of the vascular
disease temporarily, but can also include halting or reversing the
progression of the vascular disease permanently. For example, a
pharmaceutical preparation can decrease one or more symptoms of
vascular disease, for example decrease a symptom by at least 20%,
at least 50%, at least 70%, at least 90%, at least 98%, or even at
least 100%, as compared to an amount in the absence of the
pharmaceutical preparation.
[0074] Thomsen-Friedenreich Antigen: N-actetyl galactosamine linked
Galactose (Gal.beta.1-3GalNAc.alpha.1), also known as "T" antigen.
The monosialylated form of this antigen (Neu5Ac-Gal-GalNAc) is
called "ST" antigen; a disialylated form also exists. The
structures of T and ST are shown in FIG. 3 of PCT Publication No.
2014/160018, incorporated herein by reference. Methods for
detecting T and ST are disclosed, for example, in Leoyklang et al.
Biomarkers Med 2014: 8: 641-652.
[0075] Treating a disease: "Treatment" refers to a therapeutic
intervention that ameliorates a sign or symptom of a disease or
pathological condition, such a sign, parameter or symptom of
cardiovascular disease (for example, ASCVD). Treatment can also
induce remission or cure of a condition, such as a cardiovascular
disease. In particular examples, treatment includes preventing a
disease, for example by inhibiting the full development of a
disease, such as preventing development of cardiovascular disease.
Prevention of a disease does not require a total absence of the
disease. For example, a decrease of at least 50% can be
sufficient.
[0076] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of this disclosure, suitable
methods and materials are described below. The term "comprises"
means "includes." All publications, patent applications, patents,
and other references mentioned herein are incorporated by reference
in their entirety. In case of conflict, the present specification,
including explanations of terms, will control. In addition, the
materials, methods, and examples are illustrative only and not
intended to be limiting.
Methods of Treatment
[0077] The methods disclosed herein relates to compositions and
methods for preventing and/or reducing cellular and tissue damage
caused by oxidative stress. The compositions and methods disclosed
herein are useful in preventing and treating a variety of disease
states or pathological situations in which reactive oxygen species
(ROS) are produced and released. The methods include administering
to the subject a therapeutically effective amount of a sialylation
increasing therapy, such as a sialic acid precursor, sialic acid,
one or more sialylated compounds, mannosamine, or N-acetyl
mannosamine or a derivative thereof.
[0078] Methods are disclosed herein for treating a subject with a
cardiovascular disorder associated with oxidative stress. In some
embodiments, the cardiovascular disorder is associated with
hyposialylation, such as hyposialylation in the cardiac and/or
vascular tissue. In some embodiments, the subject has heart failure
or atherosclerotic cardiovascular disease. In additional
embodiments, the subject has myocardial infarction, ischemic heart
disease, stroke, cardiomyopathy, arrhythmia, restrictive
cardiomyopahyor peripheral arterial disease.
[0079] Subjects with restrictive cardiomyopathy or an arrhythmia
identified by heart function testing will be good candidates for
treatment. Such subjects have decreased cardiac output, and
increased end-diastolic volume and end-systolic volumes. Exemplary
decreased cardiac output, and increased end-diastolic volume and
end-systolic volumes are shown in the GNE myopathy mouse model with
oxidative-stress related cardiomyopathy (see the Examples
section).
[0080] In some embodiments, to select a subject with cardiac
impairment for sialylation-increasing therapy, levels of plasma
oxidative stress markers and/or plasma T/ST ratios can be
evaluated. When oxidative stress markers are increased compared to
an unaffected individual and/or plasma T/ST ratios are increased
beyond the normal range, the individual can be administered the
sialylation increasing therapy, such as a sialic acid precursor,
sialic acid, one or more sialylated compounds, mannosamine, or
N-acetyl mannosamine or a derivative thereof.
[0081] In some embodiments, the subject has a hyposialylation
disorder, such as, but not limited to GNE myopathy. The subject can
have a cardiovascular disorder associated with oxidative stress,
and GNE myopathy. In specific examples, the subject has cardiac
impairment and GNE myopathy. A subject can be selected that has
signs or symptoms of restrictive cardiomyopathy, an arrhythmia,
decreased cardiac output, increased end-diastolic volume, decreased
end-systolic volume, or a combination thereof. The method can
include selecting this subject for treatment.
[0082] In further embodiments, the subject does not have GNE
myopathy. Thus, in some examples, the subject has a cardiovascular
disorder associated with oxidative stress, but does not have GNE
myopathy. The method can include selecting this subject for
treatment.
[0083] In yet other embodiments, the subject has GNE myopathy.
Thus, in some examples, the subject has a cardiovascular disorder
associated with oxidative stress, and has GNE myopathy. The method
can include selecting this subject for treatment.
[0084] In yet other embodiments, the subject has been determined to
be at risk for cardiovascular disease based on risk factors, such
as, but not limited to, Framingham risk factors, or guidelines
jointly issued by the American Heart Association and American
College of Cardiology. In specific non-limiting examples, the
method can include evaluating a subject to determine if the subject
is at risk for cardiovascular disease using Framingham risk
factors. These risk factors include age, gender, whether the
subject smokes, blood pressure, total cholesterol level, and high
density lipoprotein cholesterol level (see above).
[0085] The Framingham Risk Score is a gender-specific algorithm
used to estimate the 10-year cardiovascular risk of a subject using
specific factors. The Framingham Risk Score was first developed
based on data obtained from the Framingham Heart Study, to estimate
the 10-year risk of developing coronary heart disease (see Third
Report of the National Cholesterol Education Program (NCEP) Expert
Panel on Detection, Evaluation, and Treatment of High Blood
Cholesterol in Adults (Adult Treatment Panel III) final report,
Circulation 2002 Dec. 17; 106(25):3143-421, incorporated herein by
reference). The method can include evaluation of a subject to
determine if the subject is at risk for cardiovascular disease
using risk factors, such as, but not limited to, Framingham risk
factors and/or guidelines jointly issues by the American Heart
Association and American College of Cardiology.
[0086] Framingham risk factors include age, gender, low density
lipoprotein (LDL) cholesterol level, whether the subject smokes,
blood pressure (and whether the subject is receiving
pharmacological treatment for hypertension), total cholesterol
level, and high density lipoprotein (HDL) cholesterol level.
Programs for this evaluation are available on the internet, such as
at the U.S. National Heart, Lung, and Blood Institute (NHLBI)
website. The disclosed methods can include (a) selecting a subject
for treatment based on the Framingham risk factor and/or (b)
evaluating the Framingham risk factors as part of the treatment
protocol.
[0087] In some embodiments, the subject has heart failure (HF). HF
is a generally progressive, life threatening condition in which
myocardial contractility is depressed such that the heart is unable
to adequately pump the blood returning to it, also referred to as
decompensation. Symptoms include breathlessness, fatigue, weakness,
leg swelling, and exercise intolerance. On physical examination,
patients with heart failure often have elevated heart and
respiratory rates (an indication of fluid in the lungs), edema,
jugular venous distension, and enlarged hearts. The most common
cause of HF is atherosclerosis, which causes blockages in the
coronary arteries that provide blood flow to the heart muscle.
Ultimately, such blockages may cause myocardial infarction with
subsequent decline in heart function and resultant heart failure.
Other causes of HF include valvular heart disease, hypertension,
viral infections of the heart, alcohol consumption, and diabetes.
Some cases of HF occur without clear etiology and are called
idiopathic.
[0088] There are several types of HF. Two types of HF are
identified according to which phase of the cardiac pumping cycle is
more affected. Systolic heart failure occurs when the heart's
ability to contract decreases. The heart cannot pump with enough
force to push a sufficient amount of blood into the circulation
leading to a reduced left ventricular ejection fraction. Lung
congestion is a typical symptom of systolic heart failure.
Diastolic heart failure refers to the heart's inability to relax
between contractions and allow enough blood to enter the
ventricles. Higher filling pressures are required to maintain
cardiac output, but contractility as measured by left ventricular
ejection fraction is typically normal. Swelling (edema) in the
abdomen and legs is a typical symptom of diastolic heart failure.
The disclosed methods are of use in treating subject with systolic
or diastolic heart failure. HF is also classified according to its
severity. The New York Heart Association classification classifies
CHF into four classes: [0089] Class I--no obvious symptoms, with no
limitations on physical activity; [0090] Class II--some symptoms
during or after normal activity, with mild physical activity
limitations; [0091] Class III--symptoms with less than ordinary
activity, with moderate to significant physical activity
limitations; [0092] Class IV--significant symptoms at rest, with
severe to total physical activity limitations. The disclosed
methods can be used to treat a subject that has class I, class II,
class III or class IV heart failure.
[0093] The disclosed methods are also of use to treat a subject
that has acute HF. Acute HF can be caused by acute myocardial
injury that affects either myocardial performance, such as
myocardial infarction, or valvular/chamber integrity, such as
mitral regurgitation or ventricular septal rupture, which leads to
an acute rise in left ventricular and diastolic pressure resulting
in pulmonary edema, and dyspnea.
[0094] The subject with HF can be administered an additional
therapeutic agent, such as, but not limited to vasodilators,
positive inotropes, and/or diuretics. In some embodiments, the
subject is administered a beta-antagonists. The subject can be
administered dopamine, dobutamine, dopexamine, or isoproterenol.
The subject can be administered a therapeutically effective amount
of an angiotensin-converting enzyme (ACE) inhibitor, a beta
blocker, an aldosterone antagonist, a diuretic, an angiotensin
receptor blocker (ARB), and/or a vasodilator.
[0095] In some embodiments, the disclosed methods are of use to
treat a subject with atherosclerosis. The subject can have
atherosclerotic heart disease. In some embodiments the subject can
also be administered a therapeutically effective amount of a
statin, niacin, a fibrate, a bile acid binding resin, a cholesterol
absorption inhibitor, a PCSK9-targeting drug, an LDL-targeting drug
or an HDL-targeting drug.
[0096] In some embodiments, the disclosed methods are of use to
treat a subject who has a myocardial infarction, or previously had
a myocardial infarction. Generally, these subjects have cardiac
tissue death caused by ischemia. Acute myocardial infarction (AMI),
or a "heart attack," occurs when localized myocardial ischemia
causes the development of a defined region of tissue death. AMI is
most often caused by rupture of an atherosclerotic lesion in a
coronary artery. This causes the formation of a thrombus that plugs
the artery, stopping it from supplying blood to the region of the
heart that it supplies.
[0097] The disclosed methods are of use to treat a subject that has
cardiac ischemia. Severe and prolonged ischemia produces a region
of necrosis spanning the entire thickness of the myocardial wall.
Such a transmural infarct usually causes ST segment elevation. Less
severe and protracted ischemia can arise when coronary occlusion is
followed by spontaneous reperfusion; an infarct-related artery is
not completely occluded; occlusion is complete, but an existing
collateral blood supply prevents complete ischemia; or the oxygen
demand in the affected zone of myocardium is smaller. Under these
conditions, the necrotic zone may be mainly limited to the
subendocardium, typically causing non-ST segment elevation MI. A
subject with any of these changes can be selected for
treatment.
[0098] The subject can have a myocardial infarction or cardiac
ischemia, and can also be administered a therapeutically effective
amount of an antiplatelet agent, an anticoagulation agent, or a
lipid or blood pressure regulating agent. Exemplary lipid
regulating agents are statin, niacin, PCSK9-targeting drug, bile
acid binding resin, or HDL-cholesterol targeting drug.
[0099] In other embodiments the disclosed methods are of use to
treat subject has a vascular disorder, thrombotic stroke,
peripheral vascular disease, restenosis, acute coronary syndrome,
or reperfusion myocardial injury. The subject can also have chronic
kidney disease associated with a heart condition, such as diabetic
neuropathy. The disclosed methods can also be used to treat a
subject with diastolic dysfunction, restrictive cardiomyopathy,
and/or and arrhythmia.
[0100] In any embodiment disclosed herein, the subject can be
administered a therapeutically effective amount of an anti-oxidant,
such as N-acetylcysteine, vitamin C, beta carotene, or vitamin E.
In a specific non-limiting example, the subject can be administered
a therapeutically effective amount of N-acetylcysteine.
[0101] In additional embodiments, the method includes selecting a
subject with GNE myopathy who has not been identified as having
impaired cardiac function and testing the subject's cardiac
function. The method can also include administering to the subject
at therapeutically effective amount of an anti-oxidant.
Sialylation Increasing Therapies
[0102] The methods disclosed herein include administering to the
subject a therapeutically effective amount of a sialylation
increasing therapy. Thus, a therapeutically effective amount of a
sialic acid precursor, sialic acid, or one or more sialylated
compounds, mannosamine, or N-acetyl mannosamine, a derivative
thereof, or any combination of these sialylation increasing agents,
can be administered to the subject.
[0103] Sialic acids are sugars found on many cellular and tissue
components. For example, sialic acids are present on most cell
surfaces, and on proteins and lipids and are involved in cell to
cell interactions. Sialic acid-rich oligosaccharides on the
glycoconjugates found on surface membranes help keep water at the
surface of cells. The sialic acid-rich regions also contribute to
creating a negative charge on the cells surface. Since water is a
polar molecule, it is attracted to cell surfaces and membranes.
Thus, sialic acids contribute to cellular hydration and fluid
uptake. Sialic acid is also a vital component of many body fluids
including, serum, cerebrospinal, saliva, amniotic, and mother's
milk. Any therapeutic agent that increases sialylation can be used
in the methods disclosed herein. In some embodiments, the subject
is administered a sialic acid precursor, sialic acid, or one or
more sialylated compounds.
[0104] The subject can be administered intravenous immunoglobulin
(IVIG) or sialyllactose. Intravenous immunoglobulin is pooled,
polyvalent immunoglobulin G (IgG) extracted from donors. In some
embodiments, WIG is administered at a high dosage, such as about
100 to 400 mg per kg of body weight, or about 1 to about 2 grams
IVIG per kg body weight.
[0105] The therapeutic agent can be mannosamine or a derivative
thereof. See also European Patent No. EP 1521761, which is
incorporated herein by reference.
[0106] I. N-acetyl-mannosamine
[0107] N-acetyl-mannosamine and derivatives thereof are useful for
treating a variety of diseases and cardiovascular disorders
associated with oxidative stress, as disclosed herein, including.
N-acetyl-D-mannosamine is a key compound in the sialic acid
biosynthetic pathway. In particular, there is a regulated,
rate-limiting enzymatic step in the pathway that leads to sialic
acid formation, and this rate-limiting step gives rise to
N-acetyl-D-mannosamine. Hence, once N-acetyl-D-mannosamine is
formed or administered, no other enzymatic step leading to the
formation of sialic acid is subject to feedback inhibition. Thus,
administration of N-acetyl-D-mannosamine will lead to increased
amounts of sialic acid. The structure of N-acetyl-mannosamine is
shown below.
##STR00002##
[0108] Therefore, administration of N-acetyl mannosamine (ManNAc)
and/or its derivatives promotes formation of sialic acid
(N-acetylneuramic acid).
[0109] II. N-Acetylmannosamine Derivatives
[0110] N-acetylmannosamine and derivatives thereof can also be used
in the therapeutic methods and compositions of the invention. The
structures of such N-acetylmannosamine derivatives of use are shown
by Formula I.
##STR00003##
wherein:
[0111] R.sub.1, R.sub.3, R.sub.4, or R.sub.5 is hydrogen, lower
alkanoyl, carboxylate or lower alkyl; and
[0112] R.sub.2 is lower alkyl, lower alkanoylalkyl, lower alkyl
alkanoyloxy.
[0113] The following definitions are used, unless otherwise
described: Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both
straight and branched groups; but reference to an individual
radical such as "propyl" embraces only the straight chain radical,
a branched chain isomer such as "isopropyl" being specifically
referred to.
[0114] Lower alkyl refers to (C.sub.1-C.sub.6)alkyl. Such a lower
alkyl or (C.sub.1-C.sub.6)alkyl can be methyl, ethyl, propyl,
isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;
(C.sub.3-C.sub.6)cycloalkyl can be cyclopropyl, cyclobutyl,
cyclopentyl, or cyclohexyl;
(C.sub.3-C.sub.6)cycloalkyl(C.sub.1-C.sub.6)alkyl can be
cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,
cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl,
2-cyclopentylethyl, or 2-cyclohexylethyl; (C.sub.1-C.sub.6)alkoxy
can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy,
sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy;
(C.sub.2-C.sub.6)alkenyl can be vinyl, allyl, 1-propenyl,
2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl,
2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl,
3-hexenyl, 4-hexenyl, or 5-hexenyl; (C.sub.2-C.sub.6)alkynyl can be
ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl,
1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl,
2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl;
(C.sub.1-C.sub.6)alkanoyl can be acetyl, propanoyl or butanoyl;
halo(C.sub.1-C.sub.6)alkyl can be iodomethyl, bromomethyl,
chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl,
2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl;
hydroxy(C.sub.1-C.sub.6)alkyl can be hydroxymethyl, 1-hydroxyethyl,
2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl,
1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl,
1-hydroxyhexyl, or 6-hydroxyhexyl; (C.sub.1-C.sub.6)alkoxycarbonyl
can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl,
isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or
hexyloxycarbonyl; (C.sub.1-C.sub.6)alkylthio can be methylthio,
ethylthio, propylthio, isopropylthio, butylthio, isobutylthio,
pentylthio, or hexylthio; (C.sub.2-C.sub.6)alkanoyloxy can be
acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy,
or hexanoyloxy.
[0115] III. Formulations and Administration
[0116] N-acetyl mannosamine and/or derivatives thereof or any
sialylation increasing therapeutic agent are administered so as to
achieve a reduction in at least one symptom associated with an
indication or disease. For example, administration of N-acetyl
mannosamine and/or derivatives thereof or any sialylation
increasing therapeutic agent can lead to an improvement in vascular
function, an improvement in cardiac function, and/or increased
oxygenation of the blood. In additional embodiments, administration
of N-acetyl mannosamine and/or derivatives thereof or any
sialylation increasing therapeutic agent results in re-sialylating
hyposialylated heart tissue, reducing ractive oxygen species in the
heart and/or blood vessels, and/or improving vascular function.
[0117] To achieve the desired effect(s), N-acetyl mannosamine
and/or derivatives thereof, or any sialylation increasing
therapeutic agent, can be administered as single or divided
dosages, for example, of at least about 0.01 mg/kg to about 500 to
750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg,
at least about 0.1 mg/kg to about 200 to 400 mg/kg or at least
about 1 mg/kg to about 25 to 200 mg/kg of body weight, although
other dosages may provide beneficial results. The amount
administered will vary depending on various factors including, but
not limited to the disease, the weight, the physical condition, the
health, the age of the mammal, whether prevention or treatment is
to be achieved. Such factors can be readily determined by the
clinician employing animal models or other test systems that are
available in the art.
[0118] Administration of the therapeutic agents can be in a single
dose, in unit dosage form, in multiple doses, in a continuous or
intermittent manner, depending, for example, upon the recipient's
physiological condition, whether the purpose of the administration
is therapeutic or prophylactic, and other factors known to skilled
practitioners. The administration of N-acetyl mannosamine and/or
derivatives thereof, or any sialylation increasing therapeutic
agent, may be essentially continuous over a pre-selected period of
time or may be in a series of spaced doses. Both local and systemic
administration is contemplated.
[0119] To prepare the composition, N-acetyl mannosamine and/or one
or more derivatives thereof and/or or any sialylation increasing
therapeutic agent are synthesized or otherwise obtained, and
purified as necessary or desired. N-acetyl mannosamine (and/or
derivatives thereof, or any sialylation increasing therapeutic
agent) can then be added to a composition (or food product),
adjusted to the appropriate concentration, and optionally combined
with other agents. The absolute weight of N-acetyl mannosamine
and/or its derivatives, or any sialylation increasing therapeutic
agent, that is included in a unit dose can vary widely. For
example, about 0.01 to about 2 g, or about 0.1 to about 1 g of
N-acetyl mannosamine and/or derivatives thereof (or any sialylation
increasing therapeutic agent) are often used in compositions.
Alternatively, the unit dosage can vary from about 0.01 g to about
50 g, from about 0.01 g to about 35 g, from about 0.1 g to about 25
g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g,
from about 0.5 g to about 4 g, or from about 0.5 g to about 2
g.
[0120] Daily doses of N-acetyl mannosamine and/or derivatives
thereof (or any sialylation increasing therapeutic agent) can vary
as well. Such daily doses can range, for example, from about 0.01
g/day to about 50 g/day, from about 0.02 g/day to about 25 g/day,
from about 0.03 g/day to about 12 g/day, from about 0.04 g/day to
about 10 g/day, from about 0.05 g/day to about 8 g/day, and from
about 0.07 g/day to about 6 g/day.
[0121] In some non-limiting example, a dose of dose 2 g/kg/day
N-acetyl mannosamine is administered. In another embodiment, a dose
of 2 g/kg/day Neu5Ac (sialic acid) is administered.
[0122] Thus, one or more suitable unit dosage forms comprising
N-acetyl mannosamine and/or derivatives thereof, or any sialylation
increasing therapeutic agent, can be administered by a variety of
routes including oral, parenteral (including subcutaneous,
intravenous, intramuscular and intraperitoneal), rectal, dermal,
transdermal, intrathoracic, intrapulmonary and intranasal
(respiratory) routes. The therapeutic agents may also be formulated
for sustained release (for example, using microencapsulation, see
WO 94/07529, and U.S. Pat. No. 4,962,091). The formulations may,
where appropriate, be conveniently presented in discrete unit
dosage forms and may be prepared by any of the methods well known
to the pharmaceutical arts. Such methods may include the step of
mixing N-acetyl mannosamine and/or derivatives thereof with liquid
carriers, solid matrices, semi-solid carriers, finely divided solid
carriers or combinations thereof, and then, if necessary,
introducing or shaping the product into the desired delivery
system.
[0123] When N-acetyl mannosamine and/or its derivatives, or any
sialylation increasing therapeutic agent, is prepared for oral
administration, it is generally combined with a pharmaceutically
acceptable carrier, diluent or excipient to form a pharmaceutical
formulation, or unit dosage form. For oral administration, N-acetyl
mannosamine (and/or derivatives thereof or any other sialylation
increasing therapeutic agent) may be present as a powder, a
granular formulation, a solution, a suspension, an emulsion or in a
natural or synthetic polymer or resin for ingestion of N-acetyl
mannosamine (and/or derivatives thereof or any other sialylation
increasing therapeutic agent) from a chewing gum. The active
ingredients may also be presented as a bolus, electuary or paste.
Orally administered N-acetyl mannosamine and/or derivatives
thereof, or any sialylation increasing therapeutic agent, can also
be formulated for sustained release. For example, N-acetyl
mannosamine and/or derivatives thereof or any sialylation
increasing therapeutic agent, can be coated, can be
micro-encapsulated, or otherwise placed within a sustained delivery
device, for example, in order to avoid salivary bacteria
degradation. The total N-acetyl mannosamine and its derivatives, or
any other sialylation increasing therapeutic agent, in such
formulations comprises from 0.1 to 99.9% by weight of the
formulation.
[0124] By "pharmaceutically acceptable" it is meant a carrier,
diluent, excipient, and/or salt that is compatible with the other
ingredients of the formulation, and not deleterious to the
recipient thereof.
[0125] Pharmaceutical formulations containing N-acetyl mannosamine
and/or derivatives thereof, or any sialylation increasing
therapeutic agent, can be prepared by procedures known in the art
using well-known and readily available ingredients. For example,
N-acetyl mannosamine and/or its derivatives, or any sialylation
increasing therapeutic agent, can be formulated with common
excipients, diluents, or carriers, and formed into tablets,
capsules, solutions, suspensions, powders, aerosols and the like.
Examples of excipients, diluents, and carriers that are suitable
for such formulations include buffers, as well as fillers and
extenders such as starch, cellulose, sugars, mannitol, and silicic
derivatives. Binding agents can also be included such as
carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl
methylcellulose and other cellulose derivatives, alginates,
gelatin, and polyvinyl-pyrrolidone. Moisturizing agents can be
included such as glycerol, disintegrating agents such as calcium
carbonate and sodium bicarbonate. Agents for retarding dissolution
can also be included such as paraffin. Resorption accelerators such
as quaternary ammonium compounds can also be included. Surface
active agents such as acetyl alcohol and glycerol monostearate can
be included. Adsorptive carriers such as kaolin and bentonite can
be added. Lubricants such as talc, calcium and magnesium stearate,
and solid polyethyl glycols can also be included. Preservatives may
also be added. The compositions of the invention can also contain
thickening agents such as cellulose and/or cellulose derivatives.
They may also contain gums such as xanthan, guar or carbo gum or
gum arabic, or alternatively polyethylene glycols, bentones and
montmorillonites, and the like.
[0126] For example, tablets or caplets containing N-acetyl
mannosamine (and/or its derivatives or any sialylation increasing
therapeutic agent) can include buffering agents such as calcium
carbonate, magnesium oxide and magnesium carbonate. Caplets and
tablets can also include inactive ingredients such as cellulose,
pre-gelatinized starch, silicon dioxide, hydroxy propyl methyl
cellulose, magnesium stearate, microcrystalline cellulose, starch,
talc, titanium dioxide, benzoic acid, citric acid, corn starch,
mineral oil, polypropylene glycol, sodium phosphate, zinc stearate,
and the like. Hard or soft gelatin capsules containing N-acetyl
mannosamine (and/or its derivatives or any sialylation increasing
therapeutic agent) can contain inactive ingredients such as
gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch,
talc, and titanium dioxide, and the like, as well as liquid
vehicles such as polyethylene glycols (PEGs) and vegetable oil.
Moreover, enteric-coated caplets or tablets containing N-acetyl
mannosamine and/or its derivatives, or any sialylation increasing
therapeutic agent, are designed to resist disintegration in the
stomach and dissolve in the more neutral to alkaline environment of
the duodenum.
[0127] N-acetyl mannosamine and/or its derivatives, or any
sialylation increasing therapeutic agent, can also be formulated as
an elixir or solution for convenient oral administration or as a
solution appropriate for parenteral administration, for instance by
intramuscular, subcutaneous, intraperitoneal or intravenous routes.
The pharmaceutical formulations of N-acetyl mannosamine and/or its
derivatives, or any sialylation increasing therapeutic agent, can
also take the form of an aqueous or anhydrous solution or
dispersion, or alternatively the form of an emulsion or suspension
or salve.
[0128] Thus, N-acetyl mannosamine and/or its derivatives, or any
sialylation increasing therapeutic agent, may be formulated for
parenteral administration (e.g., by injection, for example, bolus
injection or continuous infusion) and may be presented in unit dose
form in ampoules, pre-filled syringes, small volume infusion
containers or in multi-dose containers. As noted above,
preservatives can be added to help maintain the shelve life of the
dosage form. The N-acetyl mannosamine, its derivatives, or any
sialylation increasing therapeutic agent, and other ingredients may
form suspensions, solutions, or emulsions in oily or aqueous
vehicles, and may contain formulatory agents such as suspending,
stabilizing and/or dispersing agents. Alternatively, the N-acetyl
mannosamine, its derivatives, or any sialylation increasing
therapeutic agent, and other ingredients may be in powder form,
obtained by aseptic isolation of sterile solid or by lyophilization
from solution, for constitution with a suitable vehicle, e.g.,
sterile, pyrogen-free water, before use.
[0129] These formulations can contain pharmaceutically acceptable
carriers, vehicles and adjuvants that are well known in the art. It
is possible, for example, to prepare solutions using one or more
organic solvent(s) that is/are acceptable from the physiological
standpoint, chosen, in addition to water, from solvents such as
acetone, ethanol, isopropyl alcohol, glycol ethers such as the
products sold under the name "Dowanol," polyglycols and
polyethylene glycols, C.sub.1-C.sub.4 alkyl esters of short-chain
acids, ethyl or isopropyl lactate, fatty acid triglycerides such as
the products marketed under the name "Miglyol," isopropyl
myristate, animal, mineral and vegetable oils and
polysiloxanes.
[0130] It is possible to add other ingredients such as
antioxidants, surfactants, other preservatives, film-forming,
keratolytic or comedolytic agents, perfumes, flavorings and
colorings. Antioxidants such as t-butylhydroquinone, butylated
hydroxyanisole, butylated hydroxytoluene and .alpha.-tocopherol and
its derivatives can be added. In one embodiment, N-acetylcysteine
is added to the composition.
[0131] Additionally, N-acetyl mannosamine and/or derivatives
thereof, or any sialylation increasing therapeutic agent, are well
suited to formulation in a sustained release dosage form. Thus,
such formulations can be so constituted that they release the
N-acetyl mannosamine and/or its derivative, for example, in a
particular part of the intestinal, urogenital or respiratory tract,
over a period of time. Coatings, envelopes, and protective matrices
may be made, for example, from polymeric substances, such as
polylactide-glycolates, liposomes, microemulsions, microparticles,
nanoparticles, or waxes. These coatings, envelopes, and protective
matrices are useful to coat indwelling devices, e.g., stents,
catheters, peritoneal dialysis tubing, draining devices and the
like.
[0132] For topical administration, N-acetyl mannosamine and/or its
derivative(s), or any sialylation increasing therapeutic agent, may
be formulated as is known in the art for direct application to a
target area. Forms chiefly conditioned for topical application take
the form, for example, of creams, milks, gels, dispersion or
microemulsions, lotions thickened to a greater or lesser extent,
impregnated pads, ointments or sticks, aerosol formulations (e.g.,
sprays or foams), soaps, detergents, lotions or cakes of soap.
Other conventional forms for this purpose include wound dressings,
coated bandages or other polymer coverings, ointments, creams,
lotions, pastes, jellies, sprays, and aerosols. Thus, N-acetyl
mannosamine and/or its derivatives, or any sialylation increasing
therapeutic agent, can be delivered via patches or bandages for
dermal administration. Alternatively, N-acetyl mannosamine and/or
its derivatives, or any sialylation increasing therapeutic agent,
can be formulated to be part of an adhesive polymer, such as
polyacrylate or acrylate/vinyl acetate copolymer. For long-term
applications it might be desirable to use microporous and/or
breathable backing laminates, so hydration or maceration of the
skin can be minimized. The backing layer can be any appropriate
thickness that will provide the desired protective and support
functions. A suitable thickness will generally be from about 10 to
about 200 microns.
[0133] Ointments and creams may, for example, be formulated with an
aqueous or oily base with the addition of suitable thickening
and/or gelling agents. Lotions may be formulated with an aqueous or
oily base and will in general also contain one or more emulsifying
agents, stabilizing agents, dispersing agents, suspending agents,
thickening agents, or coloring agents. The therapeutic agents can
also be delivered via iontophoresis, e.g., as disclosed in U.S.
Pat. Nos. 4,140,122; 4,383,529; or 4,051,842. The percent by weight
of a therapeutic agent of the invention present in a topical
formulation will depend on various factors, but generally will be
from 0.01% to 95% of the total weight of the formulation, and
typically 0.1-85% by weight.
[0134] Drops, such as eye drops or nose drops, may be formulated
with N-acetyl mannosamine and/or derivatives thereof, or any
sialylation increasing therapeutic agent, in an aqueous or
non-aqueous base also comprising one or more dispersing agents,
solubilizing agents or suspending agents. Liquid sprays are
conveniently delivered from pressurized packs. Drops can be
delivered via a simple eye dropper-capped bottle, or via a plastic
bottle adapted to deliver liquid contents dropwise, via a specially
shaped closure.
[0135] N-acetyl mannosamine and/or its derivatives, or any
sialylation increasing therapeutic agent, may further be formulated
for topical administration in the mouth or throat. For example,
N-acetyl mannosamine and/or its derivatives, or any sialylation
increasing therapeutic agent, may be formulated as a lozenge
further comprising a flavored base, usually sucrose and acacia or
tragacanth; pastilles comprising the composition in an inert base
such as gelatin and glycerin or sucrose and acacia; and mouthwashes
comprising the composition of the present invention in a suitable
liquid carrier.
[0136] The pharmaceutical formulations may include, as optional
ingredients, pharmaceutically acceptable carriers, diluents,
solubilizing or emulsifying agents, and salts of the type that are
available in the art.
[0137] Furthermore, N-acetyl mannosamine and/or its derivatives, or
any sialylation increasing therapeutic agent, may also be used in
combination with other therapeutic agents, for example, pain
relievers, anti-inflammatory agents, and the like, whether for the
conditions described or some other condition.
[0138] The present invention further pertains to a packaged
pharmaceutical composition such as a kit or other container for
increasing production of sialic acid in a mammal. The kit or
container holds a therapeutically effective amount of a
pharmaceutical composition for increasing intracellular production
of sialic acid and instructions for using the pharmaceutical
composition for increasing production of sialic acid in the mammal.
The pharmaceutical composition includes N-acetyl mannosamine and/or
its derivatives in a therapeutically effective amount such that
sialic acid production is increased.
[0139] IV. Food Supplement
[0140] N-acetyl mannosamine and/or its derivatives, or any
sialylation increasing therapeutic agent, can be administered as a
food supplement or incorporated into food or drink item such as a
nutritional bar, snack bar, cookie, candy, cereal, pudding, ice
cream, frozen confectionary, chewing gum, drink mix, soda pop,
liquid supplement, sauce, salad dressing, gravy, jelly, jam,
spread, margarine, peanut butter, nut spread, frosting, and the
like. In essence, it can be used in any food, composition or
supplement in which sugar is employed. Hence, N-acetyl mannosamine
and/or derivatives thereof, or any sialylation increasing
therapeutic agent, can be used as a partial or full substitute for
sugar.
[0141] Such food supplements, drinks and food items can include any
other food ingredient including, for example, flour, oil, cream,
butter, sugar, salt, spices and the like. In addition, the food
supplements, drinks and food items can include vitamins and
nutrients commonly found in other nutritional supplements.
Methods for Diagnosis
[0142] Methods are disclosed herein for diagnosing a disorder
associated with oxidative stress, including confirming a diagnosis
of a disorder associated with oxidative stress, such as a clinical
diagnosis. In some embodiments, disorder is a cardiovascular
disorder associated with oxidative stress (see above). These
methods include obtaining a biological sample from a subject of
interest, such as a subject suspected of having a cardiovascular
disorder associated with oxidative stress, and measuring
monosialylated Thomsen-Friedenreich (ST) antigen and measuring
non-sialylated Thomsen-Friedenreich antigen (T). The ratio of T to
ST (T/ST), in the biological sample is determined.
[0143] In some embodiments, a method is provided for diagnosing a
cardiovascular disorder associated with oxidative stress, for
example confirming the diagnosis of a cardiovascular disorder
associated with oxidative stress, that includes measuring an amount
of monosialylated Thomsen-Friedenreich (ST) antigen in a biological
sample from the subject and measuring an amount of non-sialylated
Thomsen-Friedenreich antigen (T) in the biological sample from the
subject. The ratio of T to ST is determined. The ratio of T to ST
can be compared to a control, such as a standard value. In some
embodiments, a subject is selected that is suspected of having the
cardiovascular disorder, such as based on clinical symptoms.
[0144] In specific non-liming examples method can be used to
diagnose heart failure or atherosclerotic cardiovascular disease.
In other non-limiting examples, the method can be used to diagnose
myocardial infarction, ischemic heart disease, stroke, or
peripheral arterial disease. In further non-limiting examples the
method can be used to diagnose decreased cardiac function
associated with a hyposialylation disorder, such as GNE myopathy.
Thus, the method can be used to detect cardiomyopathy and/or
arrhythmia.
[0145] In some embodiments, a ratio of T to monosialylated ST
(T/ST) in a plasma or serum sample of greater than about 0.051 to
greater than about 0.062, greater than about 0.052 to greater than
about 0.06, greater than about 0.058 to greater than about 0.062,
such as about 0.052 or greater, about 0.053 or greater, about 0.054
or greater, about 0.055 or greater, about 0.056 or greater, about
0.057 or greater, about 0.058 or greater, about 0.059 or greater,
or about 0.06 or greater indicates that the overall sialylation of
O-linked glycoproteins in the plasma or serum is below 95% of the
population and that the subject has the cardiovascular disorder,
and/or confirms the diagnosis of the cardiovascular disorder. In
some embodiments, a T/ST ratio in serum or plasma of greater than
about 0.07, about 0.08, about 0.09, or about 0.1 indicates that the
subject has a cardiovascular disorder. In other embodiments, a
ratio of T to ST (T/ST) of less than about 0.06, less than about
0.059, less than about 0.058, less than about 0.057, less than
about 0.056, less than about 0.054, less than about 0.053, less
than about 0.052, or less than about 0.051, in plasma or serum
indicates that the subject does not have the cardiovascular
disorder. In some embodiments, a ratio of T to ST in plasma or
serum of less than about 0.05, about 0.04 or about 0.03 indicates
that the subject does not have a cardiovascular disorder. In this
context, "about" indicates within about 0.005.
[0146] In some specific non-limiting examples, the biological
sample is a serum or plasma sample. In other embodiments, the
biological sample is a heart tissue sample or a sample that
includes blood vessels.
[0147] In some embodiments, the methods also include administering
to the subject a therapeutic agent for the treatment of the
cardiovascular disorder associated with oxidative stress, such as
if the ratio of T to ST in a serum or plasma sample from the
subject is greater than about 0.051 to greater than about 0.062,
greater than about 0.052 to greater than about 0.06, greater than
about 0.058 to greater than about 0.062, such as about 0.052 or
greater, about 0.053 or greater, about 0.054 or greater, about
0.055 or greater, about 0.056 or greater, about 0.057 or greater,
about 0.058 or greater, about 0.059 or greater, or about 0.06 or
greater. Suitable therapeutic agents are disclosed above. In
specific non-liming examples, the subject has heart failure or
atheroscleoric cardiovascular disease. In other non-limiting
examples, the subject has myocardial infarction, ischemic heart
disease, stroke, or peripheral arterial disease. In further
non-limiting examples the subject has cardiac function associated
with a hyposialylation disorder, such as GNE myopathy.
[0148] A ratio of T to monosialylated ST (T/ST) can also be
measured in biological samples other than serum or plasma,
including, but not limited to platelets, red cells, white cells,
cerebrospinal fluid, cell extracts (such as cell culture extracts)
urine or a biopsy sample, such as a cardiac biopsy. In some
embodiments, T and monosialylated ST are measured in biological
samples from subjects known not to have the cardiovascular disorder
associated with oxidative stress, and a control ratio of the T to
ST is established.
[0149] T and ST are measured in a biological sample from a subject
of interest, to determine if the subject has the cardiovascular
disorder associated with oxidative stress. In some embodiments, a T
to ST ratio of greater than two standard deviations greater than
the control ratio of T to ST diagnoses the cardiovascular disorder
associated with oxidative stress. In additional embodiments, a
ratio of T to ST of greater than three standard deviations than the
control ratio of T to ST diagnoses the cardiovascular disorder
associated with oxidative stress. In some embodiments, the methods
also include administering to the subject a therapeutic agent for
the treatment of the disorder associated with oxidative stress,
such as if the T to ST in a tissue sample other than serum or
plasma is greater than two standard deviations, such as three
standard deviations greater than the ratio of T to ST for the
control, such as the mean T/ST ratio for biological samples from
subjects without the disorder associated with oxidative stress
(and/or without any disorder associated with oxidative stress).
Suitable therapeutic agents are disclosed herein.
[0150] Methods are also disclosed herein for determining the
effectiveness of a first dosage, or the duration of a dosage, of a
therapeutic agent for treatment of a cardiovascular disorder
associated with oxidative stress in a subject. The method can
determine if a therapeutic agent of interest is of use for treating
the cardiovascular disorder associated with oxidative stress in a
subject, or if the therapeutic agent has been administered for a
sufficient period of time to treat the subject. The methods can be
used to determine the lowest effective therapeutic dosage of an
agent for the treatment of a subject. These methods include
measuring monosialylated ST antigen and T antigen in a biological
sample from the subject administered the therapeutic agent. In some
embodiments, the methods include administering the therapeutic
agent to the subject. The ratio of T to monosialylated ST is
determined.
[0151] In some embodiments, a ratio of T to ST in a plasma or serum
sample of less than about 0.06, less than about 0.059, less than
about 0.058, less than about 0.057, less than about 0.056, less
than about 0.054, less than about 0.053, less than about 0.052, or
less than about 0.051 indicates that the first dosage of the
therapeutic agent is effective for the treatment of the
cardiovascular disorder associated with oxidative stress, and/or
that the therapeutic agent has been administered for a sufficient
duration of time to treat the subject. In additional embodiments, a
ratio of T to ST of less than about 0.05, about 0.04 or about 0.03
indicates that the first dosage of the therapeutic agent is
effective for the treatment of the cardiovascular disorder
associated with oxidative stress, and/or that the therapeutic agent
has been administered for a sufficient duration of time to treat
the subject. Biological samples other than serum or plasma can also
be used.
[0152] In additional embodiments, a ratio of T to ST of greater
than about 0.051, greater than about 0.052, greater than about
0.053, greater than about 0.054, greater than about 0.055, greater
than about 0.056, greater than about 0.057 or greater, greater than
about 0.058, greater than about 0.059, or greater than about 0.06,
such as in serum or plasma, indicates that the first dosage of the
therapeutic agent is not effective for the treatment of the
cardiovascular disorder associated with oxidative stress and/or
that the therapeutic agent has not been administered for a
sufficient duration of time to treat the subject. In some
embodiments, a serum or plasma T/ST ratio of greater than about
0.07, about 0.08, about 0.09, or about 0.1 indicates that the first
dosage of the therapeutic agent is not effective for treating the
subject, and/or that the therapeutic agent has not been
administered for a sufficient duration of time to treat the
subject. Biological samples other than serum or plasma can also be
used. In some non-limiting examples, for any of the methods
disclosed herein, the biological sample can be a sample other than
serum or plasma.
[0153] In some embodiments, a ratio of T to monosialylated ST of at
least two standard deviations less than a control ratio of T to ST
indicates that the first dosage of the therapeutic agent is
effective for the treatment of the cardiovascular disorder
associated with oxidative stress and/or that the therapeutic agent
has not been administered for a sufficient duration of time to
treat the subject. In yet other embodiments, a ratio of T to
monosialylated ST of at least three standard deviations less than a
control ratio of T to ST for a control indicates that the first
dosage of the therapeutic agent is effective for the treatment of
the cardiovascular disorder associated with oxidative stress and/or
that the therapeutic agent has been administered for a sufficient
duration of time to treat the subject. In further embodiments, the
control ratio is the mean ratio of T to ST in biological samples
from subjects that do not have the cardiovascular disorder
associated with oxidative stress. The biological sample can be any
biological sample of interest, such as blood, an extract from a
biopsy, such as an extract of platelets, white blood cell, red
blood cells, kidney cells, muscle cells, heart cells, brain cells,
lung cells, or liver cells. The biological sample can be urine or
cerebrospinal fluid.
[0154] In certain aspects, these assays are performed at a
diagnostic laboratory, and the information is then provided to the
subject or a physician or other healthcare provider. In some
embodiments, the dosage of the therapeutic agent is decreased, and
a second lower dosage of the therapeutic agent is administered to
the subject. In additional embodiments, these methods can be used
to determine the lowest effective dosage of the therapeutic agent
of use to treat the subject. In yet other embodiments, the dosage
of the therapeutic is increased and administered to the subject. In
other examples, and additional dosage of the therapeutic agent is
administered to the subject.
[0155] Thus, in additional embodiments, the method can include
administering to the subject a second dosage of the therapeutic
agent, wherein the second dosage is the same, greater, or less than
the first dosage of the therapeutic agent. Monosialylated ST
antigen and T antigen are measured in a biological sample from the
subject, and the ratio of T to ST is determined.
[0156] In some embodiments, a ratio of T to monosialylated ST in
serum or plasma samples of less than about 0.0521, less than about
0.052, less than about 0.053, less than about 0.054, less than
about 0.055, less than about 0.056, less than about 0.057, less
than about 0.058, less than about 0.059, or less than about 0.06,
indicates that the second dosage of the therapeutic agent is
effective for the treatment of the cardiovascular disorder
associated with oxidative stress and/or has been administered for a
sufficient duration. In some embodiments, a ratio of T to ST of
less than about 0.05, about 0.04 or about 0.03 in the plasma or
serum sample indicates that the second dosage of the therapeutic
agent is effective for the treatment of the cardiovascular disorder
associated with oxidative stress and/or has been administered for a
sufficient duration. A ratio of T to ST of greater than about
0.051, greater than about 0.052, greater than about 0.053, greater
than about 0.054, greater than about 0.055, greater than about
0.056, greater than about 0.057 or greater, greater than about
0.058, greater than about 0.059, or greater than about 0.06 in the
plasma or serum sample indicates that the second dosage of the
therapeutic agent is not effective for the treatment of the
cardiovascular disorder associated with oxidative stress and/or has
not been administered for a sufficient duration. In other
embodiments, a ratio of T to ST of greater than about 07, about
0.08, about 0.09, or about 0.1 in the plasma or the serum sample
indicates that the second dosage of the therapeutic agent is not
effective for the treatment of the cardiovascular disorder
associated with oxidative stress. Thus, in some embodiments, the
methods disclosed herein can be repeated to determine the lowest
dosage of an agent that is effective for the treatment of the
subject. Biological samples other than serum or plasma can also be
used.
[0157] In some embodiments, in other samples than plasma or serum,
a ratio of T to monosialylated ST of at least two standard
deviations less than a control ratio of T to ST for a control
indicates that the second dosage of the therapeutic agent is
effective for the treatment of the cardiovascular disorder
associated with oxidative stress and/or is administered for a
sufficient duration to treat the subject. In yet other embodiments,
a ratio of T to monosialylated ST of at least three standard
deviations less than a control ratio of T to monosialylated ST for
a control indicates that the second dosage of the therapeutic agent
is effective for the treatment of the cardiovascular disorder
associated with oxidative stress and/or that the therapeutic agent
has been administered for a sufficient duration of time to treat
the subject. In further embodiments, the control ratio is the mean
ratio of T to monosialylated ST in biological samples from subjects
that do not have the cardiovascular disorder associated with
oxidative stress. Thus, the methods can be repeated to determine
the lowest dosage of an agent that is effective for the treatment
of the subject. The biological sample can be any biological sample
of interest, such as an extract from a tissue biopsy, such as an
extract of platelets, white blood cell, red blood cells, kidney
cells, muscle cells, heart cells, brain cells, lung cells, or liver
cells. The biological sample can be blood, urine or cerebrospinal
fluid.
[0158] The methods can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more times to determine the lowest dosage of a therapeutic agent
that is effective for treating the subject, and/or the shortest
duration of administration that is effective for treating the
subject. The methods can also be used over the course of a
therapeutic regimen to monitor the efficacy of a therapeutic agent
for the treatment of the subject.
[0159] The disclosed methods can include comparing the ratio of T
to monosialylated ST to a control. The control can be a standard
value, or the ratio of T to monosialylated ST in a biological
sample from a subject known not to have the disorder associated
with oxidative stress
[0160] For any and all of the methods disclosed herein, the
cardiovascular disorder associated with oxidative stress can be any
disorder of interest. In some embodiments, the disorder is
associated with hyposialylation in the cardiac or vascular tissue.
The disorder can be heart failure or atheroscleoric cardiovascular
disease. In additional embodiments, the disorder is myocardial
infarction, ischemic heart disease, stroke, or peripheral arterial
disease.
[0161] In further embodiments, the subject does not have GNE
myopathy. Thus, in some examples, the subject has a cardiovascular
disorder associated with oxidative stress, but does not have GNE
myopathy. In other examples, the subject has a vascular or cardiac
disorder is associated with hyposialylation, such as
hyposialylation in the cardiac or vascular tissue, but does not
have GNE myopathy.
[0162] The method can include purifying O-glycans from the
biological sample. Thus, the method can include releasing
O-glycans, such as by treating the biological sample with sodium
hydroxide and sodium borohydrate. Suitable concentrations of sodium
hydroxide and sodium borohydrate are, for example, about 1M sodium
borohydrate in 0.05M sodium hydroxide. In some embodiments,
O-glycans are purified from the biological sample. Methods for
purifying O-glycans include organic solvent extraction with
methanol, and ion-exchange chromatography, such as with AG 50W-X8
resin (Bio-Rad, Hercules, Calif.). Exemplary non-limiting methods
are disclosed in the examples section.
[0163] Disclosed herein are methods of detecting biomarkers for a
cardiovascular disorder associated with oxidative stress in order
to detect the cardiovascular disorder associated with oxidative
stress or to determine if a therapeutic agent is effective for the
treatment of this disorder. The monosialylated ST antigen and T
antigen biomarkers may be detected using any means known to those
of skill in the art, including the use of antibodies that
specifically bind T antigen, antibodies that specifically bind ST
antigen (see, for example, Cao et al. Histochem. Cell Biol. 106,
197-207 (1996)), and/or the use of lectins that bind T and/or ST
antigen, see for example, Almogren et al. Front Biosc S4: 840-863
(2012), incorporated herein by reference. These methods include
fluorescence activated cell sorting (FACS) and enzyme linked
immunosorbent assays (ELISA), Western blotting and 2D gel
electrophoresis. These methods can utilize both lectins and
antibodies; suitable antibodies are disclosed, for example, in
Published U.S. Patent Application No. 2012/0294859. In some
embodiments, these methods are used to detect monosialylated ST
antigen and T antigen on white blood cells, platelets, red blood
cells, or other tissues. Generally, the monosialylated ST antigen
and T antigen biomarkers are quantitated.
[0164] In particular disclosed embodiments of the method, the
biomarkers are detected as a ratio using mass spectrometry. Any
mass spectrometry technique known to those of ordinary skill in the
art to be suitable for analyzing biological molecules can be
utilized. For example, mass spectrometric techniques contemplated
herein include mass spectrometry techniques using various
ionization techniques (such as, but not limited to, matrix-assisted
laser desorption/ionization (MALDI), electrospray, thermospray, and
the like) coupled with one or more mass analyzer components (such
as, but not limited to, time-of-flight 66BTOF], quadrupole, and ion
traps). Any of the mass spectrometry detection methods used herein,
may also be modified to perform tandem mass spectrometry, and/or
may be modified to employ additional analytical techniques, such as
liquid chromatography, gas chromatography, and ion mobility.
[0165] Mass spectrometry has been used as a powerful tool to
characterize polymers such as glycans because of its accuracy
(.+-0.1 Dalton) in reporting the masses of fragments generated
(e.g., by enzymatic cleavage), and also because only pM sample
concentrations are required. For example, matrix-assisted laser
desorption ionization mass spectrometry (MALDI-MS) has been
described for identifying the molecular weight of polysaccharide
fragments in publications such as Rhomberg et al. PNAS USA 95,
4176-4181 (1998); Rhomberg et al. PNAS USA 95, 12232-12237 (1998);
and Ernst et al. PNAS USA 95, 4182-4187 (1998). Other types of mass
spectrometry known the art, such as electron spray-MS, fast atom
bombardment mass spectrometry (FAB-MS) and collision-activated
dissociation mass spectrometry (CAD) can also be used. However, the
disclosed methods are not limited to the use of mass spectrometry.
Other methods of use include, but are not limited to, capillary
electrophoresis (CE), NMR, and HPLC with fluorescence
detection.
[0166] The techniques, including mass spectrometry techniques
disclosed herein may be used to determine the ratio of biomarkers
present in a biological sample. For example, particular embodiments
concern the corel monosialylated ST antigen and the T antigen. The
ratio of these two antigens within a particular biological sample
may be determined by using the disclosed mass spectrometry
techniques to produce one or more ions identifying the particular
antigen. For example, a sample may be added to a mass spectrometer,
which promotes fragmentation of the components within the sample to
produce various different ions associated with each component.
[0167] Multiple reaction monitoring may be used to produce a unique
fragment ion that can be monitored and quantified. In particular
disclosed embodiments, the parent mass of the compound is specified
and the sample comprising the compound is monitored for the unique
fragment ion. Typically, the parent mass/ion of the compound is
selected and fragmented and either a particular fragment, the
unique fragment ion, is analyzed or all fragments from the parent
ion are analyzed. The ratio of each compound can be determined
using quantitative mass spectrometry, such as by using an internal
standard. In particular disclosed embodiments, the monosialylated
ST and T antigen have different mass transitions, which can be
determined in order to quantify the ratio of the two antigens in a
biological sample. Typically, the monosialylated ST antigen will
have a parent mass (or parent ion m/z) of 895 and the fragment ion
is 520. The T antigen can have a parent mass (or parent ion m/z) of
534 and the fragment ion is 298. The concentration of each of the
monosialylated antigen and the T antigen can be measured by
comparing the signals from the internal standard with that produced
by either the ST or T antigens. In particular disclosed
embodiments, one or more calibration curves may be produced using
various different concentrations of either antigen.
[0168] According to one embodiment of the disclosed methods, a
biological sample (e.g., a blood sample, plasma sample, tissue
extract etc.) is collected and prepared for analysis. As an
example, an internal standard may be added to the biological sample
in solution (e.g., aqueous solution). The biological sample may
then be treated with a buffered base solution (e.g., an aqueous
solution of sodium borate and sodium hydroxide) in order to promote
denaturation of the serum proteins. The solution may be neutralized
using an appropriate neutralizing solution (e.g., acetic acid in
methanol), and the desired glycans extracted using methanol. The
extracted glycans may be desalted using an ion exchange resin and
then dried.
[0169] Once the desired biological sample is obtained, it may be
manipulated in order to promote analysis using the disclosed mass
spectrometric method. In particular disclosed embodiments, desalted
glycans may be permethylated using a base and appropriate
methylating agent. Solely by way of example, the glycan may be
exposed to an aqueous solution of sodium hydroxide in
dimethylsulfoxide (DMSO) and then treated with methyl iodide. After
extraction, the permethylated glycans are purified, such as by a
SPE C18 column, and used in the disclosed mass spectrometric
analysis.
[0170] According to one embodiment, the permethylated glycans are
analyzed using tandem mass spectrometry coupled with
high-performance liquid chromatography (HPLC-MS/MS); however, any
suitable mass spectrometric methods may be used as disclosed
herein. In particular disclosed embodiments, a suitable
buffer/solvent system is selected for the HPLC analysis portion of
the analytical technique. For example, a two-buffer system may be
used. Particular disclosed embodiments concern using a first buffer
of acetonitrile/formic acid/water having ratios of 1:0/1:99
(v:v:v), respectively, and a second buffer of acetonitrile/formic
acid/water having ratios of 99:0/1:1 (v:v:v), respectively.
Exemplary flow rate protocols are provided herein. In particular
disclosed embodiments, mass spectrometry analysis is conducted
using an enhanced product ion source in the positive mode and one
or more quadrupole mass analyzers. Exemplary non-limiting methods
are disclosed in PCT Application No. PCT/US2014/025633, which is
incorporated by reference herein.
EXAMPLES
[0171] GNE myopathy, which is also known as distal myopathy with
rimmed vacuoles or hereditary inclusion body myopathy, is a
debilitating, autosomal recessive myopathy due to mutations in GNE,
a gene that encodes critical enzymes in sialic acid biosynthesis.
It is clinically characterized by skeletal muscle atrophy and
weakness that preferentially involve the distal muscles. It was
demonstrated that sialic acid deficiency is a key factor in the
pathomechanism of GNE Myopathy, since prophylactic
sialylation-increasing therapies prevented the onset of muscle
athrophy and weakness in this animal model. However, there was an
unexplained link between hyposialylation due to GNE mutations and
the pathognomonic findings in the muscle. The GNE Myopathy mice
muscle disease presents in early stages as muscle atrophy, which in
later stages progressively contributes to muscle weakness and
degeneration. Several phenomena were examined that could possibly
contribute to the muscle atrophy in GNE Myopathy muscles. It was
found that reactive oxygen species (ROS) generation is increased in
both in vitro and in vivo models of GNE Myopathy and an
antioxidant, N-acetylcysteine, successively ameliorated myopathic
phenotypes of GNE Myopathy mice. This implicates that GNE mutation
may induce oxidative stress in GNE myopathy skeletal muscle tissue
and that excessive produced ROS were scavenged by NAC therapy and
prevent severe myopathic features from developing. These findings
also implicate that sialylation-increasing therapies, previously
shown to ameliorate myopathic phenotypes of GNE Myopathy mice may
actually function as antioxidant therapy. Previous in vitro studies
suggested antioxidant features of sialic acid.
[0172] As disclosed herein, cardiac impairment is prevalent in GNE
myopathy patients. Therefore a systematic evaluation was performed
of the cardiac involvement in GNE myopathy by analyzing the mouse
model Gne-/-hGNED176VTg. Cardiac function testing on GNE myopathy
mice (Gne-/-hGNED176VTg), including MRI and ECG imaging, showed
that GNE myopathy mutant mice have decreased cardiac output, and
increased end-diastolic and end-systolic volumes. These finding
suggest that GNE myopathy mutant mice have restrictive
cardiomyopathy. Moreover, hyposialylation of left ventricular
cardiac muscle was demonstrated. These findings suggest that the
cardiac muscle pathology in GNE myopathy is similar to its skeletal
muscle pathology. Antioxidant therapy and/or sialylation-increasing
therapies can be used to treat the skeletal muscle impairment as
well as the cardiac impairment of GNE myopathy patients. In
addition, antioxidant therapies and/or sialylation-increasing
therapies can be of use in related disorders with reactive oxygen
species, such as vascular disorders and myocardial disease.
Example 1
Materials and Methods
[0173] Mice:
[0174] Generation of Gne-knockout mice that express the human GNE
mutation D176V (Gne.sup.-/-hGNED176V-Tg) was described previously
(Malicdan et al., Hum Mol Genet. 2007; 16:2669-2682). Same line of
GNE Myopathy mice (Gne.sup.-/-hGNED176V-Tg) and littermate mice
(Gne.sup.+/-hGNED176V-Tg) as controls were used throughout the
study.
[0175] Quantitative RT-PCR and Microarray Analysis:
[0176] Total RNA was extracted from triceps brachii muscles using
TRIZOL.RTM. reagent and subsequently treated with DNase I
(Invitrogen). cDNA was synthesized using SUPERSCRIPT.RTM. VILO.TM.
cDNA Synthesis Kit (Invitrogen).
[0177] Microarray experiments were carried out using a CODELINK.TM.
Mouse Whole Genome Bioarray (Applied Microarrays Inc.) at Filgen
Inc. The arrays were scanned using a GENEPIX.RTM. 4000A Array
Scanner (Molecular Devices Inc.). The data were analyzed by using
Microarray Data Analysis Tool version 3.2 (Filgen Inc.).
[0178] For quantitative PCR, TAQMAN.RTM. probes were used in
combination with the TAQMAN.RTM. Gene Expression Master Mix
(Applied Biosystems) in a total reaction of 20 .mu.l.
STEPONEPLUS.TM. Real-Time PCR System (Applied Biosystems) was used
to quantify mRNA expression. mRNA relative expression was
normalized to internal control (GAPDH) and determined as fold
change in the average expression value of littermate muscles. The
TAQMAN.RTM. probes used were as follows: F-box protein 32 (Fbxo32),
Mm00499523_m1; tripartite motif-containing 63 (Trim63),
Mm01185221_m1; Sulfiredoxin1 homolog (Srxn1), Mm00769566_m1;
metallothionein 1 (Mt1), Mm00496660_g1; metallothionein 2 (Mt2),
Mm00809556_s1; metallothionein 3 (Mt3), Mm00496661_g1; LC3b,
Mm00782868_sH.
[0179] In Vivo Hydroxyl Radical Measurement:
[0180] Hydroxyl radicals in living mice muscles were measured by
salicylate trapping method combined with microdialysis (Close et
al., Free Radic Biol Med. 2005; 39:1460-14671; Patwell et al., Free
Radic Biol Med. 2001; 30:979-985). 2,5-dihydroxybenzoic acid
(2,5-DHBA) generated from the salicylate in the microdialysis
fluids were measured as an index of reaction with hydroxyl radicals
(Richmond et al., Anal Biochem. 1981; 118:328-335). Anesthesia was
induced in mice with intraperitoneal sodium pentobarbital (50 mg
per kg body weight) and was maintained with supplemental doses.
Microdialysis probes OP-100-075 (Eicom) were placed into the
gastrocnemius muscle of the left limb and perfused with 5 mM
salicylate in Ringer's solution (8.6 g NaCl, 0.25 g CaCl.sub.2 and
0.3 g KCl in 1 liter of ultra pure water) at a flow rate of 1
.mu.l/min. All the liquid flow lines were shielded from light
exposure to avoid oxidation. Microdialysates were collected every
20 min resulting in a total of 20 .mu.l of dialysate per
collection. Following 80 to 100 min of baseline microdialysis
collections, the left gastrocnemius muscles were subjected to
contract by electrical stimulation of surface electrodes. Muscles
were stimulated to contract at 40 Hz with 3 ms pulses for 300
trains at 50 V. Following the contractions, at least 5 further
20-min microdialysate collections were taken. 2, 5-DHBAs in
microdialysates were detected by HPLC-electrochemical detection
system (Eicom) and the chromatograms were analyzed using PowerChrom
software (eDAQ).
[0181] Cell Cultures and Myotubes Analysis:
[0182] Myocytes from Gne.sup.-/-hGNED176V-Tg mice and littermate
controls were proliferated in DMEM containing 20% fetal bovine
serum and 1% chicken embryo extracts in a 5% CO.sub.2 and induced
to differentiation in a serum free medium, OPTIPRO.TM. SFM (Gibco),
to make the cells hyposialylated. Where indicated, myotubes were
treated with 5 mM NeuAc (Japan Food and Liquor Alliance) or 5 mM
NAC (Sigma) for 72 h.
[0183] For the analysis of intracellular ROS, a popular
fluorescence-based probe, 2',7'-dichlorodihydrofluorescein
diacetate (DCFH-DA) (Wang et al., Free Radical Biology and
Medicine. 1999; 27:612-616; Halliwell and Whiteman, Br J Pharmacol.
2004; 142:231-255) was used. Cells were loaded with 5 .mu.M DCFH-DA
(Invitrogen) in PBS for an hour and recovered with phenol red-free
DMEM (Gibco) for 30 min at 37.degree. C. in a 5% CO.sub.2
incubator. Then the cells were placed in a CYTOFLUOR.RTM. Series
4000 multi-well fluorescence plate reader (PerSeptive Biosystems)
with temperature maintained at 37.degree. C. The excitation filter
was set at 485 nm and the emission filter was set at 530 nm. The
fluorescence from each well was captured and recorded.
[0184] To evaluate the ROS generation under pro-oxidants, myotubes
cultured in each condition were loaded with 5 .mu.M DCFH-DA for 1 h
and recovered in the different concentrations of H.sub.2O.sub.2 or
menadione-containing medium. After 30 min incubation in dark at
37.degree. C., the microplate containing cells with pro-oxidants
were placed in a plate reader and fluorescence was captured at
every 5 min until it reached a plateau. Maximum fluorescence of
each well was used for analysis.
[0185] For the cell viability assay, myotubes were exposed to
H.sub.2O.sub.2 for 24 h and to menadione for 6 h before staining.
Nuclear staining with Hoechst 33342 (Thermo Scientific) and
propidium iodide (PI) (Dojindo) was used for morphological
assessment of apoptosis by fluorescence microscopy (Lee and
Shacter, Journal of Biological Chemistry. 1999; 274:19792-19798).
Minimum 200 nuclei were counted and cell viability was calculated
by the exclusion of PI-stained nuclei from Hoechst 33342-stained
nuclei.
[0186] N-acetylcysteine (NAC) Treatment Protocols:
[0187] Gne.sup.-/-hGNED176V-Tg mice were randomly divided into high
dose NAC (n=13), low dose NAC (n=13), or untreated control (n=17)
groups. For NAC (Sigma) treatment, mice were provided ad libitum
access to drinking water containing high dose NAC (1.0% w/v) or low
dose NAC (0.1% w/v), which gives an average dose of 1.5 g/kg for
high dose group and of 0.15 g/kg for low dose group per day. There
was no difference in water consumption between mice receiving NAC
supplemented water or regular drinking water. Treatment was
initiated from the average age of 26 weeks until the mice reached
at least 55 weeks. Three littermates groups (high dose NAC; n=7,
low dose NAC; n=7, or untreated control; n=6) were treated in the
same way.
[0188] Motor Performance Analysis:
[0189] The motor performance was evaluated using treadmill exercise
as previously reported (9, 10) with minor modification. Briefly,
after 7 days of acclimation on the treadmill, two exercise tests
were performed on separate days. The performance test started with
a speed of 10 m/min for 5 minutes and the speed was gradually
increased by 10 m/min every min until the mouse was exhausted and
could no longer run. The time of exhaustion was used to calculate
the total distance of the mice ran during the exercise. The
endurance exercise consisted of a 60-min treadmill run at 20 m/min
with a 7.degree. incline, during which the number of beam breaks or
rests were recorded. Both tests were done twice with 2 days rest in
between.
[0190] Muscle Contractile Properties Analysis:
[0191] The contractile properties of the gastrocnemius and tibialis
anterior muscles were measured according to previous protocol
(Malicdan et al., Nat Med. 2009; 15:690-695; Malicdan et al.,
Physiol Genomics. 2008; 35:106-115). After the mouse was
anesthetized with intraperitoneal sodium pentobarbital (50 mg per
kg body weight), the entire skeletal muscles were isolated along
with the proximal bone of origin intact. Then the muscle was
mounted in a vertical chamber and connected to a force-displacement
and length servo system. Square wave pulses 0.2 ms in duration were
generated by a stimulator and amplified, and subsequently muscle
length was adjusted to the length (L.sub.0) that resulted in
maximal isometric twitch force (Pt) as the muscles were bathed in a
physiologic solution. With the muscle held at L.sub.0 and duration
changed to 3 ms, the force developed during trains of stimulation
pulses (10 to 100 Hz) was recorded and the maximum absolute tetanic
force (Po) was determined. Absolute force was normalized with the
physiologic cross-sectional area (CSA; muscle weight divided by the
product of L.sub.0 and 1.066 g/cm.sup.3) to obtain specific force
(Pt/CSA and Po/CSA).
[0192] Fiber Diameter Analysis:
[0193] Muscle tissues were processed according to previous
protocols (8-10). For the morphometric analysis, we stained frozen
transverse sections (6 .mu.m) of gastrocnemius muscles with rabbit
polyclonal antibody to caveolin 3 (BD Transduction Laboratories),
followed by Alexa fluor-conjugated goat IgG antibody to rabbit
(Invitrogen). Five randomly selected images per mouse were used to
measure fiber diameters using ImageJ software (NIH). Minimal inner
diameters of 1,000 myofibers from each mouse were measured.
[0194] Statistics:
[0195] Statistics were calculated using GraphPad Prism 5 software
(GraphPad). Quantitative RT-PCR data were analyzed using
Mann-Whitney test. Between-group comparison for in vivo NAC
treatment was performed using one-way analysis of variance (ANOVA)
with Dunnett's post-test. All values are expressed as means.+-.SEM.
We performed two-sided tests with a p<0.05 level of
significance.
Example 2
Atrogins and Oxidative Stress Related Genes are Upregulated in the
GNE Myopathy Muscles
[0196] Two muscle-specific ubiquitin ligases, atrogin-1/FBxo32 and
MuRF1/Trim63, are upregulated in various models of muscle atrophy
(Bodine et al., Science Signaling. 2001; 294:1704; Gomes et al.,
Proc Natl Acad Sci USA. 2001; 98:14440-14445). Quantitative RT-PCR
revealed these two skeletal muscle atrophy markers are highly
expressed with a 1.8-fold increase in atrogin-1 expression and a
2.0-fold increase in MuRF1 in Gne.sup.-/-hGNED176V-Tg mice (n=17,
58.0.+-.0.2 wk) muscles as compared to littermates (n=6,
57.9.+-.0.2 wk) muscles (FIG. 1A). These results are in line with
myopathic phenotypes in model mice (Malicdan et al., Physiol
Genomics. 2008; 35:106-115) and suggest that common proteolytic
systems of muscle atrophy are involved in the pathomechanism of GNE
Myopathy.
[0197] In order to determine the pathologic pathways related with
loss of GNE function, the expression of genes expected to be
deregulated in GNE Myopathy was evaluated with microarray data
analysis (FIGS. 1, B-E; FIG. 6, Tables 1-4).
[0198] Through genetic ontology profiling, several genes related
with muscle atrophy, redox homeostasis, autophagy, and collagen
organization were found that were highly expressed in GNE Myopathy
(n=9, 57.7.+-.0.1 wk) muscles when compared to littermates (n=3,
57.8.+-.0.2 wk) muscles. Among these genes, RT-PCR verified that
oxidative stress responsive genes, MTs (metallothioneins) and Srxn1
(sulfiredoxin1), were upregulated. The expression of MT1, MT2, and
MT3 were increased by 3.8-, 3.4-, and 4.4-fold respectively and
Srxn1 expression was 1.5-fold increased in GNE Myopathy muscles
(FIG. 1A).
Example 3
Oxidative Stress is Increased in GNE Myopathy Muscles
[0199] To directly establish that oxidative stress is associated
with the pathomechanism of GNE Myopathy, the levels of ROS was
analyzed in skeletal muscles in vivo (FIG. 2). Baseline hydroxyl
radicals in microdialysate from resting muscles were similar in
both GNE Myopathy mice (2.3.+-.0.8 pM; n=9) and littermates
(2.4.+-.1.2 pM; n=5). Then ROS production was monitored during and
after muscle contraction by electrical stimulation of gastrocnemius
muscles; considerable increases of hydroxyl radicals after muscle
contraction were found. Notably, the degree of ROS increments was
significantly greater in GNE Myopathy mice (14.5.+-.8.8 pM) than
those in littermates (6.3.+-.2.8 pM; p<0.05) (FIGS. 2, C and D),
implying oxidative stress is indeed increased in the affected
muscles.
Example 4
Antioxidant Capacity is Impaired in Hyposialylated Myotubes of GNE
Myopathy
[0200] To further explore oxidative stress and hyposialylation,
intracellular ROS production was measured in cultured GNE Myopathy
myotubes. Intracellular ROS generation, quantified by
dichlorofluorescein diacetate (DCF-DA) labeling, was increased in
GNE Myopathy myotubes compared to controls (FIGS. 3, A and B). Mean
DCF fluorescence measured in GNE Myopathy was 1113.+-.30 AFU, which
was ten times higher than fluorescence measured in littermate
controls (115.+-.15 AFU, p<0.01). By giving NeuAC in the medium,
the increased DCF fluorescence levels in GNE Myopathy myotubes were
significantly reduced to near-normal levels (349.+-.19 AFU,
p<0.01), suggesting that sialylation is important for
maintenance of the redox homeostasis in skeletal muscle cells.
[0201] Then the susceptibility of GNE Myopathy myotubes to
oxidative stress when exposed to increasing concentration of
pro-oxidants was investigated (FIG. 3, C-F). Exposure of cells to
H.sub.2O.sub.2 (FIG. 3C) or menadione (FIG. 3D) led to increased in
intracellular ADCF fluorescence (fluorescence at each
concentration-fluorescence at zero concentration) in a dose
dependent manner. Hyposialylated GNE Myopathy myotubes showed more
accelerated increase in .DELTA.DCF fluorescence levels compared to
littermate cells (p<0.01). When GNE Myopathy myotubes were
incubated with NeuAc containing media before and during exposure to
pro-oxidants, the .DELTA.DCF fluorescence increments were
significantly decreased in both H.sub.2O.sub.2 and menadione
(p<0.01). In the same manner, relative cell viability decreased
sharply in GNE Myopathy myotubes, whereas NeuAc treated myotubes
were more resistant to the same concentrations of H.sub.2O.sub.2 or
menadione (FIGS. 3, E and F).
[0202] To determine whether pharmacological antioxidants are
protective against oxidative stress in GNE Myopathy myotubes, cells
were incubated with N-acetylcysteine (NAC); a remarkable decrease
in DCF fluorescence level was found (191.+-.10 AFU, p<0.01)
(FIGS. 3, A and B). NAC also worked effectively in the high
oxidative stress condition and the protective effect appeared to be
more evident for menadione (FIG. 3, C-F). NAC treated GNE Myopathy
myotubes showed comparable ADCF fluorescence and even better cell
viability compared to those of littermate myotubes under menadione
exposure. The data suggest that impaired antioxidant capacity
possibly due to a decreased ROS scavenging activity in
hyposialylated GNE Myopathy myotubes can be recovered by
normalization of sialylation levels or administration of exogenous
antioxidants.
Example 5
NAC Treatment Restores Muscle Weakness and Atrophy in GNE Myopathy
Model Mice
[0203] To clarify the implication of impaired antioxidant capacity
in the myopathic phenotype of GNE Myopathy, NAC was administered to
the model mouse. GNE Myopathy mice were treated continuously from
20-35 wk to 55-57 wk of age with two doses of NAC (0.15 g/kg and
1.5 g/kg per day) and analyzed motor performance, muscle force
generation, and changes in muscle histology with emphasis on
myofiber atrophy.
[0204] In high dose (HD) treatment group, both treadmill
performance and endurance tests were significantly improved and low
dose (LD) group also showed better motor function compared to
untreated group (FIGS. 4, A and B). Consistently, gastrocnemius
contractile properties were remarkably improved with NAC treatment.
Peak isometric twitch force (Pt) and maximum tetanic force (Po)
were 228.5.+-.15.0 mN and 687.5.+-.52.4 mN in HD group,
233.6.+-.10.0 mN and 735.0.+-.43.0 mN in LD group, and 167.1.+-.6.0
mN and 526.7.+-.24.1 mN in untreated group. Specific isometric
force (Pt/CSA) and specific tetanic force (Po/CSA) increased as
well in treated groups (HD: Pt/CSA=22.5.+-.5.1 and
Po/CSA=67.5.+-.15.7 mN/mm.sup.2; LD: Pt/CSA=23.4.+-.3.3 and
Po/CSA=73.5.+-.13.5 mN/mm.sup.2) compared to those in untreated GNE
Myopathy mice (Pt/CSA=16.3.+-.2.6 and Po/CSA=51.4.+-.8.9
mN/mm.sup.2). Some littermate mice also presented increased
absolute contractile forces (Pt and Po) with NAC supplement (FIG.
4, C-F), but the increase was not statistically significant. With
sialic acid supplement, muscle force generation in the treated mice
increased in proportion to muscle fiber diameter (Malicdan et al.,
Nat Med. 2009; 15:690-69; Malicdan et al., Physiol Genomics. 2008;
35:106-115). To determine the effects of NAC on muscle atrophy,
myofiber diameters were analyzed in gastrocnemius from the treated
mice (FIG. 5B). Mean fiber diameters of NAC treated mice were
significantly increased in both HD (35.2.+-.1.2 p<0.05) and LD
(35.7.+-.0.8 p<0.01) groups compared to untreated group
(31.7.+-.0.8 .mu.m). From the analysis of muscle biopsy images
(FIG. 5A) and histograms of myofiber diameters (FIG. 5C), it was
noted that the increase of average fiber diameters in treated mice
was attributed to the decreased number of atrophic fibers.
[0205] Taken together, the data revealed the ability of NAC to
recover muscle strength and myofiber size in GNE Myopathy mice. In
an attempt to understand the therapeutic effects of NAC at
molecular level, we then performed gene expression analysis with HD
(n=6, 56.7.+-.1.2 wk) and LD (n=6, 57.7.+-.1.7 wk) treated muscles.
As expected, the upregulation of atrogenes (atrogin-1 and MuRF1)
and oxidative stress responsive genes (MT1 and MT2) in the GNE
Myopathy muscle was recovered with NAC treatment (FIG. 1A). LC3b, a
marker used for the presence of autophagy, was recovered as well
(FIGS. 1, A and D) and .beta.-amyloid expression by ELISA in muscle
homogenates was decreased after treatment (FIG. 6).
[0206] No adverse side effects were found in long term NAC
treatment. Serum aspartate aminotransferase, alkaline phosphatase,
and blood urea nitrogen showed no difference between treated and
untreated mice.
[0207] Thus, increased ROS was demonstrated in both in vitro and in
vivo models of GNE Myopathy and proved this phenomenon is
associated with cellular hyposialylation, which is a key pathogenic
factor of this disease. Upregulation of ROS responsive genes such
as MTs and Srxn1 suggests that the affected muscles are highly
exposed to oxidative stress. Metallothioneins belong to the group
of intracellular cysteine-rich proteins and the synthesis of them
was known to be increased by several-fold under oxidative stress
(Ruttkay et. al., Int J Mol Sci. 2013; 14:6044-606; Sato et al.,
Free Radic Biol Med. 1993; 14:325-337). Sulfiredoxin1 is an
endogenous antioxidant protein that was initially identified by its
H.sub.2O.sub.2-induced upregulation (Biteau et al., Nature. 2003;
425:980-984) and a recent study showed that Sulfiredoxin1 is
critical to maintaining redox balance in cells, especially under
exposure to low steady-state levels of H.sub.2O.sub.2 (Baek et al.,
J Biol Chem. 2012; 287:81-89). It is shown herein that contraction
induced ROS increment was remarkable in the GNE Myopathy muscles,
suggesting that the ROS increase may either be a consequent of
muscle contraction or caused by decreased overall antioxidant
capacity in hyposialylated muscles. In the cell culture
experiments, however, acceleration in ROS increase was noted with
the same amount of H.sub.2O.sub.2, indicating the reduction of ROS
scavenging capacity in GNE Myopathy myotubes. Based on the data, it
can be implicated that chronic deregulation of redox homeostasis in
hyposialylated muscles directs cells into a catabolic state and
ultimately leads to muscle atrophy in GNE Myopathy. The fact that
the effect of NAC treatment and NeuAc treatment similarly reduces
oxidative stress likely supports the antioxidant property of sialic
acid.
[0208] The role of oxidative stress in disease has been described
in other muscle diseases. In muscular dystrophy, deregulated ROS
production might be caused by constitutive defects or tissue
responses to the primary pathology (Menazza et al., Hum Mol Genet.
2010; 19:4207-4215; Tidball and Wehling-Henricks, Journal of
Applied Physiology. 2007; 102:1677-1686; Lawler, Journal of
Physiology-London. 2011; 589:2161-2170; Terrill et al., Neuromuscul
Disord. 2012; 22:427-434). In contrast, deregulated ROS production
in GNE Myopathy is not a consequence of tissue injury, but rather
an upstream phenomenon to muscle atrophy. Regardless of the primary
etiology and mechanism of disease, however, increased radical
injury can trigger a vicious cycle that can amplify or accelerate
disease progression and thus remains as a common and rational
therapeutic target in diseases of skeletal muscle.
[0209] From these results, NAC can ameliorate myopathic phenotypes
of GNE Myopathy model mice. As NAC has free radical scavenging
properties and is able to increase the pool of glutathione in the
body, it has been regarded as a powerful antioxidant (Aruoma et
al., Free Radical Biology and Medicine. 1989; 6:593-597; Zafarullah
et al., Cellular and Molecular Life Sciences CMLS. 2003; 60:6-20;
Cotgreave, Advances in Pharmacology. 1996; 38:205-227; Parasassi et
al., Scientific World Journal. 2010; 10:1192-1202). In the skeletal
muscle, it has been reported that NAC can protect an isolated
muscle preparation from contraction-induced oxidative stress
(Sandstrom et al, J Physiol. 2006; 575:251-262) and can inhibit
muscle fatigue in humans (Reid et al., Journal of Clinical
Investigation. 1994; 94:2468). In the current study, decreased
expression of oxidative stress responsive genes in NAC treated mice
suggests that NAC is able to counteract the oxidative stress in GNE
Myopathy muscles.
[0210] The improvement of muscle atrophy and force generation with
NAC treatment supports the hypothesis that ROS are associated with
muscle atrophy and weakness in GNE Myopathy. The muscle force
generation in NAC treated GNE Myopathy GM mice was very similar to
littermates, supporting the theory that muscle force generation is
proportional to and influenced by myofiber diameter (Malicdan et
al., Physiol Genomics. 2008; 35:106-115). On the other hand, even
after corrected with CSA, contractile forces of untreated GM mice
were still lower than those of littermates, suggesting the
existence of other factors contributing to muscle weakness in GM
mice. Structural changes including intracellular protein deposits
and rimmed vacuoles formation start to affect muscle contraction
after 40 weeks of age in GM model mice (Malicdan et al., Physiol
Genomics. 2008; 35:106-115). The data disclosed herein suggest that
the abnormalities in muscle pathology, which appeared in advanced
stages of GM, are associated with oxidative stress, and can be
prevented by NAC treatment. Decrease of .beta.-amyloid expression
by ELISA and LC3b expression by RT-PCR and microarray after NAC
treatment in this study provides additional evidence relating to
redox imbalance to autophagy deregulation which is important in the
pathomechanism of various rimmed vacuolar myopathies (Kiffin et
al., Antioxidants & redox signaling. 2006; 8:152-162; Mammucari
et al., Cell metabolism. 2007; 6:458-471; Tresse et al., IBMPFD.
Autophagy. 2010; 6:217-227; Malicdan et al., Autophagy. 2007;
3:396-398).
[0211] NAC supplement also increased average fiber diameters and
absolute contractile forces in some healthy control mice. A
possible explanation for this finding is that NAC has a preventive
effect on age-related redox imbalance and associated sarcopenia in
littermate mice. Skeletal muscle is known to be particularly
affected by age-related loss of function, to which the oxidative
stress has been claimed to be relevant (Terman and Brunk,
Experimental gerontology. 2004; 39:701-705; Cadenas and Davies,
Free Radic Biol Med. 2000; 29:222; Bonetto et al., Free Radic Biol
Med. 2009; 47:906-916).
[0212] Although the relation between sialic acid and oxidative
stress has not been completely clarified, few studies infer to the
roles of sialic acid in redox balance as ROS scavenger or by
reciprocal action with glycoproteins. It has been shown that sialic
acid has a biologic function as direct scavenger of radicals such
as H.sub.2O.sub.2 (Iijima et al., FEBS Lett. 2004; 561:163-166;
Ogasawara et al., FEBS Lett. 2007; 581:2473-2477). Moreover, it has
been found that non-reducing sialic acid residue can be a target of
ROS and the sialic acid content of some glycoproteins are
apparently linked with oxidative stress (Goswami et al., Clin Chim
Acta. 2003; 337:163-168; Rajendiran et al., Am J Trop Med Hyg.
2008; 79:372-377). One of those glycoproteins being associated with
diseases of free radical etiology is transferrin, a metal binding
monomeric protein, of which the degree of sialylation has been
suggested to play a role in the pathophysiology of Parkinson's
disease and Alzheimer's disease (van Rensburg et al., Metab Brain
Dis. 2004; 19:89-96; van Kamp et al., Clin Chin Acta. 1995;
235:159-167). The results disclosed herein support an important
biologic function of sialic acid, i.e., its antioxidative activity,
especially in a GM, a disease with a hyposialylation as a primary
defect.
[0213] In summary, the data presented herein provide insight into
the pathomechanism of by revealing an important role of oxidative
stress. Increased oxidative stress in hyposialylated muscles
invariably leads to skeletal muscle atrophy and weakness in GNE
Myopathy mice. The success of NAC treatment in normalizing this
phenomenon suggests that a similar approach may benefit human GNE
Myopathy patients.
Example 6
Cardiac Impairment in GNE Myopathy
[0214] GNE myopathy is an adult-onset distal myopathy caused by
mutations in the GNE gene, which codes for a bifunctional enzyme
important in sialic acid biosynthesis. GNE myopathy is
characterized by gradually progressive weakness and atrophy that
preferentially involves distal extremities. Muscle degeneration
occurs, with accumulation of inclusion bodies and rimmed vacuoles
in muscle fibers. It was found that cardiac involvement in GNE
myopathy patients may have a higher occurrence than expected.
[0215] A systematic evaluation of the cardiac involvement in GNE
myopathy was established by analyzing the mouse model
Gne.sup.-/-hGNED176VTg. Histopathology showed the presence of
rimmed vacuoles and disorganization of cardiac myofibrils. Lectin
staining array corroborated the hyposialylation of O-linked
glycoproteins. Echocardiogram revealed decreased ejection fraction
and fractional shortening, and increased left ventricle mass,
indicating a decrease of cardiac function. These data were also
confirmed by functional MRI on the mouse model.
[0216] These findings provide evidence that cardiac muscles are
involved in GNE myopathy. Hyposialylation of cardiac muscles can
lead to impaired cardiac muscle contractility, and can be improved
with sialylation-increasing therapies. It is important for
clinicians to be aware of the possible occurrence of cardiac
disease in GNE myopathy, so that cardiac function can be monitored
and the subjects can be treated.
Example 7
GNE Myopathy Patients with Cardiac Impairment
[0217] Although GNE myopathy is known to preferentially affect
skeletal muscles, there have been a few reports demonstrating
involvement of the cardiac muscles (Chai et al., Muscle Nerve. 2011
January; 43(1):133-6; Kimpara et al., Rinsho Shinkeigaku. 1993
August; 33(8):886-90). It was observed that cardiac involvement in
GNE myopathy patients may have a higher occurrence than expected.
To rule out the effect of different GNE mutations, cardiac findings
of 7 GNE myopathy patients that are all homozygous mutated for the
most prevalent GNE variant p.M712T are listed (Table 1).
TABLE-US-00001 TABLE 1 Cardiac features of 7 GNE myopathy patients
(homozygous for the GNE p.M712T mutation). Patient No. ECG
Echocardiogram Holter monitoring 1 NSR, prolonged Normal, EF 65%
None available QTc > 460 msec 2 NSR Normal, EF 65% None
available 3 NSR Normal, EF 60 .+-. 5%, tiny Recorded for 25 hours,
NSR with color flow across intra-atrial episodes of sinus tach,
sinus brady and septumsuggestive of ectopic atrial rhythm. Ectopics
consisted patent foramen ovale of 69 SVEs including one couplet. 4
NSR Normal, EF 65% Recorded for almost 45 hours, NSR with episodes
of sinus tachycardia (maximum 111 bpm). Ectopics consisted of 6
SVEs. 5 Sinus bradycardia, Mildly dilated LA, and Recorded for 48
hours, sinus bradycardia HR 50-60 bpm. ascending aorta. LV with
episodes of NSR. 4,725 ventricular Otherwise normal normal size and
systolic ectopics, including 10 runs of ECG. On Beta function. EF
65% idioventricular rhythm with the longest blocker for HTN lasting
4 beats, and 42 runs of ventricular bieminy. 9 premature atrial
contractions. 6 None available Normal, EF 65% Recorded for 21
hours, NSR with episodes of sinus bradycardia and sinustachycardia.
No ectopy. 7 Sinus arrhythmia, Normal, EF 65% Recorded for 23
hours, NSR with HR 64 bpm episodes of sinus tachycardia (150 bpm
highest) and sinus bradycardia (42 bpm lowest). 1 SVE, no
ventricular ectopy. Abbreviations: bpm = beats per minute; EF =
Ejection Fraction; HR = Heart Rate; HTN = Hypertension; LA = Left
Atrium; LV = Left Ventricle; NSR = Normal Sinus Rhythm; QTc =
corrected QT; SVE = SupraVentricular Ectopic
Methods:
[0218] Cardiac function data, including Holter monitoring,
electrocardiograms (ECG) and echocardiograms on GNE myopathy
patients were collected through the NIH clinical study (11-HG-0218;
see the internet at clinicaltrials.gov/, trial NCT01417533) "A
Natural History Study of Patients with GNE Myopathy."
Results:
[0219] Electrocardiogram (ECG) Findings:
[0220] Three out of seven patients (all homozygous mutated for GNE
variant p.M712T) showed abnormal ECG findings: Patient 1 had
prolonged corrected QT (QTc) of >460 msec, Patient 5 had sinus
bradycardia, with a heart rate of 50-60 bpm, and Patient 7 had
sinus arrhythmia with a heart rate of 64 bpm.
[0221] Echocardiogram Findings:
[0222] Five out of seven patients had normal echocardiogram
findings, with a ejection fraction between 60% and 66%. Patient 3's
echocardiogram showed tiny color flow across intra-atrial septum
suggestive of patent foramen ovale. Patient 5's echocardiogram
suggests mildly dilated left atrium and ascending aorta, normal
left ventricle size and systiolic function.
[0223] Holter Monitoring Findings:
[0224] Based on Holter monitoring, five out of seven patients
displayed arrhythmia, ranging from episodes of sinus tachycardia,
sinus bradycardia, ectopic atrial rhythm, and ventricular
ectopics.
[0225] GNE myopathy is known to preferentially affect skeletal
muscles. These studies demonstrate that cardiac impairment in GNE
myopathy is not rare, as it was recorded it in 18% of our GNE
myopathy cohort. Severity of cardiac impairment in GNE myopathy
varies from mild to life-threatening arrhythmias. Additionally, GNE
myopathy patients can present with no apparent cardiac
abnormalities at onset of disease symptoms; some developed
cardiomyopathy 20-26 years after disease onset (Chai et al., Muscle
Nerve. 2011 January; 43(1):133-6).
[0226] The observation of cardiac impairment in GNE myopathy was
further pursued by performing cardiac function testing on newly
admitted GNE myopathy patients.
[0227] The findings provide evidence that cardiac muscles are
involved in GNE myopathy. It is important for clinicians to be
aware of the possible occurrence of cardiac disease in GNE myopathy
for careful examination of the cardiac function in patients and
treatment.
Example 8
Mouse Model of GNE Myopathy Shows Hyposialylation of Cardiac
Muscle
[0228] Patients with GNE myopathy are deficient in the GNE gene,
encoding the key enzyme of sialic acid synthesis (Eisenberg et al.,
Nature Genet 2001; 29: 83-7). Skeletal muscle of GNE myopathy mice
and patients show hyposialylation of muscle membrane proteins and
lipids by lectin staining (Niethamer et al., Mol Genet Metab 2012;
107: 748-55; Leoyklang et al., Biomarkers Med 2014; 8: 641-52). The
sialylation status of heart tissue in GNE myopathy mice was
determined.
[0229] Methods:
[0230] The transgenic Gne-/-hGNED176V-Tg mouse model was sued
(Malicdan et al., Hum Mol Genet 2007; 16; 2669-82). All animals
were housed in a barrier-protected, specific pathogen-free-grade
facility.
[0231] Mouse hearts or skeletal muscle (gluteus) were harvested and
fixed in 4% paraformaldehyde for 48 hours at room temperature,
dehydrated in 70% ethanol at 4.degree. C., and paraffin-embedded
for sectioning (Histoserv, Gaithersburg, Md.). Tissue sections (5
um) were stained with a variety of lectins, as previously described
for mouse kidneys and muscle (Kakani et al., Am J Pathol 2012; 180:
1431-40; Niethamer et al., Mol Genet Metab 2012; 107: 748-55) as
well as for GNE myopathy patients' muscle (Leoyklang et al.,
Biomarkers Med 2014; 8: 641-52). Lectin histochemistry was
performed with fluorescein isothiocyanate (FITC) labeled lectins
WGA and VVA from EY Laboratories (San Mateo, Calif.) and SNA from
Vector Laboratories (Burlingame, Calif.). Each slide was also
stained with the nuclear dye DAPI. Fluorescence imaging of multiple
sections of heart per slide was performed under a Zeiss 510 META
confocal laser-scanning microscope. All fluorescent images
represent collapsed stacks of confocal Z-sections, imaged at
63.times. magnification.
[0232] Results:
[0233] Heart and skeletal muscle sections from GNE myopathy mutant
mice (GNE -/-) and unaffected littermates (GNE +/+) were stained
with 3 lectins: WGA, SNA and VVA (FIG. 7A). WGA (wheat germ
agglutinin from Triticum vulgaris) predominantly recognizes
terminal sialic acid (Neu5Ac; independent of the linkage to the
underlying glycan) and N-acetylglucosamine (GlcNAc) on glycans
(Sharon, J Biol Chem 2007; 282: 2753-64; Iskratsch et al., Anal
Biochem 2009; 386: 133-46). SNA (elderberry bark agglutinin from
Sambucus nigra) predominantly recognizes terminal sialic acid
(Neu5Ac) in an .alpha.(2,6)-linkage with either galactose
(prevalent in N-linked glycans) or with N-acetylgalactosamine
(GalNAc) (found in O-linked glycans) (Iskratch et al. supra, 2009;
Shibuya et al., J Biol Chem 1987; 262: 1596-601). VVA (hairy vetch
agglutinin from Vicia villosa) predominantly binds GalNAc O-linked
to serine or threonine residues of proteins (Shibuya et al., supra,
1987).
[0234] Lectin imaging studies were focused on the left ventricle,
because this section of the heart is expected to be especially
affected (by hyposialylation) in GNE myopathy since it is composed
of thicker walls of cardiac muscle tissue and needs to produce the
strongest muscle contraction force (to pump blood throughout the
body), as compared to other heart compartments.
[0235] Wild type mouse hearts (GNE +/+) exhibited similar WGA
staining intensities as mutant mouse hearts (GNE -/-),
demonstrating equal total sialylation. However, SNA staining
intensities were significantly lower in mutant (GNE -/-) hearts,
indicating hyposialylation of 2,6-linked sialic acids. The WGA and
SNA findings are comparable to previously described GNE myopathy
human and mouse skeletal muscle lectin staining (FIG. 7B; Niethamer
et al., Mol Genet Metab 2012; 107: 748-55; Leoyklang et al.,
Biomarkers Med 2014; 8: 641-52). VVA staining did not show an
apparent difference in intensity in wild type and mutant heart; it
is possible that skeletal muscle (showing intense VVA signal) is
more affected/hyposialylated than heart muscle in this disease
(i.e., a larger degree of desialylation of O-linked glycoproteins
with a more intense VVA signal).
[0236] Thus, it was demonstrated that heart muscle, in particular
cardiac muscle in the left ventricle, in GNE myopathy mice is
hyposialylated in 2,6-linked sialic acids, similar to skeletal
muscle tissue in GNE myopathy mice and patients. These mouse
findings together with the finding of heart impairment in GNE
myopathy patients indicate that hyposialyation (of predominantly
2,6-linked sialic acids) is involved in the cardiac impairment
associated with GNE myopathy.
[0237] It is likely that increased oxidative stress in GNE myopathy
heart tissue (as demonstrated for the muscle phenotype in GNE
myopathy mice) increased local levels of reactive oxygen species
(ROS). This increase could not be rescued due to a deficit of local
antioxidants. It was demonstrated that antioxidant therapy could
rescue the skeletal muscle impairment in GNE myopathy mice.
[0238] The sialylation-increasing therapies ManNAc, sialic acid or
mannosamine were shown to rescue hyposialylation of GNE myopathy
mice skeletal muscle tissue (FIG. 7B; Niethamer et al., Mol Genet
Metab 2012; 107: 748-55. In addition, sialylation increasing
therapies ManNAc, sialic acid and sialyllactose were also able to
prophylactically prevent muscle disease in GNE myopathy mice
(Malicdan et al., Nature Med 2009; 15: 690-5).
Sialylation-increasing therapies improve the sialylation status of
GNE myopathy heart tissue (see FIG. 11), indicating resialylation
of glycans in heart tissue after these sialyl