U.S. patent application number 14/181525 was filed with the patent office on 2014-07-03 for agents for preventing and treating disorders involving modulation of the ryanodine receptors.
This patent application is currently assigned to The Trustees of Columbia University in the City of New York. The applicant listed for this patent is The Trustees of Columbia University in the City of New York. Invention is credited to Shixian Deng, Donald W. Landry, Andrew Robert Marks.
Application Number | 20140187536 14/181525 |
Document ID | / |
Family ID | 44258988 |
Filed Date | 2014-07-03 |
United States Patent
Application |
20140187536 |
Kind Code |
A1 |
Marks; Andrew Robert ; et
al. |
July 3, 2014 |
AGENTS FOR PREVENTING AND TREATING DISORDERS INVOLVING MODULATION
OF THE RYANODINE RECEPTORS
Abstract
Methods for reducing toxicity or side effects caused by
administration of a rycal compound for repairing ryanodine receptor
channel leaks when treating a disorder in a subject caused by such
leaks. These methods are based upon the selection for
administration of those rycal compounds having properties including
an EC.sub.50 value of 102 nM when assayed for its ability to
facilitate the rebinding of FKBP12.6 to PKA-phosphorylated RyR2 or
less and less than 50% inhibition of the hERG K.sup.+ channel
(I.sub.Kr) when administered at 10 .mu.M to reduce compound
toxicity or side effects after administration compared to other
rycal compounds not having those properties.
Inventors: |
Marks; Andrew Robert;
(Larchmont, NY) ; Landry; Donald W.; (New York,
NY) ; Deng; Shixian; (White Plains, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees of Columbia University in the City of New
York |
New York |
NY |
US |
|
|
Assignee: |
The Trustees of Columbia University
in the City of New York
New York
NY
|
Family ID: |
44258988 |
Appl. No.: |
14/181525 |
Filed: |
February 14, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12938098 |
Nov 2, 2010 |
8710045 |
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14181525 |
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11506285 |
Aug 17, 2006 |
7879840 |
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12938098 |
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11809470 |
Jun 1, 2007 |
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11506285 |
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11212309 |
Aug 25, 2005 |
8022058 |
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11809470 |
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10809089 |
Mar 25, 2004 |
7718644 |
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11212309 |
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10763498 |
Jan 22, 2004 |
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10809089 |
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11212309 |
Aug 25, 2005 |
8022058 |
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11506285 |
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60810748 |
Jun 2, 2006 |
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60904348 |
Feb 28, 2007 |
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Current U.S.
Class: |
514/211.09 ;
540/552 |
Current CPC
Class: |
C07D 417/14 20130101;
A61P 25/28 20180101; C07D 417/12 20130101; A61P 3/10 20180101; A61P
13/10 20180101; C07D 281/10 20130101; A61P 9/04 20180101; A61P 9/12
20180101; A61P 9/00 20180101; A61P 9/06 20180101 |
Class at
Publication: |
514/211.09 ;
540/552 |
International
Class: |
C07D 281/10 20060101
C07D281/10; C07D 417/12 20060101 C07D417/12 |
Goverment Interests
GOVERNMENT INTERESTS
[0002] This invention was made with government support under grant
number ARMY W911NF-05-1-0462 awarded by Defense Advanced Research
Projects Agency (DARPA), Department of Defense. The government has
certain rights in the invention.
Claims
1. in a method of treating a disorder in a subject caused by a
ryanodine receptor channel leak by administering to the subject a
therapeutically effective amount of a rycal compound to repair the
channel leak to treat the disorder, the improvement which comprises
selecting for administration a rycal compound having properties
including an EC.sub.50 value of 102 nM or less when assayed for its
ability to facilitate the rebinding of FKBP12.6 to
PKA-phosphorylated RyR2 and less than 50% inhibition of the hERG
K.sup.+ channel (I.sub.Kr) when administered at 10 .mu.M to reduce
compound toxicity or side effects after administration compared to
other rycal compounds not having those properties.
2. The method of claim 1, wherein the disorder is selected from the
group consisting of cardiac disorders or diseases, skeletal
muscular disorders or diseases, cognitive disorders or diseases,
malignant hyperthermia, diabetes, and sudden infant death
syndrome.
3. The method of claim 2, wherein the cardiac disorders and
diseases are selected from the group consisting of irregular
heartbeat disorders or diseases; exercise-induced irregular
heartbeat disorders or diseases; heart failure, congestive heart
failure; chronic obstructive pulmonary disease; and high blood
pressure; the skeletal muscular disorders or diseases are selected
from the group consisting of skeletal muscle fatigue,
exercise-induced skeletal muscle fatigue, muscular dystrophy,
bladder disorders, and incontinence; and the cognitive disorders or
diseases are selected from the group consisting of Alzheimer's
Disease, forms of memory loss, and age-dependent memory loss.
4. The method of claim 1, wherein the compound is a benzothiazepine
compound.
5. The compound of claim 4, wherein the benzothiazepine compound is
a 1,4-benzothiazepine compound
6. The compound of claim 5, having the formula: ##STR00038##
wherein R' and R'' are independently selected from the group
consisting of H, halogen, --OH, --NH.sub.2, --NO.sub.2, --CN,
--CF.sub.3, --OCF.sub.3, --N.sub.3, --SO.sub.3H,
--S(.dbd.O).sub.2alkyl, --OS(.dbd.O).sub.2CF.sub.3, acyl, alkyl,
alkoxyl, alkylamino, alkylthio, cycloalkyl, aryl, heterocyclyl,
heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl,
(hetero-)arylthio, and (hetero-)arylamino; and wherein each acyl,
alkyl, alkoxyl, alkylamino, cycloalkyl, aryl, heterocyclyl,
heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)
arylthio may be substituted or unsubstituted; R.sub.15 and R.sub.16
independently are selected from the group consisting of H, acyl,
alkenyl, alkoxyl, OH, NH.sub.2, alkyl, alkylamino, aryl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl;
wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl
may be substituted or unsubstituted; optionally R.sub.15 and
R.sub.16 together with the N to which they are bonded may form a
heterocycle which may be substituted or unsubstituted; R.sub.17 is
H, --NR.sub.15R.sub.16, --NHNR.sub.15R.sub.16, --NHOH, --CH.sub.2X,
alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl,
and heterocyclylalkyl; wherein each alkenyl, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be
substituted or unsubstituted; and n is 0, 1, or 2; or an
enantiomer, diastereomer, tautomer or pharmaceutically acceptable
salt thereof.
7. The compound of claim 6, wherein the compound is selected from
the group consisting of: ##STR00039## ##STR00040##
8. The compound of claim 7, wherein the benzodiazepine compound is
S36, S57, S59, S76 or S107 or a pharmaceutically acceptable salt
thereof.
9. The compound of claim 6, wherein R' and R'' are independently
selected from the group consisting of H, halogen, --OH, --OMe,
--NH.sub.2, --NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
morpholinyl and propenyl; and n is 1 or 2.
10. A method for reducing toxicity or side effects associated with
treatment of a disorder in a subject caused by a ryanodine receptor
channel teak, which comprises: identifying a rycal compound that
has the ability when administered to the subject in a
therapeutically effective amount to repair the channel leak to
treat the disorder; testing the compound to determine properties
including EC.sub.50 when assayed for its ability to facilitate the
rebinding of FKBP12.6 to PKA-phosphorylated. RyR2 and hERG K.sup.+
channel (I.sub.Kr) inhibition when administered at 10 .mu.M; and
selecting for the treatment a rycal compound that has an EC.sub.50
value of 102 or less and less than 50% inhibition of the hERG
K.sup.+ channel (I.sub.Kr) in order to reduce toxicity or side
effects after administration compared to other rycal compounds not
having those properties.
11. The method of claim 10, wherein the disorder is selected from
the group consisting of cardiac disorders or diseases, skeletal
muscular disorders or diseases, cognitive disorders or diseases,
malignant hyperthermia, diabetes, and sudden infant death
syndrome;
12. The method of claim 11, wherein the cardiac disorders and
diseases are selected from the group consisting of irregular
heartbeat disorders or diseases; exercise-induced irregular
heartbeat disorders or diseases; heart failure, congestive heart
failure; chronic obstructive pulmonary disease; and high blood
pressure; the skeletal muscular disorders or diseases are selected
from the group consisting of skeletal muscle fatigue,
exercise-induced skeletal muscle fatigue, muscular dystrophy,
bladder disorders, and incontinence; and the cognitive disorders or
diseases are selected from the group consisting of Alzheimer's
Disease, forms of memory loss, and age-dependent memory loss.
13. The method of claim 10, wherein the compound is a
benzothiazepine compound.
14. The compound of claim 13, wherein the benzothiazepine compound
is a 1,4-benzothiazine compound
15. The compound of claim 14, having the formula: ##STR00041##
wherein R' and R'' are independently selected from the group
consisting of H, halogen, --OH, --NH.sub.2, --NO.sub.2, --CN,
--CF.sub.3, --OCF.sub.3, --N.sub.3, --SO.sub.3H,
--S(.dbd.O).sub.2alkyl, --OS(.dbd.O).sub.2CF.sub.3, acyl, alkyl,
alkoxyl, alkylamino, alkylthio, cycloalkyl, aryl, heterocyclyl,
heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl,
(hetero-)arylthio, and (hetero-)arylamino; and wherein each acyl,
alkyl, alkoxyl, alkylamino, cycloalkyl, aryl, heterocyclyl,
heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl, (hetero-)
arylthio may be substituted or unsubstituted; R.sub.15 and R.sub.16
independently are selected from the group consisting of H, acyl,
alkenyl, alkoxyl, OH, NH.sub.2, alkyl, alkylamino, aryl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl;
wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl
may be substituted or unsubstituted; optionally R.sub.15 and
R.sub.16 together with the N to which they are bonded may form a
heterocycle which may be substituted or unsubstituted; R.sub.17 is
H, --NR.sub.15R.sub.16, --NHNR.sub.15R.sub.16, --NHOH, --OR.sub.15,
--CH.sub.2X, alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, and heterocyclylalkyl; wherein each alkenyl, aryl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl
may be substituted or unsubstituted; and n is 0, 1, or 2; or an
enantiomer, diastereomer, tautomer or pharmaceutically acceptable
salt thereof.
16. The compound of claim 15, wherein the compound is selected from
the group consisting of: ##STR00042## ##STR00043##
17. The compound of claim 16, wherein the benzodiazepine compound
is S36, S57, S59, S76 or S107 or a pharmaceutically acceptable salt
thereof.
18. The compound of claim 15, wherein R' and R'' are independently
selected from the group consisting of H, halogen, --OH, --OMe,
--NH.sub.2, --NO.sub.2, --CN, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, morpholinyl and
propenyl; and n is 1 or 2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application 12/938,098
filed Nov. 2, 2010, which is a continuation-in-part of (a) U.S.
patent application Ser. No. 11/506,285, filed Aug. 17, 2006, now
U.S. Pat. No. 7,879,840, and (b) a continuation-in-part of U.S.
patent application Ser. No. 11/809,470 filed Jun. 1, 2007,
abandoned. Each of (a) and (b) is a continuation-in-part of U.S.
patent application Ser. No. 11/212,309, filed Aug. 25, 2005, now
U.S. Pat. No. 8,022,058, which is a continuation-in-part of U.S.
patent application Ser. No. 10/809,089, filed Mar. 25, 2004, now
U.S. Pat. No. 7,718,644, which is a continuation-in-part of U.S.
patent application Ser. No. 10/763,498, filed on Jan. 22, 2004,
abandoned. Application (b) also claims the benefit of U.S.
Application Nos. 60/810,748 filed Jun. 2, 2006 and 60/904,348 filed
Feb. 28, 2007. The contents of each application mentioned above are
expressly incorporated by reference herein.
FIELD OF THE INVENTION
[0003] This invention relates to compounds and their use to treat
and prevent disorders and diseases associated with the ryanodine
receptors that regulate calcium channel functioning in cells. More
particularly, the invention discloses compounds that are related to
1,4-benzothiazepines and are useful to treat cardiac diseases and
disorders, skeletal muscle diseases and disorders, as well as
diseases, disorders and conditions affecting the nervous system,
such as neuropathies, seizures, and disorders affecting cognitive
functioning. The invention also discloses pharmaceutical
compositions comprising the compounds and articles of manufacture
comprising the pharmaceutical compositions.
BACKGROUND OF THE INVENTION
[0004] The sarcoplasmic reticulum (SR) functions, among other
things, as a specialized intracellular calcium (Ca.sup.2+) store.
Channels in the SR called ryanodine receptors (RyRs) open and close
to regulate the release of Ca.sup.2+ from the SR into the
intracellular cytoplasm of the cell. Release of Ca.sup.2+ into the
cytoplasm from the SR increases cytoplasmic Ca.sup.2+
concentration. Open probability (Po) of the RyR receptor refers to
the likelihood that the RyR channel is open at any given moment,
and therefore capable of releasing Ca.sup.2+ into the cytoplasm
from the SR.
[0005] There are three types of ryanodine receptors, all of which
are Ca.sup.2+ channels: RyR1, RyR2, and RyR3. RyR1 is found
predominantly in skeletal muscle as well as other tissues, RyR2 is
found predominantly in the heart as well as other tissues, and RyR3
is found in the brain as well as other tissues. The RyR channels
are formed by four RyR polypeptides in association with four FK506
binding proteins (FKBPs), specifically FKBP12 (calstabin1) and
FKBP12.6 (calstabin2). Calstabin1 binds to RyR1, calstabin2 binds
to RyR2, and calstabin1 binds to RyR3. The FKBP proteins
(calstabin1 and calstabin2) bind to the RyR channel (one molecule
per RyR subunit), stabilize RyR-channel functioning, and facilitate
coupled gating between neighboring RyR channels, thereby preventing
abnormal activation of the channel during the channel's closed
state.
[0006] Protein kinase A (PKA) binds to the cytoplasmic surface of
the RyR receptors. PKA phosphorylation of the RyR receptors causes
partial dissociation of calstabins from RyRs. Dissociation of
calstabin from RyR causes increased open probability of RyR, and
therefore increased Ca.sup.2+ release from the SR into the
intracellular cytoplasm.
[0007] Ca.sup.2+ release from the SR in skeletal muscle cells and
heart cells is a key physiological mechanism that controls muscle
performance, because increased concentration of Ca.sup.2+ in the
intracellular cytoplasm causes contraction of the muscle.
[0008] Excitation-contraction (EC) coupling in skeletal muscles
involves electrical depolarization of the plasma membrane in the
transverse tubule (T-tubule), which activates voltage-gated L-type
Ca.sup.2+ channels (LTCCs). LTCCs trigger Ca.sup.2+ release from
the SR through physical interaction with RyR1. The resulting
increase in cytoplasmic Ca.sup.2+ concentration induces
actin-myosin interaction and muscle contraction. To enable
relaxation, intracellular Ca.sup.2+ is pumped back into the SR via
SR Ca.sup.2+-ATPase pumps (SERCAs), which is regulated by
phospholamban (PLB) depending on the muscle fiber type.
[0009] It has been shown that disease forms that result in
sustained activation of the sympathetic nervous system and
increased plasma catecholamine levels cause maladaptive activation
of intracellular stress pathways resulting in destabilization of
the RyR1 channel closed state and intracellular Ca.sup.2+ leak. SR
Ca.sup.2+ leak via RyR1 channels was found to deplete intracellular
SR calcium stores, to increase compensatory energy consumption, and
to result in significant acceleration of muscle fatigue. The
stress-induced muscle defect permanently reduces isolated muscle
and in vivo performance particularly in situations of increased
demand.
[0010] It also has been shown that destabilization of the RyR1
closed state occurs under pathologic conditions of increased
sympathetic activation and involves depletion of the stabilizing
calstabin1 (FKBP12) channel subunit. Proof-of-principle experiments
have shown that PKA activation as an end effector of the
sympathetic nervous systems increases RyR1 PKA phosphorylation at
Ser-2843 which decreases the binding affinity of calstabin1 to RyR1
and increases channel open probability.
[0011] In cardiac striated muscle, RyR2 is the major
Ca.sup.2+-release channel required for EC coupling and muscle
contraction. During EC coupling, depolarization of the
cardiac-muscle cell membrane during phase zero of the action
potential activates voltage-gated Ca.sup.2+ channels. Ca.sup.2+
influx through the open voltage-gated channels in turn initiates
Ca.sup.2+ release from the SR via RyR2. This process is known as
Ca.sup.2+-induced Ca.sup.2+ release. The RyR2-mediated,
Ca.sup.2+-induced Ca.sup.2+ release then activates the contractile
proteins in the cardiac cell, resulting in cardiac muscle
contraction.
[0012] Phosphorylation of cardiac RyR2 by PKA is an important part
of the "fight or flight" response that increases cardiac EC
coupling gain by augmenting the amount of Ca.sup.2+ released for a
given trigger. This signaling pathway provides a mechanism by which
activation of the sympathetic nervous system, in response to
stress, results in increased cardiac output. PKA phosphorylation of
RyR2 increases the open probability of the channel by dissociating
calstabin2 (FKBP12.6) from the channel complex. This, in turn,
increases the sensitivity of RyR2 to Ca.sup.2+-dependent
activation.
[0013] Despite advances in treatment, heart failure remains an
important cause of mortality in Western countries. An important
hallmark of heart failure is reduced myocardial contractility. In
heart failure, contractile abnormalities result, in part, from
alterations in the signaling pathway that allows the cardiac action
potential to trigger Ca.sup.2+ release via RyR2 channels and muscle
contraction. In particular, in failing hearts, the amplitude of the
whole-cell Ca.sup.2+ transient is decreased and the duration
prolonged.
[0014] Cardiac arrhythmia, a common feature of heart failure,
results in many of the deaths associated with the disease. Atrial
fibrillation (AF) is the most common cardiac arrhythmia in humans,
and represents a major cause of morbidity and mortality. Structural
and electrical remodeling--including shortening of atrial
refractoriness, loss of rate-related adaptation of refractoriness,
and shortening of the wavelength of re-entrant wavelets--accompany
sustained tachycardia. This remodeling is likely important in the
development, maintenance and progression of atrial fibrillation.
Studies suggest that calcium handling plays a role in electrical
remodeling in atrial fibrillation.
[0015] Approximately 50% of all patients with heart disease die
from fatal cardiac arrhythmias. In some cases, a ventricular
arrhythmia in the heart is rapidly fatal--a phenomenon referred to
as "sudden cardiac death" (SCD). Fatal ventricular arrhythmias and
SCD also occur in young, otherwise-healthy individuals who are not
known to have structural heart disease. In fact, ventricular
arrhythmia is the most common cause of sudden death in
otherwise-healthy individuals.
[0016] Catecholaminergic polymorphic ventricular tachycardia (CPVT)
is an inherited disorder in individuals with structurally normal
hearts. It is characterized by stress-induced ventricular
tachycardia--a lethal arrhythmia that causes SCD. In subjects with
CPVT, physical exertion and/or stress induce bidirectional and/or
polymorphic ventricular tachycardias that lead to SCD even in the
absence of detectable structural heart disease. CPVT is
predominantly inherited in an autosomal-dominant fashion.
Individuals with CPVT have ventricular arrhythmias when subjected
to exercise, but do not develop arrhythmias at rest. Studies have
identified mutations in the human RyR2 gene, on chromosome
1q42-q43, in individuals with CPVT.
[0017] Failing hearts (e.g., in patients with heart failure and in
animal models of heart failure) are characterized by a maladaptive
response that includes chronic hyperadrenergic stimulation. In
heart failure, chronic beta-adrenergic stimulation is associated
with the activation of beta-adrenergic receptors in the heart,
which, through coupling with G-proteins, activate adenylyl cyclase
and thereby increase intracellular cAMP concentration. CAMP
activates cAMP-dependent PKA, which has been shown to induce
hyperphosphorylation of RyR2. Thus, chronic heart failure is a
chronic hyperadrenergic state that results in several pathologic
consequences, including PKA hyperphosphorylation of RyR2.
[0018] The PKA hyperphosphorylation of RyR2 has been proposed as a
factor contributing to depressed contractile function and
arrhythmogenesis in heart failure. Consistent with this hypothesis,
PKA hyperphosphorylation of RyR2 in failing hearts has been
demonstrated, in vivo, both in animal models and in patients with
heart failure undergoing cardiac transplantation.
[0019] In failing hearts, the hyperphosphorylation of RyR2 by PKA
induces the dissociation of FKBP 12.6 (calstabin2) from the RyR2
channel. This causes marked changes in the biophysical properties
of the RyR2 channel, including increased open probability (Po) due
to an increased sensitivity to Ca.sup.2+-dependent activation;
destabilization of the channel, resulting in subconductance states;
and impaired coupled gating of the channels, resulting in defective
EC coupling and cardiac dysfunction. Thus, PKA-hyperphosphorylated
RyR2 is very sensitive to low-level Ca.sup.2+ stimulation, and this
manifests itself as a diastolic SR Ca.sup.2+ leak through the PKA
hyperphosphorylated RyR2 channel.
[0020] The maladaptive response to stress in heart failure results
in depletion of FKBP12.6 from the channel macromolecular complex.
This leads to a shift to the left in the sensitivity of RyR2 to
Ca.sup.2+-induced Ca.sup.2+ release, resulting in channels that are
more active at low-to-moderate Ca.sup.2+ concentrations. Over time,
the increased "leak" through RyR2 results in resetting of the SR
Ca.sup.2+ content to a lower level, which in turn reduces EC
coupling gain and contributes to impaired systolic
contractility.
[0021] Additionally, a subpopulation of RyR2 that are particularly
"leaky" can release SR Ca.sup.2+ during the resting phase of the
cardiac cycle, diastole. This results in depolarizations of the
cardiomyocyte membrane known as delayed after-depolarizations
(DADs), which are known to trigger fatal ventricular cardiac
arrhythmias.
[0022] In patients with CPVT mutations in their RyR2 and otherwise
structurally-normal hearts, a similar phenomenon is at work.
Specifically, it is known that exercise and stress induce the
release of catecholamines that activate beta-adrenergic receptors
in the heart. Activation of the beta-adrenergic receptors leads to
PKA hyperphosphorylation of RyR2 channels. Evidence also suggests
that the PKA hyperphosphorylation of RyR2 resulting from
beta-adrenergic-receptor activation renders mutated RyR2 channels
more likely to open in the relaxation phase of the cardiac cycle,
increasing the likelihood of arrhythmias.
[0023] Cardiac arrhythmias are known to be associated with
diastolic SR Ca.sup.2+ leaks in patients with CPVT mutations in
their RyR2 and otherwise structurally-normal hearts. In these
cases, the most common mechanism for induction and maintenance of
ventricular tachycardia is abnormal automaticity. One form of
abnormal automaticity, known as triggered arrhythmia, is associated
with aberrant release of SR Ca.sup.2+, which initiates DADs. DADs
are abnormal depolarizations in cardiomyocytes that occur after
repolarization of a cardiac action potential. The molecular basis
for the abnormal SR Ca.sup.2+ release that results in DADs has not
been fully elucidated. However, DADs are known to be blocked by
ryanodine, providing evidence that RyR2 plays a key role in the
pathogenesis of this aberrant Ca.sup.2+ release.
[0024] It has been shown that exercise-induced arrhythmias and
sudden death (in patients with CPVT) result from a reduced affinity
of FKBP12.6 (calstabin2) for RyR2. Additionally, it has been
demonstrated that exercise activates RyR2 as a result of
phosphorylation by adenosine 3',5'-monophosphate (cAMP)-dependent
protein kinase (PKA). Mutant RyR2 channels, which had normal
function in planar lipid bilayers under basal conditions, were more
sensitive to activation by PKA phosphorylation--exhibiting
increased activity (open probability) and prolonged open states, as
compared with wild-type channels. In addition, PKA-phosphorylated
mutant RyR2 channels were resistant to inhibition by Mg.sup.2+, a
physiological inhibitor of the channel, and showed reduced binding
to FKBP12.6 (aka calstabin2, which stabilizes the channel in the
closed state). These findings indicate that, during exercise, when
the RyR2 are PKA-phosphorylated, the mutant CPVT channels are more
likely to open in the relaxation phase of the cardiac cycle
(diastole), increasing the likelihood of arrhythmias triggered by
SR Ca.sup.2+ leak.
[0025] Fatigue is the process whereby skeletal muscles become
weaker with repeated or intense use such as exercise, or as a
result of an illness, disorder or disease. Fatigue can result in
task failure and it can be a pronounced symptom in a variety of
medical conditions including heart failure, renal failure, cancer,
and various muscular dystrophies. Over the past decade, it has
become evident that the two dominant classical explanations of
muscle fatigue, namely, accumulation of lactic acid and
intracellular acidosis, do not cause fatigue. In fact, both may be
protective mechanisms during high intensity exercise to prevent
fatigue (Allen and Westerblad 2004; Pedersen, Nielsen et al.
2004).
[0026] Muscle contraction depends on the efficient coupling of
electrical stimulation of the muscle surface to Ca.sup.2+ release
via the ryanodine receptor, the SR Ca.sup.2+ release channel, to
the generation of actinmyosin cross bridges. It is evident, then,
that a defect in excitation-contraction coupling that resulted in a
reduction in the amplitude of the Ca.sup.2+ transient would, among
other effects, result in impaired contraction and force generation
through ineffective myosin cross bridge formation. Eberstein and
Sandow suggested inhibition of Ca.sup.2+ release as a likely factor
in the fatigue process (Eberstein and Sandow 1963). Reductions in
the amplitude of Ca.sup.2+ release evoked during fatiguing
stimulation have been reported in multiple muscle preparations
(Allen, Lee et al. 1989; Westerblad and Allen 1991; Allen and
Westerblad 2001). The time course of recovery from fatigue
parallels the time course over which prolonged depression of
Ca.sup.2+ release is observed (Westerblad, Bruton et al. 2000).
[0027] SR Ca.sup.2+ leak was documented in myofibers following
intense exercise and in a model of muscular dystrophy (Wang,
Weisleder et al. 2005), possibly due to defective skeletal
ryanodine receptors (RyR1 s). Chronic activation of the sympathetic
nervous system (SNS) in the context of heart failure promotes
intrinsic skeletal muscle fatigue due to depletion of the
phosphodiesterease PDE4D3 from the RyR1 complex, RyR1 PKA
hyperphosphorylation at Serine 2844, calstabin1 depletion from the
RyR1 complex, and a gain-of-function channel defect (Reiken,
Lacampagne et al. 2003). RyR1 dysfunction in the skeletal muscle
leads to altered local subcellular Ca.sup.2+ release events and
impaired global calcium transients (Ward, Reiken et al. 2003).
JTV-519,
(4-[3-(4-benzylpiperidin-1-yl)propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-
-benzothiazepine monohydrochloride-a 1,4-benzothiazepine has been
shown to be a modulator of RyR calcium-ion channels), given in the
context of a murine model of heart failure, was able to improve
skeletal muscle function, as assessed by an ex vivo isolated muscle
fatiguing protocol, five weeks after left coronary artery ligation.
JTV-519's beneficial effect on muscle fatigue was not solely due to
cardiac improvement, as a beneficial effect was still seen when the
drug was given to calstabin2 deficient mice which derive no cardiac
benefit from treatment with JTV-519. Thus, it has been postulated
that JTV-519 directly affects muscle function (Wehrens, Lehnart et
al. 2005). In the context of chronic exercise, identical changes in
the RyR1 macromolecular complex, namely depletion of PDE4D3 from
the RyR1 complex, RyR1 PKA hyperphosphorylation at Serine 2844, and
calstabin1 depletion from the RyR1 complex are related in a
time-dependent and activity-dependent manner with repeated intense
exercise in a mouse model. These biochemical changes in the RyR1
macromolecular complex regulation and function are stable following
prolonged exercise and recover slowly over days to weeks. It has
therefore been proposed that RyR1 Ca.sup.2+ leak limits peak muscle
performance and mediates muscle damage during prolonged, stressful
exercise.
[0028] The contraction of striated muscle is initiated when calcium
(Ca.sup.2+) is released from tubules within the muscle cell known
as the sarcoplasmic reticulum (SR). Calcium release channels,
called ryanodine receptors (RyR), on the SR are required for
excitation-contraction (EC) coupling. The type 2 ryanodine receptor
(RyR2) is found in the heart, while the type 1 ryanodine receptor
(RyR1) is found in skeletal muscle. The RyR1 receptor is a tetramer
comprised of four 565,000 dalton RyR1 polypeptides and four 12,000
dalton FK-506 binding proteins (FKBP12). FKBP12s are regulatory
subunits that stabilize RyR channel function (Brillantes et al.,
1994) and facilitate coupled gating between neighboring RyR
channels (Marx et al., 1998); the latter are packed into dense
arrays in specialized SR regions that release intracellular stores
of Ca.sup.2+, thereby triggering muscle contraction. In addition to
FKBP12, the RyR1 macromolecular complex also includes the catalytic
and regulatory subunits of PKA, and the phosphatase PP1 (Marx et
al., 2001).
[0029] One FKBP12 molecule is bound to each RyR1 subunit.
Dissociation of FKBP12 significantly alters the biophysical
properties of the channels, resulting in the appearance of
subconductance states, and increased open probability (P.sub.o) of
the channels (Brillantes et al., 1994; Gaburjakova et al., 2001).
In addition, dissociation of FKBP12 from RyR1 channels inhibits
coupled gating resulting in channels that gate stochastically
rather than as an ensemble (Marx et al., 1998). Coupled gating of
arrays of RyR channels is thought to be important for efficient EC
coupling that regulates muscle contraction (Marx et al., 1998).
FKBPs are cis-trans peptidyl-prolyl isomerases that are widely
expressed and subserve a variety of cellular functions (Marks,
1996). FKBP12s are tightly bound to and regulate the function of
the skeletal (RyR1) (Brillantes et al., 1994; Jayaraman et al.,
1992) and cardiac (RyR2) (Kaftan et al., 1996) muscle Ca.sup.2+
release channels, as well as a related intracellular Ca.sup.2+
release channel known as the type 1 inositol 1,4,5-triphosphate
receptor (IP3R1) (Cameron et al., 1997), and the type I
transforming growth factor .beta. (TGF.beta.) receptor (T.beta.RI)
(Chen et al., 1997).
[0030] U.S. Pat. No. 7,312,044, the contents of which are hereby
incorporated by reference, discloses methods of treating defective
skeletal muscle function during heart failure by administering an
agent which inhibits dissociation of FKB12 binding protein from
RyR1 receptor.
[0031] Co-pending U.S. patent application Ser. No. 11/212,309 and
U.S. Pat. No. 7,704,990, the contents of which are hereby
incorporated by reference, disclose methods of making and using
novel benzothiazepine derivatives to treat and prevent disorders
and diseases associated with the RyR receptors, including skeletal
muscular disorders and diseases such as skeletal muscle fatigue,
exercise-induced skeletal muscle fatigue, muscular dystrophy,
bladder disorders, and incontinence.
[0032] There is a need to identify new agents effective for
treating or preventing muscle fatigue that is stress or exercise
induced or that results from diseases associated with the RyR
receptors that regulate calcium channel functioning in cells,
including cardiac disease or disorder, defective skeletal muscle
function, HIV Infection, AIDS, muscular dystrophy, cancer,
malnutrition, exercise-induced muscle fatigue, age-associated
muscle fatigue, renal disease, and renal failure.
SUMMARY OF THE INVENTION
[0033] In view of the foregoing, there is a need to identify new
agents effective for treating or preventing disorders and diseases
associated with the RyRs that regulate calcium channel functioning
in cells, including skeletal muscular disorders and diseases and
especially cardiac disorders and diseases. More particularly, a
need remains to identify new compounds that can be used to treat
RyR associated disorders by, for example, repairing the leak in RyR
channels, and enhancing binding of FKBP proteins (calstabin1 and
calstabin2) to PKA-phosphorylated RyR, and to mutant RyRs that
otherwise have reduced affinity for, or do not bind to, FKBP12 and
FKBP12.6. Embodiments of the invention solve some or all of these
needs.
[0034] In one embodiment, the present invention provides a compound
represented by the structure of formula I:
##STR00001##
[0035] wherein n, q, R, R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are
as defined herein.
[0036] Certain compounds of formula I are defined by any of
formulae I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k,
I-k-1, I-l, I-l-1, I-m, I-m-1, I-n, I-o and I-p as defined
herein.
[0037] Non-limiting examples of compounds of formula I are
compounds selected from the group consisting of S1, S2, S3, S4, S5,
S6, S7, S9, S11, S12, S13, S14, S19, S20, S22, S23, S36, S37, S38,
S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54,
S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S66, S67, S69,
S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83,
S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96,
S97, S98, S99, 5100, 5101, 5102, 5103, S104, S107, S108, S109,
S110, S111, S112, S113, S114, S117, S118, S119, S120, S121, S122
and S123, and salts thereof.
[0038] In other embodiments, the present invention provides methods
of treating a disorder or a disease in a subject, or reducing the
risk of sudden cardiac death in a subject who is considered to be
subject to such risk, comprising administering to the subject a
therapeutically effective amount of a compound according to claim 1
to effectuate the treatment, wherein the disorder or disease is
selected from the group consisting of cardiac disorders and
diseases, skeletal muscular disorders and diseases, cognitive
disorders and diseases, malignant hyperthermia, diabetes, and
sudden infant death syndrome; wherein the cardiac disorders and
diseases are selected from the group consisting of irregular
heartbeat disorders and diseases; exercise-induced irregular
heartbeat disorders and diseases; heart failure, congestive heart
failure; chronic obstructive pulmonary disease; and high blood
pressure; wherein the skeletal muscular disorders and diseases are
selected from the group consisting of skeletal muscle fatigue,
exercise-induced skeletal muscle fatigue, muscular dystrophy,
bladder disorders, and incontinence; and wherein the cognitive
disorders and diseases are selected from the group consisting of
Alzheimer's Disease, forms of memory loss, and age-dependent memory
loss.
[0039] In one embodiment, the compound is administered to the
subject to treat cardiac disorders and diseases selected from the
group consisting of irregular heartbeat disorders and diseases;
exercise-induced irregular heartbeat disorders and diseases;
congestive heart failure; chronic obstructive pulmonary disease;
and high blood pressure.
[0040] In some embodiments, the irregular heartbeat disorders and
diseases and exercise-induced irregular heartbeat disorders and
diseases are selected from the group consisting of atrial and
ventricular arrhythmia; atrial and ventricular fibrillation; atrial
and ventricular tachyarrhythmia; atrial and ventricular
tachycardia; catecholaminergic polymorphic ventricular tachycardia
(CPVT); and exercise-induced variants thereof.
[0041] In another embodiment, the compound is administered to the
subject to treat skeletal muscular disorders and diseases selected
from the group consisting of skeletal muscle fatigue,
exercise-induced skeletal muscle fatigue, muscular dystrophy,
bladder disorders, and incontinence.
[0042] In another embodiment, the compound is administered to the
subject to treat cognitive disorders and diseases selected from the
group consisting of Alzheimer's Disease, forms of memory loss, and
age-dependent memory loss.
[0043] Other features and advantages of the present invention will
become apparent from the following detailed description. It should
be understood, however, that the detailed description and the
specific examples while indicating various embodiments of the
invention are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0044] FIG. 1, embodiments A, B, C, and D are, respectively, (A)
immunoblots of PKA phosphorylated RyR2 in the presence of FKBP12.6
and increasing JTV-519 concentrations; (B) immunoblots of PKA
phosphorylated RyR2 in the presence of 0.5 nM S36; (C) a graph of
current through plasma membrane, voltage dependent L-type Ca.sup.2+
channels which are completely blocked by nifedipine but not by S36
in isolated mouse cardiomyocytes; and (D) a graph of the
voltage-dependence of L-type Ca.sup.2+ current in channels in the
presence of JTV-519 and S36.
[0045] FIG. 2, embodiments A, B, C, and D demonstrate the
prevention of exercise-induced ventricular arrhythmias by JTV-519
in haploinsufficient calstabin (FKBP12.6).sup.+/- mice. Embodiment
A are representative telemetric electrocardiograms (ECGs) of an
untreated calstabin2 (FKBP12.6).sup.-/- mouse (left), a
JTV-519-treated calstabin2 (FKBP12.6).sup.+/- mouse (middle), and a
calstabin2 (FKBP12.6).sup.-/- mouse (right). Embodiment B are
telemetry recordings of a sustained polymorphic ventricular
tachycardia (sVT) in (upper) an untreated haploinsufficient
calstabin2 (FKBP12.6).sup.+/- mouse and (lower) a JTV-519-treated
calstabin2 (FKBP12.6).sup.+/- mouse, each subjected to exercise
testing immediately followed by injection with 0.5 mg epinephrine
per kilogram of body weight. Embodiment C are graphs showing the
numbers of mice with cardiac death (left), sustained VTs (middle),
and nonsustained VTs (right) in experimental groups of mice
subjected to exercise testing and injection with 0.5 mg/kg
epinephrine. Embodiment D provides graphs comparing the dose
dependence of pharmacological effects of JTV-519 and S36 in regard
to sudden cardiac death (left), sustained VTs (middle), and
nonsustained VTs (right).
[0046] FIG. 3 is a graph showing fractional shortening (FS) of the
left ventricle assessed by M-mode echocardiography 2 weeks
post-myocardial infarction in mice.
[0047] FIG. 4 is a graph showing heart weight to body weight
(HW/BW) ratios and pressure-volume loops quantifications (dP/dt)
one week post-myocardial infarction of placebo and S36-treated
mice. S36 treatment results in a beneficial reduction of the HW/BW
ratio and increased velocity of pressure development in S36 as
compared to placebo treated mice.
[0048] FIG. 5 is a graph summarizing EC.sub.50 values of JTV-519
and a series of Rycal compounds indicating several compounds with a
higher biologic activity as evidenced by significantly lower
EC.sub.50 values compared to JTV-519.
[0049] FIG. 6, embodiments A, B, and C are, respectively, (A)
single-channel current traces of RyR2-P2328S and RyR2-WT; (B)
single-channel current traces of RyR2-P2328S; and (C) immunoblot
analysis of calstabin-2 binding of RyR2-P2328S.
[0050] FIG. 7, embodiments A and B, are, respectively, (A) an
immunoblot of RyR2 immunoprecipitated with an antibody against
RyR2, and immunoblots of RyR2 PKA phosphorylation at Ser-2809 and
calstabin2; and (B) a bar graph quantifying the relative amount of
PKA phosphorylated RyR2 at Ser-2808 (corresponding to human
Ser-2809) bound to RyR2 in wild-type (control) and
calstabin2-deficient (FKBP 12.6.sup.-/-) mice.
[0051] FIG. 8, embodiments A, B, and C, are, respectively, bar
graphs of (A) quantitative in vivo M-mode echocardiograms comparing
ejection fraction (EF) before and following sham operation or
permanent left anterior descending (LAD) coronary artery ligation
in wild-type and RyR2-S2808A knockin mice; (B) in vivo
pressure-volume loop quantification of maximal pressure change over
time (dP/dt) in wild-type and RyR2-S2808A knockin mice after sham
operation or permanent left anterior descending coronary artery
(LAD) ligation; and (C) quantitative M-mode echocardiographic
assessment of end-systolic diameter (ESD) in wildtype and
RyR2-S2808A knockin mice after sham operation or permanent left
anterior descending coronary artery (LAD) ligation.
[0052] FIG. 9, embodiments A and B, are, respectively, immunoblots
of RyR1, RyR1-pSer.sup.2843, and RyR1-associated calstabin1 in mdx
mice and wild-type mice; and bar graphs of the relative amounts of
RyR1-pSer.sup.2843 and calstabin1 in mdx and wild-type mice.
[0053] FIG. 10, embodiments A, B, and C, demonstrate that a SR
Ca.sup.2+ leak is detectable in the skeletal muscles of animals
with heart failure. Embodiments A and B are fluorescence line scan
images of Ca.sup.2+ sparks in myofibers from, respectively, sham
and postmyocardial infarction (PMI) rats. Embodiment C provides bar
graphs summarizing the amplitude, rise time, FDHM, and FWHM of the
Ca.sup.2+ sparks for the sham (open symbols) and PMI (closed
symbols) rats.
[0054] FIG. 11, embodiments A, B, C, and D, demonstrate that PKA
phosphorylation of Ser-2843 increases the open probability and
gating kinetics of RyR1 channels. Embodiment A provides single
channel current traces and corresponding histogram of wild-type
RyR1. Embodiment B provides single channel current traces and
corresponding histogram of wild-type RyR1 that is PKA
phosphorylated. Embodiment C provides single channel current traces
and corresponding histogram of RyR1-Ser-2843A. Embodiment D
provides single channel current traces and corresponding histogram
of RyR1-Ser-2843D.
[0055] FIG. 12, embodiments A and B, demonstrate the PKA
hyperphosphorylation and calstabin1 deficiency of RyR1 channels
after sustained exercise. Embodiment A are immunoblots of RyR1,
RyR1-pSer.sup.2844, RyR1-pSer.sup.2849, and calstabin1 for control
and swim mice following an exercise regime. Embodiment B is a bar
graph summarizing the relative amounts of the indicated compounds
following the exercise regime.
[0056] FIG. 13, embodiments A and B, demonstrate that RyR1 PKA
phosphorylation increases after exposure to increasing durations of
sustained exercise. Embodiment A provides immunoblots of RyR1 and
RyR1-pSer.sup.2844 following increasing durations of sustained
exercise. Embodiment B is a graph showing the relative PKA
phosphorylation of RyR1 for varying durations of exercise.
[0057] FIG. 14, embodiments A, B, and C, demonstrate that RyR1 PKA
phosphorylation increases with muscle fatigue. Embodiments A and B
are, respectively, fatigue time tracings and a bar graph showing
mean fatigue times for rat soleus muscle of heart failure and
control subjects. Embodiment C is a graph of PKA phosphorylation
versus fatigue time.
[0058] FIG. 15 are trichrome and hematoxylin-eosin stains of
cross-sections of the mouse M. extensor digitorum longus, and
demonstrates myofiber degeneration consistent with dystrophic
remodeling following sustained exercise.
[0059] FIG. 16 shows a sample hERG current trace before (control)
and after application of ARM036 at 100 .mu.M. Also shown is the
voltage pulse protocol used to evoke the hERG currents.
[0060] FIG. 17 shows a typical time course of the effect of ARM036
on hERG current amplitude. Application of 10 .mu.M ARM036 is
indicated by the horizontal bar.
[0061] FIGS. 18 to 34 are concentration-response graphs showing the
percent inhibition of hERG current after application of various
concentrations of ARM036, ARM036-Na, ARM047, ARM048, ARM050,
ARM057, ARM064, ARM074, ARM075, ARM076, ARM077, ARM101, ARM102,
ARM103, ARM104, ARM106 and ARM107, respectively.
[0062] FIGS. 35A and B are concentration-response graphs showing
the percent inhibition of hERG current after application of S26 (A)
or JTV-519 ("ARM00X") (B) at various concentrations.
[0063] FIG. 36 shows tracking data during swimming on the first day
of exercise for control vehicle treated (PBS) mice and mice treated
with S36. Individual mouse velocities over five minute time
intervals are shown in Embodiment A and mean velocities of each
treatment group are shown in Embodiment B (n=4 PBS, n=4 S36). After
one day of exercise, there is no substantial difference between the
treatment groups. Fg-100, fg-103, fg-105, fg-106 indicates mice
treated with S36. Fg-101, fg-102, fg-104, fg-107 indicates mice
treated with PBS.
[0064] FIG. 37 shows that there is no substantial difference
between the different treatment groups after 7 days of exercise.
Embodiments A and B show results of swimming exercise for PBS and
S36 treated mice. Individual mouse velocities over five minute time
intervals are shown in Embodiment A and mean velocities of each
treatment group are shown in FIG. 37B (n=3 PBS, n=4 S36).
Embodiments C-F show results of a treadmill running exercise on day
7. As described, mice were run on the treadmill on an exercise
protocol of increasing intensity, shown on the left marked velocity
(m/min) in grey. Embodiment C shows individual traces reflecting
the number of visits to the shocking area at the rear of the
treadmill over each three minute interval. Task failure can be
clearly appreciated from the rapid rise in visits to the shocking
area as the mice fail. Embodiment D shows the number of shocks
delivered to each mouse in each three minute interval, on an
inverted axis, plotted as points with a three point moving average
interpolation for each mouse. Embodiment E depicts quantification
of total distance run in meters before failure for PBS and S36
treatment groups (n=3 PBS, n=4 S36), and Embodiment F depicts
quantification of fatigue times, defined as time to task failure,
for PBS and S36 treatment groups. (n=3 PBS, n=4 S36). No
significant difference is observed between the different treatment
groups.
[0065] FIG. 38 shows that after 14 days of swimming, there is no
significant difference in the swimming velocities of the different
treatment groups. Embodiment A shows individual mouse velocities,
and Embodiment B shows mean velocities for each treatment group.
n=3 PBS, n=4 S36. Treadmill running on day fourteen demonstrates a
trend toward improved performance in S36 treated mice. An
increasing intensity exercise protocol was used, shown on the left
marked velocity (m/min) in grey. Embodiment C shows individual
traces reflecting the number of visits to the shocking area at the
rear of the treadmill over each three minute interval. Embodiment D
shows the number of shocks delivered to each mouse in each three
minute interval, on an inverted axis, plotted as points with a
three point moving average interpolation for each mouse. Embodiment
E depicts quantification of total distance run in meters before
failure for PBS and S36 treatment groups. (n=3 PBS, n=4 S36), and
Embodiment F depicts quantification of fatigue times, defined as
time to task failure, for PBS and S36 treatment groups. (n=3 PBS,
n=4 S36).
[0066] FIG. 39 embodiments A, B, C and D show analysis of data on
day 16 of exercise regimen. Embodiments (A) and (B) shows results
of treadmill running on day sixteen that replicates the trend
toward improved performance in S36 treated mice. An increasing
intensity exercise protocol was used, shown on the left marked
velocity (m/min) in black. Embodiment A shows individual traces,
which reflect the number of visits to the shocking area at the rear
of the treadmill over each three-minute interval. Embodiment B
shows the number of shocks delivered to each mouse in each three
minute interval, on an inverted axis, plotted as points with a
three point moving average interpolation for each mouse. Embodiment
(C) shows quantification of total distance run in meters before
failure for PBS and S36 treatment groups. (n=3 PBS, n=4 S36)
Embodiment (D) shows quantification of fatigue times, defined as
time to task failure, for PBS and S36 treatment groups. (n=3 PBS,
n=4 S36). FIG. 39 shows that the trend toward improved performance
in S36 treated mice continues on page 16.
[0067] FIG. 40 shows percent improvements in fatigue times and
distance run of S36 treated mice compared to PBS vehicle treated
mice under the same conditions at each day, as measured by the
treadmill assay.
[0068] FIG. 41 shows that in vivo treatment with S36 (ARM036)
allows RyR1 to rebind calstabin 1 despite intense chronic exercise.
RyR1 was immunoprecipitated from soleus muscle homogenates of mice
following 21 days of exercise with or without simultaneous
subcutaneous mini-osmotic pump treatment with S36 and then western
blotted back for RyR1, phospho-epitope specific RyR1-pS2844, and
calstabin1 bound to the RyR1 macromolecular complex.
[0069] FIGS. 42 A and B provide data illustrating that the RyCal
compound S107 reduces creatine kinase (A) and calpain (B) activity
in the mdx mouse muscular dystrophy model during exercise, and
indicates that RyCals are useful for treating muscle related
diseases, such as muscular dystrophies.
[0070] FIG. 43 shows that the RyR1 Macromolecular Complex Undergoes
Substantial Remodeling Following Exercise. A) Composition of the
RyR1 complex in extensor digitorum longus (EDL) muscle following an
exercise protocol (consisting of twice daily swimming) lasting the
indicated number of days by immunoprecipitation of RyR1 and
immunoblot for RyR, RyR1-pS2844, and PDE4D3 and calstabin1 bound to
the receptor. B) Densitometric quantification of A, where each
value is relative to the total RyR1 immunoprecipitated. C)
Composition of the RyR1 complex in EDL muscle following low
intensity and high intensity exercise for 5 days. D) Densitometric
quantification of C. In all cases, the product of a single RyR1
immunoprecipitation was separated on a 4-20% gradient
polyacrylamide gel, transferred, and probed for both total RyR1 and
one or more of the modifications noted. The blots shown are
representative of three independent experiments.
[0071] FIG. 44 shows that high intensity cycling exercise in humans
results in PKA phosphorylation of RyR1, and calstabin1 and PDE4D3
depletion. A) Immunoblot of the RyR1 complex immunoprecipitated
from 100 ug of muscle homogenate from human thigh biopsies before
and after exercise on day 1 and day 3 (C) of a high intensity
(three hours at 57% VO.sub.2 max) cycling protocol. Control
cyclists sat in the exercise room but did not exercise. Immunoblots
show total RyR1, RyR1-S2844 PKA phosphorylation, bound calstabin1,
and bound PDE4D3. B) and D) Quantification by densitometry of A)
and C) respectively. Bar graphs depict PKA phosphorylation,
calstabin1, and PDE4D3 levels in the RyR1 complex normalized to
total RYR1 from control (n=6) and exercise (n=12) biopsies on each
day.
[0072] FIG. 45 shows that Muscle-specific Cal1-/- Mice Have a High
Intensity Exercise Defect. A) Time to failure during a single level
treadmill assay on 2 month-old cal1-/- mice and w.t. littermates.
B) Individual treadmill failure times for each mouse separated by
gender. C) Body weights of the cal1-/- mice were reduced. D)
Scatter plot of failure time versus body weight shows no
correlation in either group of mice. E) Plasma creatine kinase
(CPK) levels at baseline and following a single downhill eccentric
treadmill run. F) RyR1 immunoprecipitated from EDL and
immunoblotted for RyR, RyR1-pS2844, PDE4D3, and calstabin1. *,
p<0.01, Wilcoxon rank-sum test.
[0073] FIG. 46 shows that PDE4D-/- mice have an exercise defect. A)
Time to failure during a single level treadmill assay on 2
month-old PDE4D-/- mice and w.t. littermates. B) Individual
treadmill failure times for each mouse. C) Body weights of the
PDE4D-/- mice. D) Scatter plot of failure time versus body weight
shows no correlation in either group of mice. E) Plasma creatine
kinase (CPK) levels at baseline and following a single downhill
eccentric treadmill run. F) RyR1 immunoprecipitated from EDL and
immunoblotted for RyR, RyR1-pS2844, PDE4D3, and calstabin1. *,
p<0.05, Wilcoxon rank-sum test.
[0074] FIG. 47 shows that pharmacologic rebinding of calstabin1 to
RyR1 improves in vivo exercise performance. A) Time to failure
during treadmill assays on indicated days of a 28 day treatment
trial with S107. B) Individual treadmill failure times for each
mouse on Day 21. C) Force-frequency curves of EDL muscle isolated
immediately following the 21st day of exercise and isometrically
stimulated in an oxygenated muscle bath. Forces (cN) are normalized
to muscle cross sectional area. D) Body weights throughout the
trial showed no treatment effect. E) Treadmill failure times from a
parallel experiment in muscle-specific cal1-/- mice. F) RyR1
immunoprecipitated from EDL and immunoblotted for RyR, RyR1-pS2844,
PDE4D3, and calstabin1. *, p<0.01, Wilcoxon rank-sum test.
[0075] FIG. 48 shows that chronic S107 treatment reduces
fatiguability of isolated FDB fibers. A) Representative trace from
a vehicle treated FDB fiber of fluo-4 fluorescence (.DELTA.F/FO)
normalized to the peak during repeated 300 ms long, 120 Hz
field-stimulated tetani at a train rate of 0.5. Hz. Isolated cells
were continuously perfused with a HEPES buffered Tyrodes solution
at room temperature. B) Representative tetanic trace from an S107
treated FDB fiber. C) Mean peak tetanic calcium, as measured by
fluo-4 fluorescence (.DELTA.F/FO) normalized to the peak during
fatiguing stimulation (n=11 vehicle, n=13 S107). *, p<0.02
unpaired t-test.
[0076] FIG. 49 shows that RyR1 from exercised muscle is leaky, with
increased Po at resting calcium. (A) Representative traces of RyR1
channel activity at 90 nM [Ca2+]cis from sedentary mice (sed, left
column), mice chronically exercised and treated with vehicle
(Ex+veh, middle column) and with S107 (Ex+S107, right column).
Single channel openings are plotted as upward deflections; the open
and closed (c) states of the channel are indicated by horizontal
bars at the beginning of the traces. Corresponding channel open
probability (Po), mean open time (To) and frequency of openings
(Fo) are shown above each group of traces and represent average
values from all experiments. (B) Average values of open probability
(left), mean open times (middle) and frequency of openings (right)
of RyR1 activity from sedentary mice (sed, n=9) and mice
chronically exercised treated either with vehicle (Ex+veh, n=9) or
S107 (Ex+S107, n=12). Error bars indicate SEM; *, p<0.005
compared to sed; #, p<0.005 compared to Ex+S107.
[0077] FIG. 50 shows that S107 protects against chronic
exercise-induced muscle damage and calpain activation. A) Plasma
creatine kinase (CPK) activity levels in sedentary and chronically
exercised mice with, and without, calstabin1 rebinding with S107.
B) Calpain activity levels in EDL homogenates measured using a
fluorogenic calpain substrate assay. *, p<0.01 unpaired t-test,
S107 vs vehicle.
[0078] FIGS. 51 A, B and C show the effect of exercise on the
composition of the RyR1 complex.
[0079] FIG. 52 shows the distribution of 50% reuptake times (tau)
in muscle fibers in the presence or absence of S107 treatment.
[0080] FIG. 53 shows the progressive phosphorylation of RyR1 and
calstabin 1 depletion from the RyR1 complex in an mdx mouse model
as a factor of time.
[0081] FIG. 54 shows the effect of S107 on exercise tolerance, body
weight, CPK and calpain levels in wt (unaffected) and mdx mice.
[0082] FIG. 55 shows histological slides of wt (unaffected) and mdx
mice which are untreated or treated with S107.
[0083] FIG. 56 provides data illustrating that the compound S107
crosses the blood brain barrier and enhances binding of calstabin
to a RyR in the brain (mid-section and cerebellum) in vivo. Data
from heart and soleus muscle are also illustrated.
[0084] FIG. 57 provides a schematic representation of an
experimental protocol used to test the effect of S107 on exercise
performance and spatial learning in mice.
[0085] FIG. 58 shows the difference in permanence time between S107
and vehicle treated mice. A: schematic representation of platform.
B: latency to target(s) for vehicle (veh) and S107 treated mice. C:
mean velocity (cmls) for vehicle (veh) and S107 treated mice.
[0086] FIG. 59 shows a trend towards altered behavior consistent
with improved learning and persistence in S107-treated mice (C), as
compared with vehicle-treated mice (B). Panel A provides a
schematic representation of the platform.
[0087] FIG. 60B is a bar graph representation showing improved
learning or increased persistence with S107 treated mice, as
compared with vehicle. FIG. 60A provides a schematic representation
of the experimental set-up.
[0088] FIG. 61 is an immunoblot showing total RyR (types 1 and 2),
phosphorylated RyR and calstabin (types 1 and 2) in control mice
and mice subjected to an exercise regimen, with or without
treatment with S107. Whole brain microsomes were obtained.
Immunoprecipitates were separated by 4-20% PAGE and analyzed for
total RyR, PKA phosphorylated RyR, and calstabin.
[0089] FIG. 62 provides a schematic representation of a protocol
for evaluating the effects of restraint stress on PKA
phosphorylation at different stress periods.
[0090] FIG. 63 shows PKA phosphorylation of RyR2 channels in brain
following restraint induced stress in mice. Mice were restrained
for time periods indicated. Ryanodine receptor (type 2) was
immunoprecipitated from whole brain microsomes. Immunoprecipitates
were separated by 4-20% PAGE and analyzed for total RyR2, PKA
phosphorylated RyR2, and calstabin2.
[0091] FIG. 64 shows the effect of chronic restraint stress (CRS)
on relative PKA phosphorylation of RyR2 in brain. Total RyR2 and
PKA phosphorylated RyR2 were quantified by densitometry of the
immunoblot shown in FIG. 63. The bar graphs represent the relative
PKA phosphorylation of the RyR2 channel, as determined by dividing
the phosphorylation signal by the RyR2 signal. (***P<0.001;
**P<0.01).
[0092] FIG. 65 shows the effects of chronic restraint stress (CRS)
on calstabin2 binding to RyR2 in the brain. Total RyR2 and
calstabin2 were quantified by densitometry of the immunoblot shown
in FIG. 63. The bar graphs represent the relative amount of
calstabin2 in the immunoprecipitates and were determined by
dividing the calstabin signal by the RyR2 signal. (*P<0.05).
DETAILED DESCRIPTION OF THE INVENTION
[0093] The following are definitions of terms used in the present
specification. The initial definition provided for a chemical group
or term herein applies to that group or term throughout the present
specification individually or as part of another group, unless
otherwise indicated.
[0094] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural references unless the
content clearly dictates otherwise. Thus, for example, reference to
"an agent" includes a plurality of such agents and equivalents
thereof known to those skilled in the art, and reference to "the
FKBP12.6 polypeptide" is a reference to one or more FKBP12.6
polypeptides (also known as calstabin2) and equivalents thereof
known to those skilled in the art, and so forth. All publications,
patent applications, patents, and other references mentioned herein
are incorporated by reference in their entirety.
[0095] As used herein the term "fatigue" refers to skeletal muscle
fatigue and/or weakness. Muscle fatigue can be due to strenuous or
repeated physical activity or exercise, chronic stress, disease,
disorder, syndrome or any other underlying pathophysiological
condition that has symptoms of fatigue, or affects myofibers and/or
muscle function. Muscle fatigue is defined as the failure of
exercise performance--this can be assessed on an exercise stress
test and quantified as the time it takes to fail at the given task
(e.g. walking/jogging/running on a treadmill). Failure at the task
is defined as termination of the exercise due to inability to
continue--this is defined as muscle fatigue.
[0096] Sustained or prolonged exercise is defined as exercise
performed over a defined and measurable time period.
[0097] Strenuous exercise is exercise to evoke muscle fatigue
within a defined time period.
[0098] Chronic stress is defined as conditions that cause muscle
fatigue chronically either due to persistent chronic exercise or
stress due to chronic diseases/disorders that are often associated
with chronic activation of the sympathetic nervous systems (e.g.,
chronic activation of the "fight or flight" response). In one
embodiment, the subject's defective skeletal muscle function occurs
during chronic obstructive pulmonary disease, hypertension, asthma,
or hyperthyroidism.
[0099] In certain aspects, the present invention is directed to
compositions and methods for the treatment and prevention of
myopathies. The term "myopathy" as used herein refers to
neuromuscular disorders caused by dysfunction in the muscle itself.
The term "myopathy", as used herein, encompasses all of the
myopathies described herein and also all other myopathies known to
those of skill in the art.
[0100] Myopathies may be inherited (such as many of the muscular
dystrophies) or acquired. Myopathic diseases and disorders include,
but are not limited to, congenital myopathies, muscular dystrophies
(characterized by progressive weakness in voluntary muscles),
mitochondrial myopathies, endocrine myopathies, muscular glycogen
storage diseases, myoglobinurias, dermatomyositis, myositis
ossificans, familial periodic paralysis, polymyositis, inclusion
body myositis, neuromyotonia, stiff-man syndrome, common muscle
cramps, and tetany.
[0101] Examples of muscular dystrophies include, but are not
limited to, Duchenne muscular dystrophy, facioscapulohumeral
dystrophy, limb girdle muscular dystrophy, and myotonic muscular
dystrophy, Becker's muscular dystrophy, congenital muscular
dystrophy, Distal muscular dystrophy, Emery-Dreifuss muscular
dystrophy, Facioscapulohumeral muscular dystrophy, Limb-girdle
muscular dystrophy, Myotonic muscular dystrophy, and
Oculopharyngeal muscular dystrophy. Examples of mitochondrial
myopathies include, but are not limited to, Kearns-Sayre syndrome,
MELAS and MERRF. MELAS which is an abbreviation of "mitochondrial
myopathy, encephalopathy, lactic acidosis, and stroke" is a
progressive neurodegenerative disorder. MELAS affects multiple
organ systems including the central nervous system (CNS), skeletal
muscle, the eye, cardiac muscle, and, more rarely, the
gastrointestinal and renal systems. MERFF, which is an abbreviation
of "myoclonus epilepsy with ragged-red fibers" may cause epilepsy,
coordination loss, dementia and muscle weakness. Examples of
glycogen storage diseases of muscle include, but are not limited
to, Pompe's disease, Andersen's disease, and Cori's diseases.
Examples of myoglobinurias include, but are not limited to
McArdle's disease, Tarui disease, and DiMauro disease.
[0102] The following are definitions of terms used in the present
specification. The initial definition provided for a group or term
herein applies to that group or term throughout the present
specification individually or as part of another group, unless
otherwise indicated.
[0103] As used herein, the term "RyCal compounds" refers to
compounds of the general Formula I, I-a, I-b, I-c, I-d, I-e, I-f,
I-g, I-h, I-i, I-j, I-k, I-k-1, I-l, I-l-1, I-m, I-m-1, I-n, I-o,
I-p or II as provided by the invention, and herein referred to as
"compound(s) of the invention".
[0104] The compounds of the invention are referred using a
numerical naming system, with compound numbers 1 to 123 provided
herein. These numbered compounds are referred to using either the
prefix "S" or the prefix "ARM." Thus, the first numbered compound
is referred to either as "S1" or "ARM001", the second numbered
compound is referred to as either "S2" or "ARM002", the third
numbered compound is referred to as either "S3" or "ARM003", and so
on. The "S" and the "ARM" nomenclature systems are used
interchangeably throughout the specification, the drawings, and the
claims.
[0105] The term "alkyl" as used herein refers to a linear or
branched, saturated hydrocarbon having from 1 to 6 carbon atoms.
Representative alkyl groups include, but are not limited to,
methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl,
pentyl, isopentyl, neopentyl, hexyl, isohexyl, and neohexyl. The
term "C.sub.1-C.sub.4 alkyl" refers to a straight or branched chain
alkane (hydrocarbon) radical containing from 1 to 4 carbon atoms,
such as methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, and
isobutyl.
[0106] The term "alkenyl" as used herein refers to a linear or
branched hydrocarbon having from 2 to 6 carbon atoms and having at
least one carbon-carbon double bond. In one embodiment, the alkenyl
has one or two double bonds. The alkenyl moiety may exist in the E
or Z conformation and the compounds of the present invention
include both conformations.
[0107] The term "alkynyl" as used herein refers to a linear or
branched hydrocarbon having from 2 to 6 carbon atoms and having at
least one carbon-carbon triple bond.
[0108] The term "aryl" as used herein refers to an aromatic group
containing 1 to 3 aromatic rings, either fused or linked.
[0109] The term "cyclic group" as used herein includes a cycloalkyl
group and a heterocyclic group.
[0110] The term "cycloalkyl group" as used herein refers to a
three- to seven-membered saturated or partially unsaturated carbon
ring. Any suitable ring position of the cycloalkyl group may be
covalently linked to the defined chemical structure. Exemplary
cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, and cycloheptyl.
[0111] The term "halogen" as used herein refers to fluorine,
chlorine, bromine, and iodine.
[0112] The term "heterocyclic group" or "heterocyclic" or
"heterocyclyl" or "heterocyclo" as used herein refers to fully
saturated, or partially or fully unsaturated, including aromatic
(i.e., "heteroaryl") cyclic groups (for example, 4 to 7 membered
monocyclic, 7 to 11 membered bicyclic, or 10 to 16 membered
tricyclic ring systems) which have at least one heteroatom in at
least one carbon atom-containing ring. Each ring of the
heterocyclic group containing a heteroatom may have 1, 2, 3, or 4
heteroatoms selected from nitrogen atoms, oxygen atoms and/or
sulfur atoms, where the nitrogen and sulfur heteroatoms may
optionally be oxidized and the nitrogen heteroatoms may optionally
be quaternized. The heterocyclic group may be attached to the
remainder of the molecule at any heteroatom or carbon atom of the
ring or ring system. Exemplary heterocyclic groups include, but are
not limited to, azepanyl, azetidinyl, aziridinyl, dioxolanyl,
furanyl, furazanyl, homo piperazinyl, imidazolidinyl, imidazolinyl,
isothiazolyl, isoxazolyl, morpholinyl, oxadiazolyl, oxazolidinyl,
oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl,
phenanthrolinyl, piperazinyl, piperidinyl, pyranyl, pyrazinyl,
pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl,
pyridoimidazolyl, pyridothiazolyl, pyridinyl, pyrimidinyl,
pyrrolidinyl, pyrrolinyl, quinuclidinyl, tetrahydrofuranyl,
thiadiazinyl, thiadiazolyl, thienyl, thienothiazolyl,
thienooxazolyl, thienoimidazolyl, thiomorpholinyl, thiophenyl,
triazinyl, and triazolyl. Exemplary bicyclic heterocyclic groups
include indolyl, isoindolyl, benzothiazolyl, benzoxazolyl,
benzoxadiazolyl, benzothienyl, quinuclidinyl, quinolinyl,
tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl,
benzopyranyl, indolizinyl, benzofuryl, benzofurazanyl, chromonyl,
coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl,
pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl,
furo[3,2-b]pyridinyl] or furo[2,3-b]pyridinyl), dihydroisoindolyl,
dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl),
triazinylazepinyl, tetrahydroquinolinyl and the like. Exemplary
tricyclic heterocyclic groups include carbazolyl, benzidolyl,
phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl and the
like.
[0113] The term "phenyl" as used herein refers to a substituted or
unsubstituted phenyl group.
[0114] The aforementioned terms "alkyl," "alkenyl," "alkynyl,"
"aryl," "phenyl," "cyclic group," "cycloalkyl," "heterocyclyl,"
"heterocyclo," and "heterocycle" may further be optionally
substituted with one or more substituents. Exemplary substituents
include but are not limited to one or more of the following groups:
hydrogen, halogen, CF.sub.3, OCF.sub.3, cyano, nitro, N.sub.3, oxo,
cycloalkyl, alkenyl, alkynyl, heterocycle, aryl, alkylaryl,
heteroaryl, OR.sub.a, SR.sub.a, S(.dbd.O)R.sub.e,
S(.dbd.O).sub.2R.sub.e, P(.dbd.O).sub.2R.sub.e,
S(.dbd.O).sub.2OR.sub.a, P(.dbd.O).sub.2OR.sub.a, NR.sub.bR.sub.c,
NR.sub.bS(.dbd.O).sub.2R.sub.e, NR.sub.bP(.dbd.O).sub.2R.sub.e,
S(.dbd.O).sub.2NR.sub.bR.sub.c, P(.dbd.O).sub.2NR.sub.bR.sub.c,
C(.dbd.O)OR.sub.a, C(.dbd.O)R.sub.a, C(.dbd.O)NR.sub.bR.sub.c,
OC(.dbd.O)R.sub.a, OC(.dbd.O)NR.sub.bR.sub.c,
NR.sub.bC(.dbd.O)OR.sub.a, NR.sub.dC(.dbd.O)NR.sub.bR.sub.c,
NR.sub.dS(.dbd.O).sub.2NR.sub.bR.sub.o
NR.sub.dP(.dbd.O).sub.2NR.sub.bR.sub.c, NR.sub.bC(.dbd.O)R.sub.a,
or NR.sub.bP(.dbd.O).sub.2R.sub.e, wherein R.sub.a is hydrogen,
alkyl, cycloalkyl, alkenyl, alkynyl, alkylaryl, heteroaryl,
heterocycle, or aryl; R.sub.b, R.sub.c and R.sub.d are
independently hydrogen, alkyl, cycloalkyl, alkylaryl, heteroaryl,
heterocycle, aryl, or said R.sub.b and R.sub.e together with the N
to which they are bonded optionally form a heterocycle; and R.sub.e
is alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, alkylaryl,
heteroaryl, heterocycle, or aryl. In the aforementioned exemplary
substitutents, groups such as alkyl, cycloalkyl, alkenyl, alkynyl,
cycloalkenyl, alkylaryl, heteroaryl, heterocycle and aryl can
themselves be optionally substituted.
[0115] Exemplary substituents may further optionally include at
least one labeling group, such as a fluorescent, a bioluminescent,
a chemiluminescent, a colorimetric and a radioactive labeling
group. A fluorescent labeling group can be selected from bodipy,
dansyl, fluorescein, rhodamine, Texas red, cyanine dyes, pyrene,
coumarins, Cascade Blue.TM., Pacific Blue, Marina Blue, Oregon
Green, 4',6-Diamidino-2-phenylindole (DAPI), indopyra dyes, lucifer
yellow, propidium iodide, porphyrins, arginine, and variants and
derivatives thereof. For example, ARM118 of the present invention
contains a labeling group BODIPY, which is a family of fluorophores
based on the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene moiety. For
further information on fluorescent label moieties and fluorescence
techniques, see, e.g., Handbook of Fluorescent Probes and Research
Chemicals, by Richard P. Haughland, Sixth Edition, Molecular
Probes, (1996), which is hereby incorporated by reference in its
entirety. One of skill in the art can readily select a suitable
labeling group, and conjugate such a labeling group to any of the
compounds of the invention, without undue experimentation.
[0116] The term "quaternary nitrogen" refers to a tetravalent
positively charged nitrogen atom including, for example, the
positively charged nitrogen in a tetraalkylammonium group (e.g.,
tetramethylammonium, N-methylpyridinium), the positively charged
nitrogen in protonated ammonium species (e.g.,
trimethyl-hydroammonium, N-hydropyridinium), the positively charged
nitrogen in amine N-oxides (e.g., N-methyl-morpholine-N-oxide,
pyridine-N-oxide), and the positively charged nitrogen in an
N-amino-ammonium group (e.g., N-aminopyridinium).
[0117] Throughout the specification, unless otherwise noted, the
nitrogen in the benzothiazepine ring of compounds of the present
invention may optionally be a quaternary nitrogen. Non-limiting
examples include ARM-113 and ARM-119.
[0118] Compounds of the present invention may exist in their
tautomeric form (for example, as an amide or imino ether). All such
tautomeric forms are contemplated herein as part of the present
invention.
[0119] The term "prodrug" as employed herein denotes a compound
that, upon administration to a subject, undergoes chemical
conversion by metabolic or chemical processes to yield compounds of
the present invention.
[0120] All stereoisomers of the compounds of the present invention
(for example, those which may exist due to asymmetric carbons on
various substituents), including enantiomeric forms and
diastereomeric forms, are contemplated within the scope of this
invention. Individual stereoisomers of the compounds of the
invention may, for example, be substantially free of other isomers
(e.g., as a pure or substantially pure optical isomer having a
specified activity), or may be admixed, for example, as racemates
or with all other, or other selected, stereoisomers. The chiral
centers of the present invention may have the S or R configuration
as defined by the IUPAC 1974 Recommendations. The racemic forms can
be resolved by physical methods, such as, for example, fractional
crystallization, separation or crystallization of diastereomeric
derivatives or separation by chiral column chromatography. The
individual optical isomers can be obtained from the racemates by
any suitable method, including without limitation, conventional
methods, such as, for example, salt formation with an optically
active acid followed by crystallization.
[0121] Compounds of the present invention are, subsequent to their
preparation, preferably isolated and purified to obtain a
composition containing an amount by weight equal to or greater than
99% of the compound ("substantially pure" compound), which is then
used or formulated as described herein. Such "substantially pure"
compounds of the present invention are also contemplated herein as
part of the present invention.
[0122] All configurational isomers of the compounds of the present
invention are contemplated, either in admixture or in pure or
substantially pure form. The definition of compounds of the present
invention embraces both cis (Z) and trans (E) alkene isomers, as
well as cis and trans isomers of cyclic hydrocarbon or heterocyclic
rings.
[0123] Metabolite as used herein refers to a byproduct produced in
vivo, for example in a subject, from a chemical compound.
[0124] As used herein, the term "RyCal compounds" refers to
compounds of the general Formula I or II as provided by the
invention, and herein referred to as compound(s) of the invention.
Such compounds include, but are not limited to, any one or more of
the compounds of formulae including, but not limited to, the
compounds of formulae 51, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11,
S12, S13, S14, S19, S20, S22, S23, S24, S25, S26, S36, S37, S38,
S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54,
S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S66, S67, S68,
S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81,
S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94,
S95, S96, S97, S98, S99, S100, S101, S102, S103, S104, S105, S107,
S108, S109, S110, S111, S112, S113, S114, S115, S116, S117, S118,
S119, S120, S121, S122, and S123, as herein defined. In certain
embodiments, the compounds are isolated and substantially pure.
[0125] A subject treated by the methods of the invention can
include a mammal. Such a mammal can include a human, primate,
canine, equine, feline, porcine, murine, bovine, foul, ungulate or
sheep. The terms "animal," "subject" and "patient" as used herein
include all members of the animal kingdom including, but not
limited to, mammals, animals (e.g., cats, dogs, horses, etc.) and
humans.
[0126] "PKA phosphorylation" means a reaction in which a phosphate
group is substituted for a hydroxyl group by the enzyme protein
kinase A (PKA).
[0127] "Back-phosphorylation" of RyR1 or RyR2 receptor means the in
vitro phosphorylation of receptor by protein kinase A.
[0128] A "pharmaceutical composition" refers to a mixture of one or
more of the compounds described herein, or pharmaceutically
acceptable salts, hydrates, polymorphs, or prodrugs thereof, with
other chemical components, such as physiologically acceptable
carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0129] A compound of the present invention also can be formulated
as a pharmaceutically acceptable salt, e.g., acid addition salt,
and complexes thereof. The preparation of such salts can facilitate
the pharmacological use by altering the physical characteristics of
the agent without preventing its physiological effect. Examples of
useful alterations in physical properties include, but are not
limited to, lowering the melting point to facilitate transmucosal
administration and increasing the solubility to facilitate
administering higher concentrations of the drug.
[0130] The term "pharmaceutically acceptable acid addition salt" as
used herein means any non-toxic organic or inorganic salt of any
base compounds represented by Formula I, I-a, I-b, I-c, I-d, I-e,
I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, I-l, I-l-1, I-m, I-m-1, I-n,
I-o, I-p, or Formula II, any of their intermediates. Illustrative
inorganic acids which form suitable acid addition salts include
hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well
as metal salts such as sodium monohydrogen orthophosphate and
potassium hydrogen sulfate. Illustrative organic acids that form
suitable acid addition salts include mono-, di-, and tricarboxylic
acids such as glycolic, lactic, pyruvic, malonic, succinic,
glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic,
benzoic, phenylacetic, cinnamic and salicylic acids, as well as
sulfonic acids such as p-toluene sulfonic and methanesulfonic
acids. Either the mono or di-acid salts can be formed and such
salts exist in either a hydrated, solvated or substantially
anhydrous form. In general, the acid addition salts of compounds of
the invention are more soluble in water and various hydrophilic
organic solvents, and generally demonstrate higher melting points
in comparison to their free base forms.
[0131] A compound of the present invention also can be formulated
as a pharmaceutically acceptable salt, e.g., acid addition salt,
and complexes thereof. The preparation of such salts can facilitate
the pharmacological use by altering the physical characteristics of
the agent without preventing its physiological effect. Examples of
useful alterations in physical properties include, but are not
limited to, lowering the melting point to facilitate transmucosal
administration and increasing the solubility to facilitate
administering higher concentrations of the drug.
[0132] The term "pharmaceutically acceptable salt" means a salt
that is suitable for, or compatible with, the treatment of a
patient or a subject such as a human or animal patient such as a
person or dog. The salts can be any non-toxic organic or inorganic
salt of any of the compounds represented by Formula I, I-a, I-b,
I-c, I-d, I-e, I-f, I-g, I-h, I-j, I-k, I-k-1, I-l, I-l-1, I-m,
I-m-1, I-n, I-o, I-p, II or any of the specific compounds described
herein, or any of their intermediates. Illustrative salt-forming
ions include, but are not limited to, ammonium (NH.sub.4.sup.+),
calcium (Ca.sup.2+), iron (Fe.sup.2+ and Fe.sup.3+), magnesium
(Mg.sup.2+), potassium (K.sup.+), pyridinium
(C.sub.5H.sub.5NH.sup.+) quaternary ammonium (NR.sub.4.sup.+),
sodium (Na.sup.+), acetate, carbonate, chloride, bromide, citrate,
cyanide, hydroxide, nitrate, nitrite, oxide, phosphate, sulfate,
maleate, fumarate, lactate, tartrate, gluconate, besylate, and
valproate. Illustrative inorganic acids that form suitable salts
include, but are not limited to, hydrochloric, hydrobromic,
sulfuric and phosphoric acids, as well as metal salts such as
sodium monohydrogen orthophosphate and potassium hydrogen sulfate.
Illustrative organic acids that form suitable acid addition salts
include, but are not limited to, mono-, di-, and tricarboxylic
acids such as glycolic, lactic, pyruvic, malonic, succinic,
glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic,
benzoic, phenylacetic, cinnamic and salicylic acids, as well as
sulfonic acids such as p-toluene sulfonic and methanesulfonic
acids. Either mono or di-acid salts can be formed, and such salts
exist in either a hydrated, solvated or substantially anhydrous
form. In general, the acid addition salts of compounds of Formula
I, I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-j, I-k, I-k-1, I-l,
I-l-1, I-m, I-m-1, I-n, I-o, or I-p, are more soluble in water and
various hydrophilic organic solvents, and generally demonstrate
higher melting points in comparison to their free base forms. The
selection of an appropriate salt can be performed by one skilled in
the art. For example, one can select salts in reference to
"Handbook of Pharmaceutical Salts: Properties, Selection, and Use"
by P. Heinrich Stahl and Camille G. Wermuth, or Berge (1977)
"Pharmaceutcial Salts" J. Pharm Sci., Vol 66(1), p 1-19. Other
non-pharmaceutically acceptable salts (e.g., oxalates) may be used,
for example, in the isolation of compounds of the invention for
laboratory use or for subsequent conversion to a pharmaceutically
acceptable acid addition salt.
[0133] The compounds of Formula I, I-a, I-b, I-c, I-d, I-e, I-f,
I-g, I-h, I-j, I-k, I-k-1, I-l, I-l-1, I-m, I-m-1, I-n, I-o, I-p,
and Formula II of the present invention may form hydrates or
solvates, which are included in the scope of the claims. When the
compounds of Formula I of the present invention exist as
regioisomers, configurational isomers, conformers or
diasteroisomeric forms all such forms and various mixtures thereof
are included in the scope of Formula I. It is possible to isolate
individual isomers using known separation and purification methods,
if desired. For example, when a compound of Formula I of the
present invention is a racemate, the racemate can be separated into
the (S)-compound and (R)-compound by optical resolution. Individual
optical isomers and mixtures thereof are included in the scope of
Formula I, I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k,
I-k-1, I-l, I-l-1, I-m, I-m-1, I-n, I-o, I-p, and Formula II.
[0134] The term "solvate" as used herein means a compound of
Formula I, I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k,
I-k-1, I-l, I-l-1, I-m, I-m-1, I-n, I-o, I-p, and Formula II or a
pharmaceutically acceptable salt of a compound of Formula I,
wherein molecules of a suitable solvent are incorporated in the
crystal lattice. A suitable solvent is physiologically tolerable at
the dosage administered. Examples of suitable solvents are ethanol,
water and the like. When water is the solvent, the molecule is
referred to as a "hydrate."
[0135] The term "polymorph" refers to a particular crystalline
state of a substance, having particular physical properties such as
X-ray diffraction, IR spectra, melting point, and the like.
[0136] The term an "effective amount," "sufficient amount" or
"therapeutically effective amount" of an agent as used herein is
that amount sufficient to effect beneficial or desired results,
including clinical results and, as such, an "effective amount"
depends upon the context in which it is being applied, whether the
response is preventative and/or therapeutic. The term "effective
amount" also includes that amount of the compound of Formula I
which is "therapeutically effective" and which avoids or
substantially attenuates undesirable side effects.
[0137] As used herein and as well understood in the art,
"treatment" is an approach for obtaining beneficial or desired
results, including clinical results. Beneficial or desired clinical
results can include, but are not limited to, alleviation or
amelioration of one or more symptoms or conditions, diminishment of
extent of disease, stabilized (i.e., not worsening) state of
disease, preventing spread of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment.
[0138] As used herein, the term "inhibiting dissociation" includes
blocking, decreasing, inhibiting, limiting or preventing the
physical dissociation or separation of an FKBP subunit from an RyR
molecule in cells of the subject, and blocking, decreasing,
inhibiting, limiting or preventing the physical dissociation or
separation of an RyR molecule from an FKBP subunit in cells of the
subject.
[0139] As used herein, the term "increasing binding" includes
enhancing, increasing, or improving the ability of phosphorylated
RyR to associate physically with FKBP (e.g., binding of
approximately two fold or, approximately five fold, above the
background binding of a negative control) in cells of the subject
and enhancing, increasing or improving the ability of FKBP to
associate physically with phosphorylated RyR (e.g., binding of
approximately two fold, or, approximately five fold, above the
background binding of a negative control) in cells of the
subject.
[0140] As used herein, the term "cardiac muscle cell" includes
cardiac muscle fibers, such as those found in the myocardium of the
heart.
[0141] The present invention provides compounds that are capable of
treating and preventing disorders and diseases associated with the
RyR receptors that regulate calcium channel functioning in cells.
More particularly, the present invention provides compounds that
are capable of treating or preventing a leak in RyR channels.
"Disorders and diseases associated with the RyR receptors" means
disorders and diseases that can be treated and/or prevented by
modulating the RyR receptors that regulate calcium channel
functioning in cells. "Disorders and diseases associated with the
RyR receptors" include, without limitation, cardiac disorders and
diseases, skeletal muscular disorders and diseases, cognitive
disorders and diseases, malignant hyperthermia, central core
disease, diabetes, and sudden infant death syndrome. Cardiac
disorder and diseases include, but are not limited to, irregular
heartbeat disorders and diseases; exercise-induced irregular
heartbeat disorders and diseases; sudden cardiac death;
exercise-induced sudden cardiac death; congestive heart failure;
chronic obstructive pulmonary disease; and high blood pressure.
Irregular heartbeat disorders and diseases include and
exercise-induced irregular heartbeat disorders and diseases
include, but are not limited to, atrial and ventricular arrhythmia;
atrial and ventricular fibrillation; atrial and ventricular
tachyarrhythmia; atrial and ventricular tachycardia;
catecholaminergic polymorphic ventricular tachycardia (CPVT); and
exercise-induced variants thereof. Skeletal muscular disorder and
diseases include, but are not limited to, skeletal muscle fatigue,
exercise-induced skeletal muscle fatigue, muscular dystrophy,
bladder disorders, and incontinence. Cognitive disorders and
diseases include, but are not limited to, Alzheimer's Disease,
forms of memory loss, and age-dependent memory loss.
[0142] As contemplated herein, the compounds of the invention are
capable of treating and preventing disorders and diseases
associated with the RyR receptors that regulate calcium channel
functioning in cells, by repairing the leak in RyR channels, and
enhancing binding of FKBP proteins (e.g., calstabin1) to
PKA-phosphorylated RyR. Thus, in one embodiment, the compounds are
useful to prevent and treat muscle fatigue that is associated with
the RyR receptors that regulate calcium channel functioning in
cells.
[0143] In one embodiment, the compounds of the invention are
effective to treat muscle fatigue that results from pathologies,
illnesses, diseases, disorders or conditions that are associated
with the RyR receptors that regulate calcium channel functioning in
cells. Examples of such disorders and conditions include, but are
not limited to, cardiac disease or disorder, defective skeletal
muscle function, HIV Infection, AIDS, muscular dystrophy, cancer,
malnutrition, exercise-induced muscle fatigue, age-associated
muscle fatigue, renal disease, and renal failure.
[0144] Examples of cardiac disorders and diseases include, but are
not limited to, irregular heartbeat disorders and diseases;
exercise-induced irregular heartbeat disorders and diseases;
congestive heart failure; chronic obstructive pulmonary disease;
and high blood pressure. Examples of irregular heartbeat disorders
and diseases and exercise-induced irregular heartbeat disorders and
diseases include, but are not limited to, atrial and ventricular
arrhythmia; atrial and ventricular fibrillation; atrial and
ventricular tachyarrhythmia; atrial and ventricular tachycardia;
catecholaminergic polymorphic ventricular tachycardia (CPTV); and
exercise-induced variants thereof.
[0145] In one embodiment, the compounds of the invention modulate
calcium-ion channels in cells of the subject. In another
embodiment, the compounds of the invention decrease the release of
calcium into cells of the subject. In another embodiment, the
compounds of the invention limit or prevent a decrease in the level
of RyR-bound FKBP in the subject. In another embodiment, the
compounds of the invention inhibit dissociation of FKBP and RyR in
cells of the subject. In another embodiment, the compounds of the
invention increase binding between FKBP and RyR in cells of the
subject. In another embodiment, the compounds of the invention
stabilize the RyR-FKBP complex in cells of a subject. In another
embodiment, the compounds of the invention prevent, or treat a leak
in a RyR receptor in the subject. In another embodiment, the
compounds of the invention modulate the binding of RyR and FKBP in
the subject. In another embodiment, the compounds of the invention
reduce the open probability of RyR by increasing the affinity of
FKBP for PKA-phosphorylated RyR. In another embodiment, the
compounds of the invention reduce or inhibit calpain activity so as
to treat muscle fatigue. In another embodiment, the compounds of
the invention reduce plasma creatine kinase levels so as to treat
muscle fatigue.
[0146] The methods of the present invention can be practiced in
vitro or in vivo. Thus, in one embodiment, the methods of the
present invention are practiced in an in vitro system (e.g., in a
test tube on isolated cellular components). In another embodiment,
the methods of the invention are practiced in vivo, e.g., in
cultured cells or tissues, or in subjects.
[0147] In another embodiment, the present invention provides use of
a compound represented by the structure of formula I, I-a, I-b,
I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, I-l, I-l-1,
I-m, I-m-1, I-n, I-o, I-p, and Formula II in the preparation of a
medicament for treating or preventing muscle disorder or disease,
for example but not limited to muscle fatigue in a subject.
[0148] In another embodiment, muscle fatigue can be caused by
increased stress such as in individuals exposed to a continued and
prolonged exercise regimen, e.g., soldiers or athletes. Thus in one
embodiment, the compounds of the invention are useful for treating
muscle fatigue in individuals exposed to stress due to, for
example, an intense exercise regimen. Skeletal muscular disorder
and diseases include, but are not limited to, stress induced
skeletal muscle fatigue, exercise-induced skeletal muscle fatigue,
muscular dystrophy, bladder disorders, and incontinence.
Skeletal Muscle Fatigue
[0149] Defects in Ca2+ release channel, for example increased
"leakiness" of the channel can lead to skeletal muscle fatigue. In
certain aspects the invention provides that RyCal compounds which
treat defects in Ca2+ release channel can be used in methods for
treating, reducing or preventing muscle disorders, muscle fatigue,
including but not limited to exercise-induced muscle fatigue or
muscle damage, muscle fatigue or damage associated with a disease
condition, for example but not limited to a myopathy, muscular
dystrophy and the like. Recent studies suggesting that lactic acid
accumulation may not be detrimental have raised questions about the
molecular basis underlying skeletal muscle fatigue. Among
hypotheses a role for defective regulation of calcium has been
proposed.
[0150] The invention provides data showing altered function of the
major calcium release channel in skeletal muscle sarcoplasmic
reticulum (SR), the type 1 ryanodine receptor (RyR1), is required
for excitation-contraction coupling (ECC), during chronic exercise.
During chronic exercise the RyR1 channel is PKA hyperphosphorylated
at Ser2844 (Ser2843 in human). The PKA hyperphosphorylation is
associated with depletion of the phosphodiesterase PDE4D3 from the
RyR1 complex. Furthermore, PKA hyperphosphorylation contribute to
depletion of the RyR1 stabilizing subunit calstabin1 (FKBP12) from
the channel macromolecular complex resulting in "leaky" channels
(increased open probability under conditions when normal channels
are not active). The degree of PKA phosphorylation, and depletion
of calstabin1 and PDE4D3 are correlated with the intensity and
duration of exercise and progressive fatigue. Mice with skeletal
muscle-specific calstabin1 deficiency and PDE4D deficient mice both
exhibited significantly impaired exercise capacity. A small
molecule, S107, that specifically causes rebinding of calstabin1 to
the RyR1 channel improved exercise capacity and force generation of
isolated muscle during a 21 day exercise protocol. S107 treated
muscle fibers exhibited reduced fatigue, as determined by
measurement of intracellular calcium during repeated tetanic
contractions. Furthermore, S107 treated chronically exercised mice
exhibited reduced levels of plasma creatine kinase, and
calcium-dependent neutral protease calpain activity in muscle
homogenates. This demonstrates the existence of a mechanism of
muscle fatigue, during chronic or high-intensity exercise, where SR
calcium leak due calstabin1 depleted RyR1 channels leads to
defective calcium signaling and skeletal muscle damage. In one
aspect, the invention provides use of RyCal compounds which target
molecular mechanisms of muscle fatigue, muscle conditions and
disorders, and provide treatments treatment thereof.
[0151] Calcium release channel stabilizing drugs prevent muscle
fatigue by preventing ryanodine receptor calcium leak during
sustained or strenuous exercise. Repeated and strenuous activity of
skeletal muscle may cause 1) weakness with intense use (also
referred to as fatigue), 2) feeling of sore and weak muscles
(referred to as perception), and 3) different degrees of muscle
degeneration (referred to as dystrophic remodeling). A dominant
theory of muscle fatigue has been that accumulation of
intracellular lactic acid resulting in intracellular acidosis
directly inhibits force production by the myofibrillar proteins
(Hill et al., 1929). Indeed, at lower than physiologic temperatures
(20.degree. C.), acidotic changes of intracellular pH were found to
accelerate fatigability of skeletal muscles (Hill at al., 1929).
However, more recent studies have challenged the significance of
acidosis for muscle fatigue by showing that repeated short tetanic
contractions which induce fatigue do not result in significant
intracellular pH changes under more physiologic conditions at
37.degree. C. (Westerblad at al., 1997) where acidosis does not
significantly effect force production. These findings are
consistent with lactic acid which is produced during fatiguing
contractions being extruded at a substantial rate by lactate
transporters. However, during very intense athletic training lactic
acid resulting from anaerobic breakdown of glycogen remains an
important limiting factor. Importantly, intracellular acidosis was
found to preserve muscle excitability and relaxation of the
myofilaments during sustained increases of intracellular Ca.sup.2+
during repeated or sustained tetanic contractions, and thus
protects from muscle fatigue (Pedersen at al. 2004).
[0152] Given that changes in intracellular pH may not represent a
major fatigue mechanism, it is likely that alterations in EC
coupling contribute to fatigue. Intracellular Ca.sup.2+ release via
RyR1 channels initiates muscle contraction. Now classic physiologic
experiments suggested in 1963, that reversible alterations in
contractile activation may play an important role in muscle fatigue
(Eberstein et al., 1963). During progressive development of fatigue
resulting from repeated tetanic contractions, elevations in
intracellular Ca.sup.2+ concentrations decline which explains
reduced force production (Allen et al., 2001). However, caffeine
and other compounds, which maximally activate RyR1 channels and
cause sudden SR Ca.sup.2+ release, can briefly normalize tetanic
Ca.sup.2+ concentrations (Allen at al., 2001). Thus alterations of
RyR1-dependent SR Ca.sup.2+ release mechanisms are likely to be
involved in fatigue development.
[0153] Measurements of SR Ca.sup.2+ load have demonstrated a
decreased pool of releasable Ca.sup.2+ during skeletal muscle
fatigue, which may be one of the causes of reduced SR Ca.sup.2+
release during fatigue. (Cooke at al. 1985). Another theory
involves increased intracellular inorganic phosphate concentrations
([P.sub.i].sub.i) precipitating Ca.sup.2+ in the SR storage
organelle (Allen, 2001; Cooke, 1985). However, [P.sub.i].sub.i
increases occur during the early phase of fatigue resulting from
rapid ATP breakdown whereas the decline of tetanic
[Ca.sup.2+].sub.i occurs late in fatigue (phase iii). Moreover,
after repeated, stretched contractions resting [Ca.sup.2+].sub.i
was found elevated while stimulated, tetanic [Ca.sup.2+].sub.i
decreased (Warren et al. 1993; Balnave et al. 1995). The increased
resting [Ca.sup.2+].sub.i may initiate chronic impairment of EC
coupling for example by activating proteases that can damage the SR
Ca.sup.2+ release channel (Lamb et al., 1995; Chin et al., 1996;
Bruton et al., 1996).
[0154] Fatigue from chronically sustained exercise may be caused by
SR Ca.sup.2+ leak resulting from defective closure of the RyR1
channel and partial depletion of the SR Ca.sup.2+ store
contributing to diminished force production and increased resting
[Ca.sup.2+].sub.i interfering with muscle relaxation and when
sustained causing muscle degeneration. Recent data shows that an
evolutionary conserved stress pathway, the fight-or-flight
response, specifically controls RyR1 Ca.sup.2+ release in skeletal
muscle (Gaburjakova et al., 2001; Marx et al., 2001) and that
abnormal, chronic activation of this stress pathway causes SR
Ca.sup.2+ leak contributing to muscle fatigue (Reiken et al.,
2003).
[0155] RyR1/calcium release channels become PKA hyperphosphorylated
and depleted of the stabilizing protein calstabin1 during exercise.
RyCal compounds of the invention increase the binding affinity of
calstabin1 to PKA hyperphosphorylated RyR1. These compounds are
called "calcium channel stabilizers" or "RyCals" and are in a class
of 1,4-benzothiazepines and related structures. In a non-limiting
example, treatment with a RyCal compound improves exercise
performance of mice running on a treadmill. Furthermore, there is
evidence that a calcium leak via PKA hyperphosphorylated RyR1
channels causes muscle damage due to activation of
calcium-dependent proteases and RyCals prevent the calcium leak and
inhibit muscle damage during chronic exercise. In certain
embodiments, RyCal compound can be used to treat, prevent or
improve muscle fatigue in chronic diseases including but not
limited to heart failure, AIDS, cancer, renal failure, chronic
obstructive pulmonary disease, hypertension, asthma,
hyperthyroidism, chronic muscle fatigue. In other embodiments,
RyCal compound can be used to treat or improve muscular
dystrophies. In other embodiments, treatment with RyCal compound
can prevent or reduce muscle fatigue which can improve exercise
performance in individuals who are exposed to sustained chronic
stress and/or physical exercise. In other embodiments, treatment
with RyCals can prevent or reduce muscle fatigue which can improve
exercise performance in individuals who are exposed to strenuous
physical exercise.
[0156] Skeletal muscles become weaker with intense use also
referred to as fatigue. Moreover, repeated stretch-dependent
contractures can result in additional muscle damage and
degeneration. Although fatigue is recognized as an important
mechanism of limited peak performance and task failure during
stress, the mechanisms that promote fatigue or muscle fiber damage
are incompletely characterized. Moreover, defining molecular
fatigue mechanisms may enable targeted interventions that could
help prevent fatigue and muscle tissue damage (Wehrens, 2005). A
key physiologic mechanism that controls skeletal muscle performance
is intracellular calcium (Ca.sup.2+) release from specialized
Ca.sup.2+ stores (the sarcoplasmic reticulum, SR) via ryanodine
receptor (RyR1) Ca.sup.2+ release channels. In skeletal muscle,
plasma membrane depolarization activates voltage-gated L-type
Ca.sup.2+ channels (LTCCs; Ca.sub.V1.1) which in turn activate
RyR1s on the SR mediated by direct contact between both ion
channels.
[0157] Opening of RyR1 channels results in bulk SR Ca.sup.2+
release which activates the myofilaments and muscle contraction.
Also, disease forms associated with sustained activation of the
sympathetic nervous system and increased plasma catecholamine
levels cause maladaptive activation of intracellular stress
pathways resulting in destabilization of the RyR1 channel closed
state and intracellular Ca.sup.2+ leak (Reiken et al. 2003;
Brillantes et al. 1994). SR Ca.sup.2+ leak via RyR1 channels was
found to deplete intracellular SR calcium stores, to increase
compensatory energy consumption, and to result in significant
acceleration of muscle fatigue. The stress-induced muscle defect
limits peak performance and contributes to pathologic forms of
muscle fatigue that permanently reduce performance. Also,
destabilization of the RyR1 closed state involves depletion of the
stabilizing calstabin1 (FKBP12) channel subunit (Reiken et al.
2003; Brillantes et al. 1994). Experiments demonstrate that
increasing the binding affinity of calstabin to RyR rescues channel
function (Wehrens, 2003).
[0158] Skeletal muscle contraction is activated by SR Ca.sup.2+
release via the type 1 skeletal ryanodine receptor (RyR1).
Depolarization of the T-tubule membrane activates the
dihydropyridine receptor voltage sensor (Cav1.1) which in turn
activates RyR1 channels via a direct protein-protein interaction
causing the release of SR Ca.sup.2+ stores. Ca.sup.2+ binds to
troponin C allowing actin-myosin cross-bridging to occur and
sarcomere shortening. Ca.sup.2+ release channels comprise
macromolecular complexes consisting of a homotetramer of 560 kDa
RyR1 subunits that form scaffolds for proteins that regulate
channel function including: protein kinase A and the
phosphodiesterase PDE4D3, both of which are targeted to the channel
via the anchoring protein mAKAP, PP 1 (targeted via spinophilin),
and calstabin1 (FKBP12) (Jayaraman, Brillantes et al. 1992;
Brillantes, Ondrias et al. 1994; Marx, Reiken et al. 2000; Marx,
Reiken et al. 2001).
[0159] A defect in ECC that resulted in a reduction in the
amplitude of SR Ca.sup.2+ release would impair contraction and
force generation. Eberstein and Sandow proposed impaired SR
Ca.sup.2+ release as a likely contributor to muscle fatigue
(Eberstein and Sandow 1963). Reductions in the amplitude of SR
Ca.sup.2+ release evoked during fatiguing stimulation have been
reported (Allen, Lee et al. 1989; Westerblad and Allen 1991; Allen
and Westerblad 2001). In addition, it has been shown that Ca.sup.2+
stores decline during intense and repeated contractions (Kabbara
& Allen, 1999), and that the time course of recovery from
fatigue parallels the time course over which prolonged depression
of SR Ca.sup.2+ release is observed (Westerblad, Bruton et al.
2000). Furthermore, reduction of free SR Ca.sup.2+, due to
inorganic calcium phosphate salt precipitation during fatigue, has
been proposed (Allen and Westerblad 2001).
[0160] Evidence of defective SR Ca.sup.2+ release in fatigued
muscle prompted examination of the role of RyR1 mediated SR
Ca.sup.2+ release in skeletal muscle fatigue. The binding of
calstabin1 (FKBP12) to RyR1 stabilizes the closed state of the
channel and facilitates coupled gating between neighboring channels
(Brillantes, Ondrias et al. 1994; Marx, Ondrias et al. 1998).
Pharmacologic depletion of calstabin1 from RyR1 (with rapamycin or
FK506 both of which bind to calstabin1 and dissociate it from the
RyR1 macromolecular complex) promotes subconductance states and, in
intact skeletal muscle, can cause a rapid loss of
depolarization-induced contraction (Lamb and Stephenson 1996).
Mutation of RyR1 resulting in the loss of calstabin1 binding causes
impaired ECC with reduced maximal voltage-gated SR Ca.sup.2+
release without affecting the SR Ca.sup.2+ store content (Avila,
Lee et al. 2003). Genetic deletion of FKBP12 (calstabin1) induced
no gross histological or developmental defect in skeletal muscle,
though severe developmental cardiac defects were observed which
precluded detailed assessment of skeletal muscle function (Shou,
Aghdasi et al. 1998). Whereas skeletal muscle-specific knock-out of
FKBP12 (calstabin1) resulted in reduced voltage-gated SR Ca.sup.2+
release and increased L-type channel currents in isolated myotubes
(Tang, Ingalls et al. 2004). In extensor digitalis longus (EDL),
but not soleus or diaphragm, reduced maximal tetanic force and a
rightward shift in force-frequency relationships were observed
(Tang, Ingalls et al. 2004). These data indicated that calstabin1
modulates the gain of ECC in skeletal muscle.
[0161] The binding of calstabin1 to RyR1 is regulated by PKA
phosphorylation at RyR1-S2843 (position S2844 in the mouse RyR1
sequence) (Reiken, Lacampagne et al. 2003). Phosphorylation at
RyR1-S2843 increases the mean open probability of RyR1 in the lipid
bilayer (Reiken, Lacampagne et al. 2003). RyR1-52843A mutant
channels could not be PKA phosphorylated and did not show the same
PKA-dependent increase in open probability. An RyR1-52843D mutation
mimicked PKA phosphorylation with an increased open probability and
destabilized open and closed states (Reiken, Lacampagne et al.
2003). The role of PKA phosphorylation of RyR1 is still under
investigation as other groups have found little or no effect on
channel function (Stange, Xu et al. 2003). Other post-translational
modifications of RyR1 which might modulate calstabin1 binding to
RyR1 have been suggested, including oxidation, and
glutathionylation of the up to 50 free (reduced) thiols on each RyR
monomer.
[0162] SR Ca.sup.2+ leak has been documented as aberrant calcium
sparks in myofibers following intense exercise and in a model of
muscular dystrophy (Wang, Weisleder et al. 2005), possibly due to
defective RyR1 function. Chronic activation of the sympathetic
nervous system (SNS) during heart failure is associated with early
skeletal muscle fatigue and PKA hyperphosphorylation of RyR1 at
Ser2844 (meaning that on average 3-4 of the four PKA sites in each
homotetrameric channel are PKA phosphorylated in heart failure
skeletal muscle), calstabin1 depletion from the RyR1 complex, and a
gain-of-function channel defect (Reiken, Lacampagne et al. 2003).
RyR1 dysfunction in skeletal muscle leads to altered local
subcellular Ca.sup.2+ release events (Ward, Reiken et al.
2003).
[0163] Modifications in the RyR1 complex could alter and are likely
to limit peak muscle performance, increase muscle fatigue, and
contribute to muscle damage during prolonged or high intensity
exercise. In certain aspects the invention provides use of a mouse
model of chronic, high-intensity, forced exercise to assess the
role of the RyR1 channel in skeletal muscle fatigue. As described
herein, the RyR1 channel macromolecular complex undergoes
remodeling during exercise such that it is progressively PKA
hyperphosphorylated, and depleted of PDE4D3 and calstabin1.
Functionally, this remodeling is associated with "leaky" channels
(increased open probability) and activation of the
calcium-activated protease calpain, and leakage of creatine kinase
(CPK) into the plasma, which is consistent with muscle damage.
These changes are further associated with decreased force
production in isolated muscles and impaired exercise capacity and
are exacerbated in mice with PDE4D deficiency or muscle specific
calstabin1 deficiency. Preventing the RyR1 channel leak with a
calcium channel stabilizer S107, which enhances binding of
calstabin1 to RyR1, inhibits calpain activation, CPK leak and
improves exercise performance. Therefore, remodeling of the RyR1
channel complex that causes leaky channels, activation of the
calcium-activated protease calpain, and leakage of creatine kinase
(CPK) into the plasma, is a mechanism involved in muscle fatigue
during chronic or high intensity exercise.
[0164] Confocal imaging studies of intracellular Ca.sup.2+ release
(Ca.sup.2+ sparks) in muscle cells after mild and strenuous
treadmill exercise, showing that abnormal Ca.sup.2+ spark activity
is induced by fatiguing exercise (Wang et al. 2005). Moreover,
myofibers with abnormal Ca.sup.2+ spark activity resulting form
strenuous exercise show histological signs of degeneration from
toxic Ca.sup.2+ effects also known as `dystrophic remodeling` (Wang
et al. 2005). It is likely that sustained exercise over weeks and
months results in RyR1 dysfunction, intracellular Ca.sup.2+ leak,
depressed muscle performance, and dystrophic muscle remodeling.
Furthermore, 1,4-benzothiazepine based drugs enhance peak muscle
performance and prevent dystrophic remodeling by fixing
stress-induced intracellular Ca.sup.2+ leak.
[0165] Animal models can establish, in vivo and at the level of
isolated skeletal muscle cell, and single RyR1 channel, that
defects in RyR1 function cause muscle fatigue and dystrophic
remodeling. Use of these animal models of fatigue and the
characterized muscle, cells, and channel techniques allows tests of
therapeutic approaches based on fixing the leak in RyR1 which will
result in improved skeletal muscle performance, decreased muscle
fatigue, and reduced dystrophic remodeling during chronic forms of
exercise.
Muscular Dystrophy
[0166] Myotonic dystrophy type 1 (DM1), the most common muscular
dystrophy in adults (1 in 7,400 live births), is a multisystemic
disorder caused by a CTG trinucleotide repeat expansion in the 3'
untranslated region of the myotonic dystrophy protein kinase (DMPK)
gene which causes progressive muscle weakness, inherited muscle
hyperexitability (myotonia), cardiac conduction defect, cataract,
and insulin resistance (Bachinski L L, Udd B, Meola G, et al.
Confirmation of the type 2 myotonic dystrophy (CCTG)n expansion
mutation in patients with proximal myotonic myopathy/proximal
myotonic dystrophy of different European origins: a single shared
haplotype indicates an ancestral founder effect. Am J Hum Genet.
October 2003; 73(4):835-848; Hamshere M G, Harley H, Harper P, et
al. Myotonic dystrophy: the correlation of (CTG) repeat length in
leucocytes with age at onset is significant only for patients with
small expansions. J Med Genet. January 1999; 36(1):59-61; Liguori C
L, Ricker K, Moseley M L, et al. Myotonic dystrophy type 2 caused
by a CCTG expansion in intron 1 of ZNF9. Science. Aug. 3 2001;
293(5531):864-867). The mutant DMPK messenger RNA (mRNA) containing
an expanded CUG repeat is retained in the nucleus and protein
levels are reduced (Mankodi A, Logigian E, Callahan L, et al.
Myotonic dystrophy in transgenic mice expressing an expanded CUG
repeat. Science. Sep. 8 2000; 289(5485):1769-1773). The RNA repeat
expansion changes the chromatin structure, silences the expression
of the flanking SIX5 gene which codes for a transcription factors,
and disrupts regulation of gene expression during development and
exercise (Ebralidze A, Wang Y, Petkova V, et al. RNA leaching of
transcription factors disrupts transcription in myotonic dystrophy.
Science. Jan. 16 2004; 303(5656):383-387).
[0167] The cause of the most severe symptoms including muscle
weakness and progressive muscle wasting appear to be caused by
elevated intracellular Ca.sup.2+ concentrations and subsequent
myofiber degeneration in DM1 (Jacobs A E, Benders A A, Oosterhof A,
et al. The calcium homeostasis and the membrane potential of
cultured muscle cells from patients with myotonic dystrophy.
Biochim Biophys Acta. Nov. 14 1990; 1096(1):14-19). Moreover, a
recent study has linked disturbed Ca.sup.2+ cycling in DM1 to
aberrant splicing of RyR1 and SR Ca.sup.2+ ATPase (SERCA1) mRNAs
(Kimura T, Nakamori M, Lueck J D, et al. Altered mRNA Splicing of
the Skeletal Muscle Ryanodine Receptor and Sarcoplasmic/Endoplasmic
Reticulum Ca2+-ATPase in Myotonic Dystrophy Type 1. Hum Mol Genet.
Jun. 22 2005). A muscle-specific genetic mouse model HSA.sup.LR of
DM1 exists in which expanded CUG repeat expression results in a
DM-like phenotype (Mankodi A, Logigian E, Callahan L, et al.
Myotonic dystrophy in transgenic mice expressing an expanded CUG
repeat. Science. Sep. 8 2000; 289(5485):1769-1773). HSA.sup.LR have
a myotonic phenotype in the absence of muscle fiber necrosis and
the short--versus long-repeat expressing mouse lines show
relatively less or more histopathological signs of muscle
regeneration and repair, respectively (Mankodi A, Logigian E,
Callahan L, et al. Myotonic dystrophy in transgenic mice expressing
an expanded CUG repeat. Science. Sep. 8 2000; 289(5485):1769-1773).
Since HSA.sup.LR mice with short- or long repeat expression show no
signs of muscle weakness and since RyR1 alterations have been
linked to DM1, the HSA.sup.LR mice provide a model to study the
effects of exercise and activation of the sympathetic nervous
system in these mice. After characterizing RyR1 channel
composition, phosphorylation status, and function, the HSA.sup.LR
mice can be challenge with sustained exercise tests and treated
with RyCal compounds. Since the mechanism by which transcripts with
expanded CUG repeats cause myotonia and muscle degeneration in DM1
is not known, this would provide 1) study of a genetic animal model
of severe fatigue, 2) elucidation of the key molecular mechanism of
DM1, and 3) developing a therapeutic rationale for the most common
muscular dystrophy in adults.
Stress Pathways and Muscle Fatigue
[0168] Sustained activation of intracellular stress pathways such
as occurring during strenuous physical exercise, for example but
not limited to combat, can result in reduced muscle performance and
tissue damage. A major determinant of muscle damage may occur due
to toxicity of continuously high catecholamine levels resulting in
intracellular Ca.sup.2+ leak. This concept is supported by: 1)
physiologic (non-combat) exercise or stress was reported to induce
muscle weakness, cramps, and tissue atrophy in susceptible
individuals (stress-induced rhabdomyolysis) (Wappler et al., 2001);
2) strenuous but not mild treadmill exercise induces a
significantly elevated Ca.sup.2+ spark frequency in muscle cells
indicating intracellular Ca.sup.2+ leak (Wang et al., 2005); 3)
excess catecholamine levels were found in malignant hyperthermia
(MH) and central core disease (CCD) contributing to uncontrolled
intracellular Ca.sup.2+ release (Monnier et al. 2000; MacLennan et
al. 1995); 4) a majority of MH/CCD were linked to RyR1 missense
mutations (Loke et al., 2003); 5) a hyperadrenergic state as
occurring in heart failure causes RyR1 hyperphosphorylation,
Ca.sup.2+ leak, and skeletal muscle fatigue (Reiken et al. 2003);
and 6) excess plasma catecholamine levels by activating
.beta.-adrenergic receptors, intracellular cAMP synthesis and
protein kinase A phosphorylation results in muscle damage
(Goldspink et al., 2004; Tan et al. 2003). Stress-dependent muscle
damage and dysfunction occurs at the interface between
intracellular catecholamine effectors (protein kinase A, PKA) and
intracellular Ca.sup.2+ release. Skeletal ryanodine receptor (RyR1)
Ca.sup.2+ release channels constitute intracellular scaffolds that
integrate PKA-mediated stress signaling and regulation of
intracellular Ca.sup.2+ release and therefore determine the gain of
EC coupling and muscle function. Importantly, chronically increased
PKA phosphorylation of RyR1 occurring from a chronic
hyperadrenergic state in vivo depletes the stabilizing calstabin1
subunits resulting in SR Ca.sup.2+ leak and muscle fatigue.
Moreover, these observations extend to an in vivo animal model of
fatigue. Therefore, sustained activation of the sympathetic nervous
system during continued muscle performance contributes to increased
fatigue development and dystrophic skeletal muscle damage. Since
skeletal RyR1 Ca.sup.2+ release channel is PKA hyperphosphorylated
and depleted of the stabilizing calstabin1 subunit after 21 days of
intense exercise, wherein similar alterations result in
intracellular Ca.sup.2+ leak in animals with heart failure, it is
likely that chronic (>1 week) forms of exercise result in
adverse intracellular Ca.sup.2+ leak from defective RyR1
channels.
[0169] In one aspect, the invention establishes molecular
mechanisms of muscle fatigue occurring from chronically sustained
muscle performance. In another aspect the invention provides
methods for treating or preventing detrimental intracellular
Ca.sup.2+ leak and muscle damage by administering novel
1,4-benzothiazepine derivatives. The skeletal ryanodine receptor
(RyR1) channel is comprised of 4 RyR1 subunits and associated
proteins that bind to the cytoplasmic domain of the channel forming
a macromolecular signaling complex. Certain aspects of the
invention examine mechanisms by which allosteric modulators
regulate RyR1 function. Two specific forms of allosteric modulation
are examined: 1) regulation of the channel by cAMP-dependent
protein kinase A (PKA) that potently activates channel gating; 2)
depletion of the stabilizing subunit calstabin1 from the RyR1
channel during chronically increased PKA phosphorylation resulting
from chronic activation of the sympathetic nervous system by
strenuous, sustained exercise.
[0170] Dysregulation of RyR1 by PKA during sustained exercise
causes intracellular Ca.sup.2+ leak and may be a mechanism of
increased muscle fatigue and dystrophic remodeling. In certain
aspects, the invention determines the effects of fatiguing exercise
on 1) RyR1 PKA phosphorylation; 2) RyR1 channel function examined
using RyR1 channels reconstituted into planar lipid bilayers; 3)
Calstabin1 binding to RyR1; 4) intracellular Ca.sup.2+ sparks in
isolated myofibers; 5) isolated skeletal muscle function; 6)
mitochondrial integrity, 7) in vivo exercise performance, 8)
skeletal muscle histology, fiber type composition and oxidative
capacity, 9) creatine kinase (CK) plasma levels, 10) RyR1 PKA
phosphorylation and calstabin1 depletion in leukocytes. Calstabin1
depletion during chronically sustained exercise and PKA
hyperphosphorylation causes RyR1 hyperactivity, intracellular
Ca.sup.2+ leak, depletion of SR Ca.sup.2+ stores, accelerated
fatigue and dystrophic muscle remodeling.
[0171] In certain embodiments, treatment with any RyCal compound
normalizes RyR dysfunction and intracellular Ca.sup.2+ leak. RyR1,
which are protein kinase A (PKA) hyperphosphorylated and "leaky" in
heart failure models and during strenuous exercise, can rebind
calstabin1 after RyCal compound treatment, which normalizes single
channel and improves muscle performance in heart failure. It is
likely that RyCals in vivo and in cells treated with RyCals prevent
intracellular Ca.sup.2+ leak and normalize RyR1 channel function
during sustained exercise.
[0172] By preventing RyR1 Ca.sup.2+ leak, RyCal compounds improves
skeletal muscle fatigue and dystrophic remodeling during strenuous
exercise as occurs in combat. By using two animal models of muscle
fatigue (swimming and running on a treadmill) in mice and rats,
certain aspects of the invention show that treatment with RyCal
compounds prevents depletion of calstabin1 from RyR1, which
decreases muscle fatigue, improves performance, and inhibits
dystrophic muscle remodeling.
[0173] Certain aspects of the invention identify molecular
mechanisms of muscle fatigue in myotonic dystrophy (DM1). In
certain embodiments, by preventing RyR1 Ca.sup.2+ leak, RyCal
compounds improve skeletal muscle fatigue times and dystrophic
remodeling in mouse models of myotonic dystrophy.
[0174] Other aspects of the invention characterize molecular
fatigue mechanisms in genetic mouse models. In certain embodiments,
calstabin1 and PDE4D3 knockout mice can be used to further explore
molecular fatigue mechanisms and dysregulation during sustained
exercise states that contribute to intracellular Ca.sup.2+ leak. In
other aspects, the compounds of the invention reduce calpain
activity. In other aspects, the compounds of the invention reduce
plasma creatine kinase activity.
[0175] Other aspects of the invention, characterize mechanisms that
destabilize the closed state of intracellular calcium (Ca.sup.2+)
release channels during chronic activation of the sympathetic
nervous system as occurs during sustained exercise and/or in
combat. There are two allosteric modulators of skeletal ryanodine
receptors (RyR1s), one is PKA and the other one is a stabilizing
protein subunit of the channel (calstabin1) (Wehrens et al., 2004).
Other aspects of the invention disclose therapeutic and preventive
measures wherein a drug molecule that rebinds calstabin1 may
prevent skeletal muscle fatigue by normalizing the skeletal (RyR1)
ryanodine receptor gating. Protein kinase A (PKA)
hyperphosphorylation of RyR1 during chronic activation of the
sympathetic nervous system was shown to result in SR Ca.sup.2+ leak
as a cause of fatigue (Reiken et al., 2003) which was reversed by
JTV-519 (Wehrens et al., 2005). In certain aspects, the invention
provides, thorough characterization of the effects of strenuous
exercise on RyR1 Ca.sup.2+ leak, in vivo and ex vivo muscle
performance and energetic metabolism, methods for the use of RyCal
compounds to overcome muscle fatigue and to prevent muscle damage
and/or fatigue during strenuous, and/or sustained exercise, and
muscle damage and/or fatigue associated with defective skeletal
muscle function, or any disease condition.
[0176] Certain aspects of the invention characterize mechanisms
that destabilize the closed state of intracellular calcium
(Ca.sup.2+) release channels which is a prerequisite for muscle
relaxation to occur and prevents damage of myofibers from
uncontrolled intracellular SR Ca.sup.2+ leak. Chronic activation of
the sympathetic nervous system during forced and sustained exercise
caused RyR1 PKA hyperphosphorylation, calstabin1 depletion, and a
defective channel closed state are consistent with SR Ca.sup.2+
leak. Since stress and physical performance in combat are
considered significantly more severe compared to the animal use
protocols, it can be extrapolated that muscle fatigue and
dystrophic degeneration from RyR1 Ca.sup.2+ leak represents a more
severe phenotype in warfighters. The focus is on two allosteric
modulators of skeletal ryanodine receptors (RyR1 s), one PKA as a
key stress pathway and the other one a stabilizing protein subunit
of the channel (calstabin1).
[0177] Certain aspects of the invention address potential
therapeutic and preventive measures in that a drug molecule
rebinding calstabin1 may prevent skeletal muscle fatigue by
normalizing the skeletal (RyR1) ryanodine receptor channel gating.
Protein kinase A (PKA) hyperphosphorylation of RyR1 during chronic
activation of the sympathetic nervous system was shown to result in
SR Ca.sup.2+ leak as a cause of fatigue (Wehrens X H, Lehnart S E,
Reiken S, et al. Enhancing calstabin binding to ryanodine receptors
improves cardiac and skeletal muscle function in heart failure.
Proc Natl Acad Sci USA. Jul. 5 2005; 102(27):9607-9612; Ward C W,
Reiken S, Marks A R, et al. Defects in ryanodine receptor calcium
release in skeletal muscle from post-myocardial infarct rats. Faseb
J. August 2003; 17(11):1517-1519). Certain aspects of the invention
are directed to thorough characterization of the effects of
strenuous exercise on RyR1 Ca.sup.2+ leak, in vivo and ex vivo
muscle performance and energetic metabolism, and the use of RyCal
compounds to improve muscle fatigue times, to increase exercise
capacity, and to prevent muscle damage during and/or after
strenuous, sustained exercise.
[0178] Life quality and prognosis in heart failure patients is
decreased due to skeletal muscle dysfunction (e.g., shortness of
breath due to diaphragmatic weakness, and exercise intolerance due
to limb skeletal muscle fatigue) (Harrington et al. 1997). Recent
studies have identified dysregulation of intracellular Ca.sup.2+
release from the SR as a pathogenic mechanism underlying skeletal
muscle dysfunction in heart failure (Reiken S, Lacampagne A, Zhou
H, et al. PKA phosphorylation activates the calcium release channel
(ryanodine receptor) in skeletal muscle: defective regulation in
heart failure. J Cell Biol. Mar. 17 2003; 160(6):919-928; Ward,
2003; Perreault C L, Gonzalez-Serratos H, Litwin S E, et al.
Alterations in contractility and intracellular Ca2+ transients in
isolated bundles of skeletal muscle fibers from rats with chronic
heart failure. Circ Res. August 1993; 73(2):405-412). Heart failure
in animals with myocardial infarcts causes significantly
accelerated fatigue which is intrinsic to skeletal muscle as
measured by the time for the tetanic force to fall below 50% of the
maximal contraction (Reiken, 2003).
Skeletal RyR1 are PKA Hyperphosphorylated and Depleted of
Calstabin1
[0179] Previous studies have suggested that skeletal muscle RyR1
channels are PKA hyperphosphorylated and depleted of calstabin1 in
a pacing-induced canine model of heart failure and a rat
post-myocardial infarct model (Reiken et al., 2003; Ward et al.,
2003). Furthermore, in a mouse model of post-myocardial infarction
heart failure, RyR1 in soleus muscle is PKA-hyperphosphorylated. In
certain embodiments, treatment with a RyCal compound, allows
rebinding of calstabin1 to RyR1 despite intense chronic
exercise.
[0180] Beta-adrenergic stimulation increases the gain of the EC
coupling when enhanced muscle performance is required during
exercise or stress (fight-or-flight response). Binding of
catecholamines to .beta.-adrenoceptors activates a G-protein
coupled intracellular signaling cascade, which leads to increased
intracellular cAMP concentrations and activation of protein kinase
A (PKA). PKA is targeted to RyR1 via mAKAP forming a signaling
complex with the skeletal Ca.sup.2+ release channel (Reiken et al.
2003). RyR1 phosphorylation by PKA increases the channel open
probability and SR Ca.sup.2+ release (Reiken et al., 2003; Wehrens
et al., 2004).
[0181] Data from skeletal myofibers have confirmed intracellular
Ca.sup.2+ leak from enhanced RyR1 activity after strenuous exercise
consistent with an increased maximal rate of SR Ca.sup.2+ release.
PKA-hyperphosphorylation of RyR1 results in depletion of calstabin1
(FKBP12) from the channel complex due to a reduced binding affinity
for calstabin1. Chronic depletion of calstabin1 from the RyR1
channel complex relieves an intrinsic inhibition of the channel and
induces uncontrolled intracellular Ca.sup.2+ leak and reduced
fatigue resistance during a sustained hyperadrenergic state.
Skeletal muscle fatigue is increased in heart failure patients and
in animal models of heart failure (Reiken et al., 2003; Harrington
et al., 1997; Perreault et al., 1993; Lunde et al. 2001; Lunde et
al. 1998). In both patients and animals with heart failure the
skeletal RyR1 channel isoform was found PKA hyperphosphorylated and
depleted of the stabilizing calstabin1 subunits (Reiken et al.,
2003; Wehrens et al., 2004). Increased fatigue and RyR1
hyperphosphorylation are associated with an increased Ca.sup.2+
spark frequency and a decreased Ca.sup.2+ spark amplitude in
skeletal myofibers in heart failure animals consistent with
intracellular Ca.sup.2+ leak and decreased SR Ca.sup.2+
concentrations (Reiken et al., 2003). Therefore, most likely
increased muscle fatigue results from a chronic hyperadrenergic
state causing an intracellular Ca.sup.2+ leak via defective RyR1
channels (Reiken et al., 2003).
[0182] However, it is important to conceptualize that the role of
external Ca.sup.2+ ions in mammalian skeletal muscle contraction is
not completely understood. The LTCC and RyR isoforms in skeletal
and cardiac muscles are different, with skeletal muscle expressing
the LTCC .alpha.1.sub.S subunit (Tanabe et al., 1988) and RyR1
(Marks et al., 1989), and cardiac muscle expressing the LTCC
.alpha.1.sub.C subunit (Mikami et al., 1989) and RyR2 (Nakai et
al., 1990). RyR1 in skeletal muscle does not depend on Ca.sup.2+
influx via LTCC .alpha.1.sub.S to activate SR Ca.sup.2+ release as
evidenced by continuous EC coupling in skeletal muscle cells when
external Ca.sup.2+ is removed or when Ca.sup.2+ channel blockers
are present (Armstrong et al., 1972; Dulhunty et al., 1988;
Gonzalez-Serratos et al., 1982). Later experimental findings
support RyR1 activation by physical coupling with LTCC
.alpha.1.sub.S (Rios et al., 1987; Tanabe et al., 1990). RyR1
Ca.sup.2+ leak likely increases the cellular energy demands by
compensatory SR Ca.sup.2+ ATPase uptake consuming more ATP which
may contribute to earlier skeletal muscle fatigue. From direct
oxygen measurements on the surface of contracting muscle
preparations, it is estimated that total ATP consumption by SR
Ca.sup.2+ ATPases is significantly elevated in heart failure (Meyer
et al., 1998) likely resulting from intracellular Ca.sup.2+ leak.
In agreement with decreased SR Ca.sup.2+ concentrations due to RyR1
Ca.sup.2+ leak, muscle-specific calstabin1 knockout increases LTCC
Ca.sup.2+ influx and reduces maximal voltage-gated intracellular
Ca.sup.2+ release (Tang et al., 2004).
[0183] Recent studies have demonstrated defective function of RyR1
channels in skeletal muscle during heart failure, which were
analogous to those found in RyR2 channels in failing myocardium:
PKA hyperphosphorylation of RyR1 and depletion of calstabin1 (Marx
et al., 2000; Reiken et al., 2003; Wehrens et al., 2005). These
findings suggest that defects in RyR1 function alter intracellular
Ca.sup.2+ handling, thereby contributing to early fatigue in
skeletal muscles. Depletion of calstabin1 from the RyR1
macromolecular complex may also uncouple channels from one another
and allow stochastic as opposed to coupled gating (Marx et al.,
1998), thus providing an attractive hypothesis for explaining the
altered Ca.sup.2+ spark behaviour in skeletal muscle with reduced
fatigue resistance (Ward et al., 2003). Thus, alterations in RyR1
could play a significant role in the skeletal muscle specific force
decrements and reduced exercise-tolerance seen in models of
increased muscle fatigue.
Methods
[0184] Aerobic exercise can be defined as a form of physical
exercise that increases the heart rate and enhances oxygen intake
to improve performance. Examples of aerobic exercise are running,
cycling, and swimming. In certain embodiments, mice were challenged
by aerobic exercise (forced swimming) for 90 mins twice daily. The
animals were accustomed to swimming in preliminary training
sessions: day -3 twice 30 mins, day -2 twice 45 mins, day -1 twice
60 mins, day 0 and following twice 90 mins. Mice were then
exercised for 1, 7, or 21 additional, consecutive days for 90 mins
twice daily. Between swimming sessions separated by a 4 hour rest
period the mice are kept warm and given food and water. An
adjustable-current water pool was used to exercise mice by
swimming. An acrylic pool (90 cm long.times.45 cm wide.times.45 cm
deep) was filled with water to a depth of 25 cm. A current in the
pool was generated with a pump. The current speed during the
swimming session was at a constant speed of 1 l/min flow rate. The
water temperature was maintained at 34.degree. C. with an electric
heater. Age- and weight-matched mice are used to exclude
differences in buoyancy from body fat.
[0185] Using forced swimming as an efficient protocol to increase
skeletal muscle aerobic capacity in mice (Evangelista et al.,
2003), the composition and phosphorylation status of the skeletal
RyR1 channel complex was investigated. Unexpectedly, after 3 weeks
of 90 mins swimming twice daily, C57Bl6 wild-type mice showed
significantly increased RyR2 phosphorylation by PKA while CaMKII
phosphorylation was not changed. RyR1 protein expression was
stable, however, RyR1 channels were depleted of the stabilizing
subunit calstabin1 (FKBP12). RyR1 hyperphosphorylation and
calstabin1 depletion are consistent with leaky RyR1 channels that
cause intracellular Ca.sup.2+ leak.
[0186] To investigate the influence of the duration of sustained
exercise on the RyR1 Ca.sup.2+ release channel defect, mice were
exposed to swimming for 1, 7, or 21 days followed by immediate
sacrifice. Longer exposure to sustained exercise shows a
significant increase of RyR1 PKA hyperphosphorylation beginning at
7 days and saturating at 21 days.
[0187] Moreover, a mouse model of muscular dystrophy which is known
to result in intracellular Ca.sup.2+ leak and dystrophic muscle
changes was investigated. Surprisingly, soleus muscles of the
dystrophin-deficient mdx mouse show calstabin1 depletion in the
absence of increased RyR1-Ser2844 phosphorylation. These results
identify RyR1-Ser2844 PKA hyperphosphorylation as a specific event
in exercise-induced RyR1 dysfunction.
[0188] Additionally, the histological changes in the fast-twitch
muscles of mice exposed to 3 weeks of exercise by swimming were
characterized. Cross-sections of the mouse M extensor digitorum
longus (EDL) showed histological changes consistent with myofiber
degeneration from intracellular Ca.sup.2+ overload from defective
RyR1 channel. Therefore sustained exercise for 90 mins twice daily
triggers a distrophic phenotype in EDL muscles of normal C57Bl6
mice.
[0189] Up-regulation of intracellular SR Ca.sup.2+ release by
cAMP-dependent signaling pathways augments the gain of
excitation-contraction coupling during peak muscle performance.
(Reiken, 2003) Therefore transient PKA phosphorylation of skeletal
RyR1 Ca.sup.2+ release channels represents a key mechanism of the
fight-or-flight response. Studies have established that
dysregulation of RyR1 Ca.sup.2+ release channels occurs during
strenuous fatiguing exercise. Mice which underwent rigorous
exercise for 3 weeks showed significantly increased levels of RyR1
PKA phosphorylation, increased RyR1 channel activity, and
dystrophic histological changes. Chronic RyR1 hyperphosphorylation
results in depletion of the stabilizing calstabin1, functional
channel defects, suggesting intracellular Ca.sup.2+ leak. At the
level of the muscle cell, this model was recently confirmed by a
report that increased Ca.sup.2+ spark frequency after fatiguing
exercise in mouse skeletal muscle (Wang et al., 2005). Moreover, it
was shown that intracellular Ca.sup.2+ leak from chronic exercise
acts as dystrophic signal in mammalian skeletal muscle (Wang,
2005). This confirms previous observations in cardiac muscle, where
hormonal and structural changes contribute to intracellular
Ca.sup.2+ leak causing defective excitation-contraction coupling
(Gomez, 1997). From these studies (Wehrens et al., 2005; Reiken et
al., 2003) and the Ca.sup.2+ spark data in fatiguing muscle (Wang
et al., 2005) it is likely that intracellular Ca.sup.2+ leak
represents a key pathology that accelerates muscle fatigue and
causes dystrophic remodeling of muscles. Accordingly, extended
investigation of fatigue models at the in vivo, isolated muscle,
muscle cell, mitochondria, and single RyR1 channel level will
characterize detrimental effects during peak performance on muscle
fatigue and dystrophic remodeling.
[0190] 1,4-benzothiazepine derivatives prevent muscle dysfunction
from intracellular Ca.sup.2+ leak under conditions of sustained
sympathetic nervous system activation occurring from heart failure
(Wehrens et al., 2005). In certain aspects, the invention provides
RyCal compounds and their use to enhance skeletal muscle
performance and to reduce muscle dysfunction during sustained
stress as occurs under combat conditions. Defects in the RyR1
Ca.sup.2+ release channel due to dysregulation by PKA allow
rationalizing of a therapeutic concept: intracellular SR Ca.sup.2+
leak and muscle dysfunction can be prevented by a drug that
increases binding of the stabilizing calstabin1 subunit to the RyR1
channel complex and thereby inhibits SR Ca.sup.2+ leak and
dystrophic muscle remodeling from sustained stress or exercise.
Applying a drug to prevent SR Ca.sup.2+ leak can therefore enhance
a variety of important physiologic skills which are prone to
stress-induced dysfunction. In certain aspects, the invention
provides that enhanced binding of calstabin1 to RyR1 by RyCal
compounds prevents muscle fatigue. Using animal models of
physiologic (swimming and running on a treadmill) and/or
pharmacologic activation (.beta.-adrenergic receptor agonists) of
the sympathetic nervous system combined with different degrees of
exercise exposure, it can be tested whether RyCal compounds improve
skeletal muscle function and prevent dystrophic muscle
degeneration. This may lead to a pharmacologic approach based on
allosteric modulation of RyR1 that can result in improved human
performance during sustained stress and prevent adverse tissue
damage thereby shortening recovery times. In other aspects, the
invention provides pharmacologic approaches based on allosteric
modulation of RyR1 that can result in improved human performance
during sustained stress and prevent adverse tissue damage and
shorten recovery times.
[0191] RyR1 PKA Phosphorylation:
[0192] quantification of skeletal RyR1 channel phosphorylation by
protein kinase A (PKA) following sustained exercise for 2 days, 1
week, and 3 weeks using two independent techniques (RyR1 PKA
phospho-epitope detection by specific antibody; incorporation of
radiolabeled phosphate by backphosphorylation essay).
[0193] RyR1 CaMKII Phosphorylation:
[0194] quantification of skeletal RyR1 channel phosphorylation by
Ca.sup.2+-calmodulin protein kinase II (CaMKII) following sustained
exercise for 2 days, 1 week, and 3 weeks using RyR1 CaMKII
phospho-epitope detection by specific antibody essay developed in
our laboratory. This will allow characterizing specific defects
resulting from chronic PKA phosphorylation and/or secondary
activation of CaMKII signaling by intracellular Ca.sup.2+ leak
mechanisms.
[0195] Calstabin1 Depletion in the RyR1 Complex:
[0196] quantification of depletion of the channel stabilizing
subunit calstabin1 (FKBP12) from the RyR1 channel complex by
immunoprecipitation techniques following sustained exercise for 2
days, 1 week, and 3 weeks. Calstabin1 depletion occurs from Ser2844
phosphorylation by PKA.
[0197] RyR1 Functional Defects--
[0198] in vivo development of "leaky" RyR1 channels:
electrophysiologic characterization of RyR1 single-channel activity
and open probability following sustained exercise for 2 days, 1
week, and 3 weeks. This allows for a comprehensive and sensitive
assessment of SR Ca.sup.2+ release channel defects and leak
mechanisms that are known to contribute to muscle fatigue.
Moreover, these data will allow developing a rationale for
preventive treatment of RyR1 Ca.sup.2+ leak using a small lead
RyCal compounds.
[0199] Improvement of Fatigue from Sustained Exercise:
[0200] quantification of in vivo fatigue using two independent
exercise performance tests: swimming and running on a treadmill.
The treadmill test will be combined with electrocardiogram
telemetry in a subgroup of animals to allow for objective
correlation of increased heart rates during exercise with fatigue
symptoms. Moreover, plasma and muscle catecholamine levels will be
determined to verify sustained activation of the sympathetic
nervous system. Three weeks of maximal fatiguing swimming exercise
induces progressive RyR1 dysfunction and dystrophic skeletal muscle
changes during a low flow rate of 1 l/min (baseline condition which
prevents mice from floating passively). To objectively quantify
fatigue times during swimming exercise, video tracking system can
be used (San Diego Instruments Incorporated) which automatically
tracks and digitizes spatio-temporal movements of 8 mice.
Improvement of fatigue will be assessed by time to fatigue (defined
as significant increase in 2D distance or activity over time).
[0201] Improvement of Maximal Exercise Capacity:
[0202] To determine maximal endurance exercise capacity (i.e., time
to exhaustion), the maximum swim times are measured at a flow rate
of 7 l/min in repeated measurements three times a week. To reduce
the inherent variation in swimming capacity, mice whose mean
maximum swim times vary by more than 40% than the average swim time
will be excluded. A mouse is qualified as fatigued when it fails to
rise to the water surface to breathe and will be rescued at this
point. To verify the systemic exhaustion after swimming, plasma
lactate and pH will be determined in heparinized arterial blood
samples. To objectively quantify fatigue times during forced
swimming exercise, we will use a video tracking system (San Diego
Instruments Incorporated) which automatically tracks and digitizes
spatio-temporal movements of 8 mice in a 2D plane.
[0203] Isolated Skeletal Muscle Function:
[0204] ex vivo characterization of intrinsic muscle resistance to
fatigue stimulation or single-twitch contraction protocols
following sustained exercise with placebo or a RyCal compound
treatment for 2 days, 1 week, and 3 weeks. Two different forms of
isolated skeletal muscles will be tested: extensor digitorum longus
for a fast-twitch muscle and soleus muscle for a slow-twitch
muscle. This test can determine the effect of RyCal compounds on
skeletal muscle under fatiguing exercise, or disease conditions.
Skeletal muscle contraction and relaxation is critically dependent
on intracellular Ca.sup.2+ metabolism and RyR1 function and
therefore a highly sensitive test.
[0205] Intracellular Ca.sup.2+ Leak and SR Ca.sup.2+ Content in
Isolated Skeletal Myofibers:
[0206] assessment of isolated skeletal muscle myofibers loaded with
fluo-4 Ca.sup.2+ indicators for resting SR Ca.sup.2+ leak using
intracellular Ca.sup.2+ sparks. SR Ca.sup.2+ content will be
assessed by caffeine pulse protocols which result in complete
release of the free SR Ca.sup.2+ pool. Previous studies have
documented calcium leak resulting from a chronic hyperadrenergic
state in myofibers of rats with heart failure (Reiken 2003; Ward
2003; Gomez 2001; Cheng 1996).
[0207] Changes in Mitochondrial Integrity from SR Ca.sup.2+
Leak:
[0208] mitochondria in mouse skeletal muscle take up Ca.sup.2+
which under conditions of strong physiologic muscle stimulation
result in continuously elevated mitochondrial Ca.sup.2+ levels
which stimulates mitochondrial metabolism (Rudolf et al., 2004).
However, myotubes from dystrophic mdx mice showed significantly
elevated Ca.sup.2+ uptake. (Robert V, Massimino ML, Tosello V, et
al. Alteration in calcium handling at the subcellular level in mdx
myotubes. J Biol Chem. Feb. 16 2001; 276(7):4647-4651.) Cytosolic
Ca.sup.2+ overload is a highly toxic event that represents a common
final pathway of cell death. Mitochondria are key players in cell
death, and the spatial proximity of RyR1 Ca.sup.2+ release and
mitochondrial Ca.sup.2+ uptake suggest, that SR Ca.sup.2+ leak
during strenuous exercise can cause mitochondrial Ca.sup.2+
overload which impacts on mitochondrial structure and function and
may trigger cell death. Caspase-12 is localized in the SR, is
regulated by Ca.sup.2+, and participates in the SR stress-induced
apoptosis pathway (Yoneda et al., 2001). In certain aspects the
invention characterizes the effects of fatiguing exercise on
mitochondrial membrane potential by rhodamine 123 uptake,
mitochondrial swelling indicating mitochondrial permeability
transition by 520 nm spectrophotometry and light scatter, the
amount of intra-mitochondrial Ca.sup.2+ by organelle incubation
with .sup.45Ca.sup.2 followed by radioactivity quantification with
a liquid scintillation counter, measurement of cytochrome c from
release mitochondria coactivating caspases, and staining of muscle
preparations for TUNEL-positive cell nuclei. The in vivo effects of
RyCal compounds treatment on mitochondrial integrity and function
will be assessed.
[0209] Activation of Intracellular Proteases Fragments RyR1:
[0210] A direct link exists between the cytosolic Ca.sup.2+
elevations and the proteolysis of intracellular targets through the
activation of Ca.sup.2+-dependent proteases, including calpains and
caspases. Calpain activation is part of the apoptosis machinery.
Increased activation of the ubiquitous calpains has been found in
the mouse model of Duchenne muscular dystrophy (DMD), but null
mutations of muscle specific calpain causes limb girdle muscular
dystrophy 2A (LGMD2A) (Tidball et al., 2000). These findings
indicate that dysregulation of calpain activity contributes to
progression of muscle disease by disrupting normal regulatory
mechanisms and by a generalized, nonspecific increase of
proteolytic capacity. RyR1 and other components of the Ca.sup.2+
cycling machinery are targets of and cleaved by caspases and
calpains (Johnson et al., 2004; Shevchenko at al., 1998). We will
therefore investigate if Ca.sup.2+ dependent protease and/or
caspase activation result in RyR1 cleavage following strenuous
exercise and if JTV-519 by inhibiting SR Ca.sup.2+ leak can prevent
activation of unspecific proteolysis.
[0211] Histologic Changes of Skeletal Muscle from Strenuous,
Fatiguing Exercise:
[0212] Dystrophic changes from sustained exercise may result in
muscle fiber necrosis and progressive muscle wasting and weakness.
Histological analysis of skeletal muscles will include analysis for
eosinophilic hypercontracted muscle fibers, necrotic fibers,
ongoing muscle regeneration, and the proliferation of fibroblasts
within muscle tissue since the replacement of muscle tissue by
connective tissue (fibrosis) is a major cause of permanent muscle
weakness. Accordingly, RyCal compounds can be tested for inhibition
of these changes. Conventional histology techniques will allow to
assess variability in fiber size, split fibers, and centralized
nuclei.
[0213] Fiber Typing by Histochemistry in Skeletal Muscles:
[0214] Historically, the most widely used classification of fiber
types is based on the mATPase (myofibrillar adenosine
triphosphatase) activity by histochemistry which distinguishes
between type I (low activity) and type II (high activity) fibers
(Brooke et al., 1970). By characterizing the pH lability of the
mATPase, type II fibers were further subdivided into IIA and IIB
fibers. Additional fiber type characterization with physiologic,
histochemical, and ultrastructural methods has revealed: type I,
intermediate slow-twitch oxidative; type IIA, red fast-twitch
oxidative-glycolytic; and type IIB, white fast-twitch glycolytic
fibers. Quantitative histochemistry can be used to determine
mATPase, succinate dehydrogenase, and .alpha.-glycerophosphate
dehydrogenase and cross-sectional areas in MHC-based fiber type
changes in oxidative and glycolytic capacities resulting from
sustained exercise and/or RyCal compound treatment. To determine
myofiber ATPase activity, a protocol using 10 .mu.m thick frozen
sections which are preincubated for 5 mins under acidic or 12 mins
under alkaline conditions can be used. Succinate dehydrogenase
staining will allow characterizing activity and distinction between
muscle fibers. Changes in oxidative muscle fiber types and
improvement after treatment for myotonia have been reported in mice
previously (Reininghaus et al., 1988).
[0215] Fiber Typing by Immunocytochemistry in Skeletal Muscles:
[0216] To quantitate changes in myonuclear number and location and
to distinguish from nuclei of interstitial cells, morphometry by
fluorescence microscopy using stains for nuclei (DAPI) and basement
membrane (anti-laminin) will be applied. This technique will be
particularly important to precisely determine cross-sectional area
and number of nuclei in muscle fibers. Moreover, the technique
allows for combined immunocytochemistry with the following
affinity-purified antibodies: C-terminal RyR2-5029,
phospho-epitope-specific RyR1-p2844 (Wehrens et al., 2004),
calstabin1 (FKBP12) (Jayaraman et al., 1992), isoform-specific
myosin heavy chain (MHC) antibodies (Rivero et al. 1999), and
.alpha.-actinin (Ruehr et al., 2003) using a previously established
protocol with 10 .mu.m thick cryostat sections of EDL or soleus
muscles (Moschella et al., 1995). This approach will allow us to
classify myofibers and phenotypic changes according to MHC content,
metabolic activity, fiber size, RyR1 PKA phosphorylation, and
calstabin1 binding occurring from sustained exercise and/or RyCal
compound treatment. Further, the fiber type and immunocytochemistry
data will be correlated to isolated skeletal muscle function,
general histologic data, histochemistry, RyR1 single-channel
function, and mitochondria data. Using immunohistochemical
staining, fiber type-specific improvement of calstabin1 binding to
RyR1 and RyR1 PKA phosphorylation can be determined in the presence
or absence, or after RyCal compound treatment in .alpha.-actinin
positive compartments (Z-disk), and for increased calpain
expression in the myofibrillar area (Z disks; compartment
containing RyR1 channels) of muscle fibers.
[0217] Skeletal Muscle Oxidative Capacity:
[0218] Muscle oxidative activity correlates with skeletal muscle
adoption to aerobic exercise. As a general test, fast- and
slow-twitch skeletal muscle oxidative enzyme activity will be
assessed by spectrophotometric assessment of the citric acid cycle
and citrate synthase activity in muscle homogenates. Activity as
determined as the complex from coenzyme A and oxaloacetate is
expected to be increased as reported by a group using a similar
protocol (Evangelista et al., 2003). This test will be used to
confirm changes in oxidative capacity seen by histochemistry.
[0219] Improvement of Creatine Kinase (CK) Plasma Levels:
[0220] Creatine kinase (CK) and lactate dehydrogenase (LDH) plasma
concentrations (Santos et al., 2004; Thompson et al., 2004), can be
determined as indicators or muscle damage and inflammation after
exhaustive exercise and to determine effect due to RyCal compound
treatment.
[0221] RyR1 Composition and Function in White Blood Cells:
[0222] RyR1 in immune cells functions as a Ca.sup.2+ release
channel during B- or T-cell receptor-stimulated activation (Sei et
al., 2002; Kraev et al., 2003). For functional analysis, peripheral
white blood cells are isolated from mouse blood samples by
centrifugation. Leukocytes (10.sup.6/ml) are loaded with 1 .mu.m
acetoxy-methyl ester of fluo-3 (Molecular Probes, Eugene, Oreg.) by
incubation for 30 mins at 25.degree. C. and caffeine sensitivity of
intracellular Ca.sup.2+ release is tested. Composition and PKA
phosphorylation of the RyR1 channel complex will be characterized.
Investigating RyR1 in white blood cells will allow monitoring
temporal changes during sustained exercise and RyCal compound
treatment in vivo.
Genetically Modified Mice
[0223] The RyR1 macromolecular signaling complex plays a key role
in modulating activation of the channel and excitation-contraction
coupling by the sympathetic nervous system. In the RyR1 complex
mAKAP targets PKA and the phosphodiesterase PDE4D3 to the channel
and the phosphatase PP1 is targeted to the channel by the targeting
protein spinophilin. This signaling module controls PKA
phosphorylation of RyR2 at Ser2843 as part of the "fight-or-flight"
stress response. During normal exercise 2-3 of the four Ser2843 PKA
phosphorylation sites in each tetrameric RyR1 channel are
transiently PKA phosphorylated resulting in increased activity of
the RyR1 channel.
[0224] Activation of the RyR1 due to PKA phosphorylation occurs
because PKA phosphorylation decreases the binding affinity of the
stabilizing protein calstabin1 (FKBP12) for the channel resulting
in increased sensitivity of the channel to Ca.sup.2+-dependent
activation. PDE4D3 in the RyR1 macromolecular signaling complex
plays a protective role against PKA hyperphosphorylation and forms
a negative feedback loop during PKA activation. The
phosphodiesterases in the RyR1 complex by rapidly degrading local
cAMP and thereby terminating channel activation by PKA. Mice that
are deficient in PDE4D3 or calstabin1 in the RyR1 complex will be
tested for accelerated muscle fatigue. Thus, both calstabin1 and
PDE4D3 in the RyR2 complex can be thought of as being "protective"
against muscle dysfunction during excessive exercise or stress.
Thus additional components of the RyR1 macromolecular complex are
protective against fatigue as these molecules could potentially be
novel therapeutic targets and/or identify adverse pharmaceutical
agents for preventing fatigue during intense stress in
warfighters.
[0225] The muscle-specific genetic mouse model HSA.sup.LR of
myotonic dystrophy type 1 (DM1) has a DM-like phenotype.
Importantly, the HSA.sup.LR myotonic phenotype includes variable
degrees of histopathological signs of muscle degeneration and
repair, which correspond to the expression of more toxic, long or
less, toxic short repeat variants. In addition to wild-type mice,
susceptibility to muscle fatigue will be investigated using
swimming and treadmill running protocols. In a subgroup of mice,
implantable telemetry devices will be implanted two weeks before
the mice are subjected to an exercise-stress protocol as described.
Upon completion of the experiment, mice will be sacrificed and
muscles will be flash-frozen in liquid nitrogen or further
processed for histological assays. Specific muscle types are
carefully dissected under stereoscope vision and flash frozen in
liquid nitrogen or examined by histology and immunohistochemistry.
In all tissue samples, RyR1 PKA phosphorylation, levels of the
components of the RyR2 macromolecular complex including calstabin1,
PKA, RII, mAKAP, PP1, spinophilin, PDE4D3, CaMKII, and single
channel properties will be examined. The following properties are
determined: Ca.sup.2+ sensitivity of activation and inhibition,
Mg.sup.2+ inhibition, and changes from in vivo RyCal treatment. At
conclusion of each single channel experiment ryanodine will be
applied to the channel to confirm RyR identity. The HSA.sup.LR
mouse will allow to test beneficial effects of RyCal compounds in
an extreme model of genetic muscle fatigue and dystrophy.
[0226] Susceptibility to muscle fatigue can be investigated using
swimming and treadmill running protocols. Implantable telemetry
devices can be implanted two weeks before the mice are subjected to
an exercise-stress protocol. Upon completion of the experiment,
mice are sacrificed and muscles are flash-frozen in liquid
nitrogen. Upon completion of each experiment, muscles can be
dissected and will also be examined by histology and Western
blotting. In all tissue samples, RyR1 PKA phosphorylation, levels
of the components of the RyR2 macromolecular complex including
calstabin1, PKA, RII, mAKAP, PP1, spinophilin, PDE4D3, CaMKII, and
single channel properties can be examined. The following properties
can be determined: Ca.sup.2+ sensitivity of activation and
inhibition, Mg.sup.2+ inhibition, response to PKA phosphorylation.
At conclusion of each single channel experiment ryanodine is
applied to the channel to confirm RyR identity.
RyCal Compounds Prevent Muscle Fatigue and Muscle Degeneration
[0227] In certain aspects, the invention provides that RyCal
compounds can restore normal function to hyperphosphorylated RyR1.
Furthermore, use of PDE4D knockout and FKBP12 haploinsufficient
mice allows determination whether RyCal compound prevent muscle
fatigue.
[0228] The foregoing discussion established two animal models
(mouse and rat) of muscle dysfunction and fatiguing, which result
from sustained forms of exercise. The models can provide important
clues if chronic activation of the sympathetic nervous system
resulting from sustained exercise and stress cause a critical
defect in the RyR1 Ca.sup.2+ release channel. Previous experiments
in a heart failure model that results in chronic sympathetic
hyperactivity have established a critical defect in RyR1
contributing to accelerated muscle fatigue. RyCal compounds have
beneficial effects to inhibit muscle fatigue during sustained
exercise capacity, and isolated slow- and fast-twitch skeletal
muscle function in the investigated fatigue models.
Catecholamine-induced muscle fatigue has been established in rat
and mouse hearts failure models earlier and therefore we will be
able to apply similar techniques to test for muscle fatigue
following sustained animal exercise protocols.
[0229] Maintenance of muscle performance during sustained
activation of the sympathetic nervous system, for example but not
limited to combat, requires a maximal rate of intracellular SR
Ca.sup.2+ cycling. Chronic maximal stress results in permanent
activation of the sympathetic nervous system potentially causing
RyR1 hyperphosphorylation and intracellular Ca.sup.2+ leak. In
skeletal muscles, intracellular Ca.sup.2+ leak gradually causes a
myopathy characterized by significantly reduced duration and
maximal power of peak performance as well as accelerated fatigue by
additional ATP consumption of SR Ca.sup.2+ ATPase pumps that
compensate for uncontrolled SR Ca.sup.2+ leak. SR Ca.sup.2+ leak is
unique since it is a direct cause of muscle fatigue intrinsic to
myofibers which is not reversible in the acute setting. A drug like
any RyCal compound which fixes the SR Ca.sup.2+ leak by binding
calstabin1 to the channel and stabilizing the closed state even
during stress therefore helps to prevent accelerated fatigue
development and promotes longer performance despite sustained
stress (Wehrens, 2005). The pharmacotherapy is unique since it
targets a central fatigue mechanism and potentially prevents toxic
effects of intracellular Ca.sup.2+ leak. Moreover the molecular
mechanism of this pharmocotherapy is unique, since it treats a
specific defect contributing to muscle fatigue and since the
mechanism of RyCal action is stabilization of normal RyR1 channel
closure by increasing the calstabin1 binding affinity, which is
distinct from historical approaches that block ion channel
function.
[0230] More recently, SR Ca.sup.2+ leak was documented in myofibers
following intense exercise and in a model of muscular dystrophy,
(Wang et al., 2005), possibly due to defective skeletal ryanodine
receptors (RyR1 s). Also, chronic activation of the sympathetic
nervous system (SNS) in the context of heart failure promotes
intrinsic skeletal muscle (SM) fatigue due to depletion of the
phosphodiesterease PDE4D3 from the RyR1 complex, RyR1 PKA
hyperphosphorylation at Serine 2844, calstabin1 depletion from the
RyR1 complex, and a gain-of-function channel defect (Reiken et al.,
2003). RyR1 dysfunction in the skeletal muscle leads to altered
local subcellular Ca.sup.2+ release events and impaired global
calcium transients (Ward et al., 2003). In the context of chronic
exercise, there is evidence indicating that changes in the RyR1
macromolecular complex, namely depletion of PDE4D3 from the RyR1
complex, RyR1 PKA hyperphosphorylation at Serine 2844, and
calstabin1 depletion from the RyR1 complex are related in a
time-dependent and activity-dependent manner with repeated intense
exercise in a mouse model. These biochemical changes in the RyR1
macromolecular complex regulation and function are stable following
prolonged exercise and recover slowly over days to weeks. Thus RyR1
Ca.sup.2+ leak limits peak muscle performance and mediates muscle
damage during prolonged, stressful exercise.
Molecular Mechanisms of Muscle Fatigue
[0231] The hypotheses that muscle fatigue is due to lactic acid
accumulation in the cytoplasm and potassium ion accumulation in
T-tubules have largely been set aside and attention has shifted to
the study of metabolic and mitochondrial regulation and signaling
pathways during chronic exercise (Lin, Wu et al. 2002; Wu, Kanatous
et al. 2002; Wang, Zhang et al. 2004). These alternative
explanations, while important, are unlikely to directly address the
underlying abnormalities in ECC observed in fatigued muscle
(Berchtold, Brinkmeier et al. 2000). Described herein is the
regulation of the skeletal calcium release channel, RyR1, during
chronic or high intensity exercise. The remodeling of the RyR1
macromolecular complex during chronic exercise, consisting of PKA
hyperphosphorylation at Ser2844, PDE4D3 depletion, and calstabin1
depletion, likely plays a role in determining muscle fatigue during
chronic exercise.
[0232] Exercise promotes numerous positive effects on an organism,
from improvement in cardiovascular performance to increased glucose
uptake and normalization of fuel metabolism (Goodyear and Kahn
1998; Pollock, Franklin et al. 2000). In heart failure, light
exercise training has been shown to improve skeletal muscle
strength and reduce fatigue, perhaps through adaptation to more
aerobic muscle properties (Minotti, Johnson et al. 1990; Lunde,
Sjaastad et al. 2001; Meyer 2006). On the other hand, high
intensity exercise, such as that performed by a marathon runner or
a long distance cyclist results in significant muscle damage and
can impair task performance for days or weeks after a single event
(O'Reilly, Warhol et al. 1987; Balnave and Thompson 1993;
Komulainen and Vihko 1994).
[0233] Described herein is also a mouse model of intense
physiological exercise to examine the changes in RyR1 function and
ECC experienced by elite athletes, soldiers, or others under
intense stressful activity. By combining daily swimming with level
treadmill running assays, a physiological exercise regimen was
constructed that did not exclusively involve isometric or eccentric
contraction of the hind limb. While this resulted in less dramatic
evidence of exercise-induced muscle damage than pure eccentric
contractions, the data presented are more readily generalized.
[0234] Biochemical changes were identified in the RyR1
macromolecular complex consistent with leaky calcium release
channels. Single channel bilayer data confirmed a leaky phenotype
of the RyR1 channels from chronically exercised hind limb muscle,
with elevated open probabilities in the chronically exercised group
at resting calcium levels compared to sedentary controls. In two
mouse genetic models replicating aspects of the biochemical changes
in the RyR1 complex, namely muscle-specific deficiency of
calstabin1 (cal1-/-) and deficiency of PDE4D3 (PDE4D-/-), exercise
defects were identified. The role of calstabin1 depletion was
assessed in another way by pharmacologically rebinding the
stabilizing subunit to RyR1 with the Ca.sup.2+ channel stabilizer
S107. Calstabin1 rebinding to RyR1, induced by S107 resulted in
improved exercise capacity, as measured by treadmill failure times,
over the same 21 day time course that depleted calstabin1 in the
vehicle treated mice. The lack of an effect of S107 on cal1-/- mice
provides evidence that the molecular mechanism of S107 is indeed
through calstabin1 rebinding to RyR1.
[0235] In vitro fatigue protocols on intact isolated muscles suffer
from the limitation that force declines are largely limited by
hypoxia (Zhang, Bruton et al. 2006). Therefore, single FDB muscle
fibers were isolated from sedentary and chronically exercised mice
with and without S107 for assessment of the decline in tetanic
Ca.sup.2+ during fatigue. Exercised fibers with calstabin rebound
were relatively protected against fatigue (FIG. 48). The effect of
S107 did not appear to be due to a shift in the fiber kinetics to
slower calcium cycling.
[0236] S107 corrected the leak in RyR1 from chronically exercised
mice as measured in a lipid bilayer at low resting Ca.sup.2+
levels. Ca.sup.2+ leak resulting from the overactive skeletal
ryanodine receptors was not directly visualized in isolated muscle
fibers, as calcium sparks were infrequent under all conditions
tested, which is consistent with most reports that sparks in
skeletal muscle are rare except under certain highly pathological
conditions such as hypoosmotic shock or muscular dystrophy (Isaeva,
Shkryl et al. 2005; Rios 2005; Wang, Weisleder et al. 2005).
[0237] Numerous hypotheses present themselves for how alterations
in RyR1 Ca.sup.2+ leak could result in muscle damage. These data do
not identify one muscle damage pathway solely responsible for the
physiological effects seen, however, they implicate a role for
calpain activation during chronic, and/or high intensity exercise.
Several groups have demonstrated that calpain activation is a major
mechanism for exercise-induced muscle damage (Belcastro 1993;
Spencer and Mellgren 2002). As described herein, calpain activation
in isolated EDL muscle was elevated following chronic exercise, but
reduced by treatment with S107, suggesting that correction of the
leaky RyR1 may protect against calpain activation (FIG. 50). With a
potential contribution of other Ca.sup.2+-dependent pathways such
as caspases, calmodulin, or calmodulin-dependent kinases to the
damage induced by leaky ryanodine receptors, the present data
suggest a mechanism by which local elevation of cytosolic Ca.sup.2+
could lead to damage. The hypothesis that ryanodine
receptor-induced leak can cause muscle damage was further supported
by evidence of reduced muscle damage, as measured by serum creatine
kinase, in the S107 treated mice (FIG. 50). The data described
herein shows that changes in the RyR1 macromolecular complex
produces a leaky phenotype during chronic, high intensity exercise
which impairs exercise performance.
Neuropathies
[0238] In one aspect, the present invention is directed to
compositions and methods for the treatment and prevention of
neuropathies. The term "neuropathy" as used herein refers to
conditions characterized by damage to the nerves, including, but
not limited to, damage caused by infections, inflammatory
processes, exposure to toxins, treatment with drugs, nutritional
deficiency, trauma, pressure on a nerve, neuronal death, neuronal
degeneration, and heritable conditions. Although the term
"neuropathy" is most frequently used to refer to conditions
characterized damage to the peripheral nerves, i.e. "peripheral
neuropathies", as used herein, the term "neuropathy" includes both
peripheral neuropathies and neuropathies affecting nerves of the
central nervous system, i.e. "central neuropathies."
[0239] There are various sub-classifications of neuropathies. For
example, neuropathies may be classified as either peripheral or
central, as either acute or chronic, or as either demyelinating or
axonal. Neuropathies may also be classified according to the number
of nerves that they affect. A neuropathy may involve damage to only
a single nerve or nerve group (referred to as mononeuropathies) or
may affect multiple nerves (polyneuropathies).
[0240] Peripheral neuropathies may be caused by hereditable
disorders, systemic or metabolic disorders, dietary deficiencies,
exposure to toxic substances, treatment with drugs, infection,
inflammatory response, autoimmune diseases, and multiple other
factors. Also, many peripheral neuropathies are of unknown
etiology.
[0241] Examples of hereditable peripheral neuropathies include, but
are not limited to, Charcot-Marie-Tooth disease (CMT) and
Friedreich's ataxia.
[0242] Examples of peripheral neuropathies caused by systemic or
metabolic disorders include, but are not limited to diabetic
neuropathy.
[0243] Examples of peripheral neuropathies caused by dietary
deficiencies include, but are not limited to neuropathy caused by
vitamin B-12 deficiency, and neuropathy caused by thiamine
deficiency.
[0244] Examples of peripheral neuropathies caused by exposure to
toxic substances include, but are not limited to neuropathy caused
by excessive alcohol use ("alcoholic neuropathy"), neuropathy
caused by memia (such as in kidney failure patients), neuropathy
caused by arsenic, neuropathy caused by nitrous oxide, neuropathy
caused by industrial agents especially solvents, neuropathy caused
by heavy metal exposure (such as lead, arsenic, mercury, and the
like).
[0245] Examples of peripheral neuropathies caused by infectious
agents and/or inflammatory or autoimmune processes include, but are
not limited to, neuropathies caused by GullainBarre syndrome,
polyarteritis nodosa, sarcoidosis, systemic lupus erythematosus,
rheumatoid arthritis, sjogren syndrome, HIV infection, syphhilis
infection, herpes infection, hepatitis infection, colorado tick
fever infection, diptheria infection, leprosy, Lyme disease, and
amyloidosis.
[0246] Examples of peripheral neuropathies caused by drugs include,
but are not limited to neuropathy caused by amiodarone,
hydralazine, perhexiline, chemotherapeutic drugs, vincristine,
cisplatin, metronidazole (Flagy1), nitrofurantoin, thalidomide, INH
(isoniazid), Dapsone, anticonvulsants, Phenytoin, Disulfiram,
zidovudine, retrovir, AZT, didanosine (Videx), stavudine (Zerit),
zalcitabine (Hivid), ritonavir (Norvir), amprenavir (Agenerase),
lovastatin (Mevacor), indapamid (Lozol), gemfibrozil (Lopid).
[0247] Other miscellaneous causes or peripheral neuropathy include,
but are not limited to ischemia, prolonged exposure to cold
temperature, prolonged pressure on, or compression of a nerve, and
trauma.
[0248] Peripheral neuropathies are characterized by damage to the
either the sensory, motor, or autonomic peripheral nerves. The
symptoms and effects of peripheral neuropathies depend on the types
of nerves affected. Damage to such nerves can result in one or more
of pain (neuropathic pain), loss of sensation, and loss of muscular
control, abnormal blood pressure, abnormal heart function,
digestion problems, and the like.
[0249] Damage to sensory fibers may result in changes in sensation,
burning sensations, nerve pain (neuralgia, neuropathic pain),
tingling, numbness, inability to determine joint position, and
incoordination. Damage to the motor fibers may affect muscle
control and can cause weakness, cramps, loss of muscle bulk, and
loss of dexterity, paralysis, muscle atrophy, Muscle twitching
(fasciculation), difficulty breathing or swallowing, falling. The
autonomic nerves control involuntary and semi-voluntary functions,
such as control of the internal organs, control of breathing, and
blood pressure. Damage to autonomic nerves may cause, inability to
regulate blood pressure, respiratory problems, problems of the
digestive system (including nausea, vomiting, abdominal bloating,
early satiety, diarrhea, constipation, unintentional weight loss),
problems with the genitourinary system, (such as urinary
incontinence, other bladder-function disorders, and male
impotence.
[0250] Examples of specific nerves that may be affected in
peripheral neuropathies include, but are not limited to, the
axillary nerve, the brachial plexus, the peroneal nerve, the distal
median nerve, the facial nerves palsy, the femoral nerves, the
radial nerves, the sciatic nerve, the tibial nerves, and the ulnar
nerves.
[0251] Examples of central neuropathies include, but are not
limited to, vestibular neuropathies, optic neuropathies, optic
nerve neuropathies, and retinal neuropathies. Other types of
central neuropathy are known to those of skill in the art, and are
encompassed by the present invention.
Seizures
[0252] The term "seizure" as used herein includes epileptic
seizures and non-epileptic seizure. Epileptic seizures result from,
temporary abnormal electrical activity in the brain. They can
manifest as an alterations tonic or chronic movements, convulsions,
sudden and involuntary contraction of a group of muscles,
involuntary changes in body movement or function, numbness,
alterations in mental state, alterations in sensation, alterations
in awareness, changes in behavior, temporary loss of memory, visual
disturbances, and various other symptoms. Symptoms experienced by a
person during a seizure depend on where in the brain the
disturbance in electrical activity occurs.
[0253] There are various different types of seizures, all of which
are within the scope of the present invention. For example,
seizures may be epileptic or non-epileptic, as described below.
Seizures may also be classified according to whether the source of
the seizure within the brain is localized (partial or focal onset
seizures) or distributed (generalized seizures).
[0254] Partial seizures are further divided on the extent to which
consciousness is affected. If consciousness is unaffected the
seizure is referred to as a simple partial seizure. If
consciousness is affected, the seizure is referred to as a complex
partial seizure. A partial seizure may also spread within the
brain--a process known as secondary generalization.
[0255] Generalized seizures are divided according to the effect on
the body but all involve loss of consciousness. These include
absence, myoclonic, clonic, tonic, tonic-clonic, and atonic
seizures. In the past, seizures have also been classified as "petit
mal", "grand mal", "Jacksonian", "psychomotor", and "temporal-lobe"
seizures.
[0256] Epilepsy is a chronic neurological condition characterized
by recurrent unprovoked seizures. These seizures involve abnormal,
rhythmic discharges of cortical neurons. Epilepsy may be
symptomatic or idiopathic. Symptomatic epilepsies are caused by
structural or metabolic abnormality in the brain, which may be the
result of factors such as genetic disorders (such as tuberous
sclerosis or ring chromosome 20 syndrome), stroke, head injury,
bacterial or viral encephalitis, alcohol use. There are several
syndromes that associated with epilepsy, including, but not limited
to, infantile spasms (West syndrome), benign childhood epilepsy
with centro-temporal spikes (or benign rolandic epilepsy), benign
childhood epilepsy with occipital paroxysms, juvenile myoclonic
epilepsy (JME), temporal lobe epilepsy, frontal lobe epilepsy,
Lennox-Gastaut syndrome, occipital lobe epilepsy, and fetal alcohol
spectrum disorder (F ASD). Idiopathic seizures are those for which
no specific cause has been identified.
[0257] Certain triggers or environmental factors or can lead to an
increased likelihood of seizures in subjects with epilepsy.
Examples of such triggers include, but are not limited to, sleep,
the transition between sleep and wakefulness, tiredness, illness,
constipation, menstruation, stress, and alcohol consumption. It
should also be noted that, even in epileptic subjects, seizures may
be triggered by some of the same specific events that cause
"provoked" seizures in non-epileptic subjects.
[0258] Non-epileptic seizures appear outwardly similar to epileptic
seizures but do not involve abnormal, rhythmic discharges of
cortical neurons. Non-epileptic seizures are typically provoked by
either physiological or psychological conditions. Seizures caused
by psychological conditions are referred to as "psychogenic"
non-epileptic seizures.
[0259] Causes of non-epileptic or "provoked" seizures include, but
are not limited to, head injury, intoxication with drugs, drug
toxicity (for example aminophylline or local anaesthetic toxicity,
drugs that lower the seizure threshold (such as tricyclic
antidepressants), infection (such as encephalitis or meningitis),
fever leading to febrile convulsions, metabolic disturbances such
as hypoglycaemia or hypoxia, withdrawal from drugs (such as
anticonvulsants, sedatives, alcohol, barbiturates, and
benzodiazepines), brain tumors, other brain lesions, eclampsia
during pregnancy, photosensitivity, flashing or flickering lights
and electroconvulsive therapy (ECT). It should be noted that the
above stimuli may also trigger epileptic seizures.
Cognitive Disorders
[0260] In another aspect, the present invention is directed to the
treatment and prevention of cognitive disorders, and also to
methods and compositions for improvement of cognitive function more
generally, even in the absence of a specific cognitive disorder.
For example, improvement of cognitive function to combat the normal
cognitive decline associated with aging, or to enhance cognitive
function for other reasons, is encompassed by the present
invention.
[0261] The terms "cognitive function" and "cognitive process" as
used herein, include the mental processes of attention, learning
and memory, perception, language skills, problem solving skills,
and other type of cognitive function known to those of skill in the
art. The terms "cognitive disorder," "cognitive disease," and
"cognitive condition," as used herein, refer to situations in which
processes are disrupted or abnormal. The term "cognitive disorder,"
as used herein encompasses all of the cognitive disorders described
below and also all other cognitive disorders known to those of
skill in the art. Types of cognitive disorders that are within the
scope of the invention include, but are not limited to, dementias,
delirium, amnesias, post-traumatic stress disorder and
stress-induced cognitive dysfunction.
[0262] The term "dementia" as used herein refers to decline in
cognitive function due to damage or disease in the brain or central
nervous system beyond that which might be expected from normal
aging. Dementias typically affect cognitive functions such as
learning, memory, attention, language skills, and problem solving
skills. Types and causes of dementia include, but are not limited
to, chronic diseases such as cancer, Alzheimer's disease, vascular
dementia (also known as multi-infarct dementia), Binswanger's
disease, dementia with Lewy bodies (DLB), alcohol-induced
persisting dementia, frontotemporal lobar degenerations (FTLD),
Pick's disease, frontotemporal dementia (or frontal variant FTLD),
semantic dementia (or temporal variant FTLD), progressive
non-fluent aphasia, Creutzfeldt-lakob disease, Huntington's
disease, Parkinson's disease, and AIDS dementia complex.
[0263] Other types of cognitive disorders that may be treated with
the methods and compositions of the present invention include the
various attention disorders. Attention Deficit/Hyperactivity
Disorder (ADHD; ADH is also referred to as Attention-deficit
syndrome (ADS)) is a neurological disorder initially appearing in
childhood which manifests itself with symptoms such as
hyperactivity, forgetfulness, poor impulse control, and
distractibility. In neurological terms, ADHD is currently
considered to be a persistent and chronic syndrome for which no
medical cure is available. ADHD is believed to affect between 3-5%
of the United States population, including both children and
adults. ADH D is sometimes referred to as ADD when only
inattentiveness and distractibility are problematic. ADHD can be
classified into three subtypes: predominantly inattentive
(sometimes referred to as ADD), predominantly
hyperactive-impulsive, and combined. Those presenting impairing
symptoms of ADHD who do not fully fit the criteria for any of the
three subtypes can be diagnosed with "ADHD Not Otherwise
Specified." The symptoms of ADHD are given the name "Hyperkinetic
disorders". When a conduct disorder is present, the condition is
referred to as "Hyperkinetic conduct disorder". All of the above
conditions are within the scope of the present invention.
[0264] In one embodiment, the cognitive disorder is not Alzheimer's
Disease. In another embodiment, the cognitive disorder is not
memory loss. In another embodiment, the cognitive disorder is not
age-dependent memory loss.
Prevention and Treatment
[0265] In one embodiment, the present invention provides
compositions and methods that are useful for treating and/or
preventing conditions affecting the nervous system, such as
neuropathies, seizures and cognitive disorders.
[0266] In certain embodiments, the compositions and methods of the
present invention may be used preventively in subjects who are not
yet suffering from neuropathies, seizures or cognitive disorders,
but whom exhibit one or more "risk factors" or are otherwise
predisposed to the development of neuropathies, seizures or
cognitive disorders.
Subjects
[0267] In preferred embodiments, the compositions described herein
are administered therapeutically or prophylactically to subjects
who are suffering from, or at risk of developing a disease,
disorder or condition affecting the nervous system, such as a
neuropathy, seizures or a cognitive disorder. Such a subject may be
any animal. For example, in one embodiment, the subject is a
mammal. Examples of mammals that may be treated using the methods
and compositions of the invention include, but are not limited to,
primates, rodents, ovine species, bovine species, porcine species,
equine species, feline species and canine species. In preferred
embodiments the subjects are human.
[0268] In preferred embodiments, the methods and compositions of
the invention may be used to treat or prevent a disease, disorder
or condition affecting the nervous system, such as a neuropathy,
seizures or a cognitive disorder, in a subject having a mutation in
a ryanodine receptor gene, such as a mutation that results in
defective functioning of the ryanodine receptor, such as an
increased open probability or "leakiness" of the ryanodine
receptor. In other embodiments, the "subjects" of the present
invention may also be in vitro or in vivo systems, including,
without limitation, isolated or cultured cells or tissues, in vitro
assay systems.
[0269] Throughout the specifications, groups and substituent's
thereof may be chosen to provide stable moieties and compounds.
[0270] The present invention provides compounds that are capable of
treating and preventing disorders and diseases associated with the
RyR receptors that regulate calcium channel functioning in cells.
More particularly, the present invention provides compounds that
are capable of treating or preventing a leak in RyR channels.
"Disorders and diseases associated with the RyR receptors" means
disorders and diseases that can be treated and/or prevented by
modulating the RyR receptors that regulate calcium channel
functioning in cells. "Disorders and diseases associated with the
RyR receptors" include, without limitation, cardiac disorders and
diseases, skeletal muscular disorders and diseases, cognitive
disorders and diseases, malignant hyperthermia, diabetes, and
sudden infant death syndrome. Cardiac disorder and diseases
include, but are not limited to, irregular heartbeat disorders and
diseases; exercise-induced irregular heartbeat disorders and
diseases; sudden cardiac death; exercise-induced sudden cardiac
death; congestive heart failure; chronic obstructive pulmonary
disease; and high blood pressure. Irregular heartbeat disorders and
diseases include and exercise-induced irregular heartbeat disorders
and diseases include, but are not limited to, atrial and
ventricular arrhythmia; atrial and ventricular fibrillation; atrial
and ventricular tachyarrhythmia; atrial and ventricular
tachycardia; catecholaminergic polymorphic ventricular tachycardia
(CPVT); and exercise-induced variants thereof. Skeletal muscular
disorder and diseases include, but are not limited to, skeletal
muscle fatigue, exercise-induced skeletal muscle fatigue, muscular
dystrophy, bladder disorders, and incontinence. Cognitive disorders
and diseases include, but are not limited to, Alzheimer's Disease,
forms of memory loss, and age-dependent memory loss.
Compounds
[0271] In one embodiment, the present invention provides a method
which comprises administering compounds of Formula I:
##STR00002##
wherein, n is 0, 1, or 2; q is 0, 1, 2, 3, or 4; each R is
independently selected from the group consisting of H, halogen,
--OH, --NH.sub.2, --NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3,
--N.sub.3, --SO.sub.3H, --S(.dbd.O).sub.2alkyl, --S(.dbd.O)alkyl,
--OS(.dbd.O).sub.2CF.sub.3, acyl, --O-acyl, alkyl, alkoxyl,
alkylamino, alkylarylamino, alkylthio, cycloalkyl, alkylaryl, aryl,
heteroaryl, heterocyclyl, heterocyclylalkyl, alkenyl, alkynyl,
(hetero-)arylthio, and (hetero-)arylamino; wherein each acyl,
--O-acyl, alkyl, alkoxyl, alkylamino, alkylarylamino, alkylthio,
cycloalkyl, alkylaryl, aryl, heteroaryl, heterocyclyl,
heterocyclylalkyl, alkenyl, alkynyl, (hetero-)arylthio, and
(hetero-)arylamino may be optionally substituted; R.sub.1 is
selected from the group consisting of H, oxo, alkyl, alkenyl, aryl,
alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl; wherein each
alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and
heterocyclyl may be optionally substituted; R.sub.2 is selected
from the group consisting of H, --C(.dbd.O)R.sub.5,
--C(.dbd.S)R.sub.6, --SO.sub.2R.sub.7, --P(.dbd.O)R.sub.8R.sub.9,
--(CH.sub.2).sub.m--R.sub.10, alkyl, aryl, alkylaryl, heteroaryl,
cycloalkyl, cycloalkylalkyl, and heterocyclyl; wherein each alkyl,
aryl, alkylaryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and
heterocyclyl may be optionally substituted; R.sub.3 is selected
from the group consisting of H, --CO.sub.2Y, --C(C.dbd.O)NHY, acyl,
--O-acyl, alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl,
and heterocyclyl; wherein each acyl, alkyl, alkenyl, aryl,
alkylaryl, cycloalkyl, heteroaryl, and heterocyclyl may be
optionally substituted; and wherein Y is selected from the group
consisting of H, alkyl, aryl, alkylaryl, cycloalkyl, heteroaryl,
and heterocyclyl, and wherein each alkyl, aryl, alkylaryl,
cycloalkyl, heteroaryl, and heterocyclyl may be optionally
substituted; R.sub.4 is selected from the group consisting of H,
alkyl, alkenyl, aryl, alkylaryl, cycloalkyl, heteroaryl, and
heterocyclyl; wherein each alkyl, alkenyl, aryl, alkylaryl,
cycloalkyl, heteroaryl, and heterocyclyl may be optionally
substituted; R.sub.5 is selected from the group consisting of
--NR.sub.15R.sub.16, --(CH.sub.2).sub.tNR.sub.15R.sub.16,
--NHNR.sub.15R.sub.16, --NHOH, --OR.sub.15,
--C(.dbd.O)NHNR.sub.15R.sub.16, --CO.sub.2R.sub.15,
--C(.dbd.O)NR.sub.15R.sub.16, --CH.sub.2X, acyl, alkyl, alkenyl,
aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl,
heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkyl,
alkenyl, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl,
heterocyclyl, and heterocyclylalkyl may be optionally substituted,
and wherein t is 1, 2, 3, 4, 5, or 6; R.sub.6 is selected from the
group consisting of --OR.sub.15, --NHNR.sub.15R.sub.16, --NHOH,
--NR.sub.15R.sub.16, --CH.sub.2X, acyl, alkenyl, alkyl, aryl,
alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl,
and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl,
alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl,
and heterocyclylalkyl may be optionally substituted; R.sub.7 is
selected from the group consisting of --OR.sub.15,
--NR.sub.15R.sub.16, --NHNR.sub.15R.sub.16, --NHOH, --CH.sub.2X,
alkyl, alkenyl, alkynyl, aryl, alkylaryl, cycloalkyl,
cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl;
wherein each alkyl, alkenyl, alkynyl, aryl, alkylaryl, cycloalkyl,
cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl
may be optionally substituted; R.sub.8 and R.sub.9 independently
are selected from the group consisting of OH, acyl, alkenyl,
alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl,
cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl;
wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl,
alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl,
and heterocyclylalkyl may be optionally substituted; R.sub.10 is
selected from the group consisting of --NR.sub.15R.sub.16, OH,
--SO.sub.2R.sub.11, --NHSO.sub.2R.sub.11, C(.dbd.O)(R.sub.12),
NHC.dbd.O(R.sub.12), --OC.dbd.O(R.sub.12), and
--P(.dbd.O)R.sub.13R.sub.14; R.sub.11, R.sub.12, R.sub.13, and
R.sub.14 independently are selected from the group consisting of H,
OH, NH.sub.2, --NHNH.sub.2, --NHOH, acyl, alkenyl, alkoxyl, alkyl,
alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl,
heteroaryl, heterocyclyl, and heterocyclylalkyl; wherein each acyl,
alkenyl, alkoxyl, alkyl, alkylamino, aryl, alkylaryl, cycloalkyl,
cycloalkylalkyl, heteroaryl, heterocyclyl, and heterocyclylalkyl
may be optionally substituted; X is selected from the group
consisting of halogen, --CN, --CO.sub.2R.sub.15,
--C(.dbd.O)NR.sub.15R.sub.16, --NR.sub.15R.sub.16, --OR.sub.15,
--SO.sub.2R.sub.7, and --P(.dbd.O)R.sub.8R.sub.9; and R.sub.15 and
R.sub.16 independently are selected from the group consisting of H,
acyl, alkenyl, alkoxyl, OH, NH.sub.2, alkyl, alkylamino, aryl,
alkylaryl, cycloalkyl, cycloalkylalkyl, heteroaryl, heterocyclyl,
and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl,
alkylamino, aryl, alkylaryl, cycloalkyl, cycloalkylalkyl,
heteroaryl, heterocyclyl, and heterocyclylalkyl may be optionally
substituted; and optionally R.sub.15 and R.sub.16 together with the
N to which they are bonded may form a heterocycle which may be
substituted; the nitrogen in the benzothiazepine ring may
optionally be a quaternary nitrogen; and enantiomers,
diastereomers, tautomers, pharmaceutically acceptable salts,
hydrates, solvates, complexes, and prodrugs thereof;
[0272] provided that when q is 0 and n is 0, then R.sub.2 is not H,
Me, Et, --C(C.dbd.O)NH.sub.2, --C(C.dbd.O)NHPh,
--C(.dbd.S)NH-nButyl, --C(C.dbd.O)NHC(.dbd.O)CH.sub.2Cl,
--C(C.dbd.O)H, --C(C.dbd.O)Me, --C(C.dbd.O)Et,
--C(C.dbd.O)CH.dbd.CH.sub.2, --S(.dbd.O).sub.2Me,
--S(.dbd.O).sub.2Et, --C(C.dbd.O)OC(CH.sub.3).sub.3, or
9-.beta.-D-ribofuranosyl-9H-purin-6-yl or --C(C.dbd.O)Ph;
[0273] further provided that when q is 0 and n is 1 or 2, then
R.sub.2 is not H, --C(C.dbd.O)Me, --C(C.dbd.O)Et,
--S(.dbd.O).sub.2Me, --S(.dbd.O).sub.2Et or
--C(C.dbd.O)OC(CH.sub.3).sub.3;
[0274] further provided that when q is 1, and R is Me, Cl, CN or F
at the 6 position of the benzothiazepine ring, or Br at position 7
of the benzothiazepine ring, then R.sub.2 is not H, Me,
--C(C.dbd.O)H, --C(C.dbd.O)Me, --C(C.dbd.O)Et, --C(C.dbd.O)Ph,
--S(.dbd.O).sub.2Me, or --S(.dbd.O).sub.2Et;
[0275] further provided that when q is 1, n is 0, and R is OH,
C.sub.1-C.sub.3 alkoxyl at the 7 position of the benzothiazepine
ring, then R.sub.2 is not H, --C(C.dbd.O)CH.dbd.CH.sub.2,
--C(C.dbd.O)CH.sub.2Br, --(CH.sub.2).sub.3-4-benzylpiperidine,
##STR00003##
[0276] further provided that when q is 0, n is 0 or 2, R.sub.1 is H
or oxo, R.sub.3 is H or Me and R.sub.4 is H, then R.sub.2 is not
--C.dbd.ONHPh, --C.dbd.ONHCOCH.sub.2Cl, --C.dbd.ONH.sub.2,
--C.dbd.ONH(n-Bu), --C.dbd.S(NHPh), --C.dbd.S(NHCOCH.sub.2Cl),
--C.dbd.S(NH.sub.2), --C.dbd.SNH(n-Bu),
--CH.sub.2CH.sub.2N(Me).sub.2, --CH.sub.2CH.sub.2NH.sub.2 or
--C.dbd.OCHCl.sub.2;
[0277] further provided that when q is 2, each R is methoxy at
positions 7 and 8 of the benzothiazepine ring, R.sub.3 and R.sub.4
are each H and n is 0 or 2, then R.sub.1 is not methyl,
--CH.sub.2Ph or 3,4-dimethoxybenzyl, and R.sub.2 is not
--C(C.dbd.O)Me;
[0278] further provided that when q is 0, R.sub.1, R.sub.2 and
R.sub.4 are each H, then R.sub.3 is not H or CH.sub.3;
[0279] further provided that when q is 0, R.sub.2 is H,
--CH.sub.2C(.dbd.O)OCH.sub.3, --CH.sub.2C(.dbd.O)NH.sub.2,
--C(C.dbd.O)--C.sub.6H.sub.4--Cl, --CH.sub.2--C.sub.6H.sub.4--Cl,
--(CH.sub.2).sub.3-morpholino,
--(CH.sub.2).sub.3-4-methylpiperazino,
--(CH.sub.2).sub.2--C(C.dbd.O)OCH.sub.3,
2,2',3,3'-tetrahydro-4(5H)-1,4 benzothiazepine, or --CH.sub.2-Ph,
R.sub.3 and R.sub.4 are either H or CH.sub.3 but not both CH.sub.3,
then R.sub.1 is not oxo;
[0280] further provided that when q is 2, each R is methoxy at
positions 7 and 8 or 7 and 9 of the benzothiazepine ring, R.sub.1,
R.sub.2 and R.sub.4 are each H and n is 0, then R.sub.3 is not
H;
[0281] further provided that when q is 0, R.sub.1, R.sub.3 and
R.sub.4 are each H and n is 0, then R.sub.2 is not methyl,
benzotriazolylmethyl, 4-methoxybenzyl, Ph-C.ident.C--CH.sub.2--,
4-chlorobenzyl, ethyl, pentyl,
--CH.sub.2P(O)(OCH.sub.2CH.sub.3).sub.2, Ph-CO--CH.sub.2CH.sub.2--,
C(.dbd.O)CH.dbd.CH.sub.2 and C(.dbd.O)CH.sub.2Br; and
[0282] further provided that when q is 1, R is CH.sub.3 at position
9 of the benzothiazepine ring, R.sub.1, R.sub.3 and R.sub.4 are
each H and n is 0, then R.sub.2 is not methyl,
benzotriazolylmethyl, pentyl,
--CH.sub.2P(O)(OCH.sub.2CH.sub.3).sub.2 or 4-methoxybenzyl.
[0283] In one embodiment, the present invention provides compounds
of Formula I, as described above, with the proviso that said
compound is not S24 or S68.
[0284] In one embodiment, the present invention provides compounds
of Formula I-a:
##STR00004##
wherein: n is 0, 1, or 2; q is 0, 1, 2, 3, or 4; each R is
independently selected from the group consisting of H, halogen,
--OH, --NH.sub.2, --NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3,
--N.sub.3, --SO.sub.3H, --S(.dbd.O).sub.2alkyl, --S(.dbd.O)alkyl,
--OS(.dbd.O).sub.2CF.sub.3, acyl, alkyl, alkoxyl, alkylamino,
alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino,
alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino may be substituted or unsubstituted; R.sub.2 is
selected from the group consisting of H, --C.dbd.O(R.sub.5),
--C.dbd.S(R.sub.6), --SO.sub.2R.sub.7, --P(.dbd.O)R.sub.8R.sub.9,
--(CH.sub.2).sub.m--R.sub.10, alkyl, aryl, heteroaryl, cycloalkyl,
cycloalkylalkyl, and heterocyclyl; wherein each alkyl, aryl,
heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl may be
substituted or unsubstituted; R.sub.5 is selected from the group
consisting of --NR.sub.15R.sub.16, --NHNR.sub.15R.sub.16, --NHOH,
--OR.sub.15, --C(.dbd.O)NHNR.sub.15R.sub.16, --CO.sub.2R.sub.15,
--C(.dbd.O)NR.sub.15R.sub.16, --CH.sub.2X, acyl, alkyl, alkenyl,
alkynyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl; wherein each acyl, alkyl, alkenyl, alkynyl,
aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl may be substituted or unsubstituted; R.sub.6 is
selected from the group consisting of --OR.sub.15,
--NHNR.sub.15R.sub.16, --NHOH, --NR.sub.15R.sub.16, --CH.sub.2X,
acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl,
alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl may be substituted or unsubstituted; R.sub.7 is
selected from the group consisting of H, --OR.sub.15,
--NR.sub.15R.sub.16, --NHNR.sub.15R.sub.16, --NHOH, --CH.sub.2X,
alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, and heterocyclylalkyl; wherein each alkyl, alkenyl,
alkynyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl may be substituted or unsubstituted; R.sub.8 and
R.sub.9 independently are selected from the group consisting of
--OH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each
acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be
substituted or unsubstituted; R.sub.10 is selected from the group
consisting of --NR.sub.15R.sub.16, OH, --SO.sub.2R.sub.11,
--NHSO.sub.2R.sub.11, --C(.dbd.O)R.sub.12, --NH(C.dbd.O)R.sub.12,
--O(C.dbd.O)R.sub.12, and --P(.dbd.O)R.sub.13R.sub.14; m is 0, 1,
2, 3, or 4; R.sub.11, R.sub.12, R.sub.13, and R.sub.14
independently are selected from the group consisting of H, OH,
NH.sub.2, --NHNH.sub.2, --NHOH, acyl, alkenyl, alkoxyl, alkyl,
alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl,
alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl may be substituted or unsubstituted; X is
selected from the group consisting of halogen, --CN,
--CO.sub.2R.sub.15, --C(.dbd.O)NR.sub.15R.sub.16,
--NR.sub.15R.sub.16, --OR.sub.15, --SO.sub.2R.sub.7, and
--P(.dbd.O)R.sub.8R.sub.9; and R.sub.15 and R.sub.16 independently
are selected from the group consisting of H, acyl, alkenyl,
alkoxyl, OH, NH.sub.2, alkyl, alkylamino, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each
acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be
substituted or unsubstituted; and optionally R.sub.15 and R.sub.16
together with the N to which they are bonded may form a heterocycle
which may be substituted or unsubstituted; the nitrogen in the
benzothiazepine ring may be optionally a quaternary nitrogen; and
enantiomers, diastereomers, tautomers, pharmaceutically acceptable
salts, hydrates, solvates, complexes, and prodrugs thereof;
[0285] provided that when q is 0 and n is 0, then R.sub.2 is not H,
Me, Et, --C(C.dbd.O)NH.sub.2, --C(C.dbd.O)NHPh,
--C(.dbd.S)NH-nButyl, --C(C.dbd.O)NHC(.dbd.O)CH.sub.2Cl,
--C(C.dbd.O)H, --C(C.dbd.O)Me, --C(C.dbd.O)Et,
--C(C.dbd.O)CH.dbd.CH.sub.2, --S(.dbd.O).sub.2Me,
--S(.dbd.O).sub.2Et, --C(C.dbd.O)O(CH.sub.3).sub.3, or
9H-D-ribofuranosyl-9H-purin-6-yl or --C(C.dbd.O)Ph; further
provided that when q is 0 and n is 1 or 2, then R.sub.2 is not H,
--C(C.dbd.O)Me, --C(C.dbd.O)Et, --S(.dbd.O).sub.2Me,
--S(.dbd.O).sub.2Et or --C(C.dbd.O)O(CH.sub.3).sub.3;
[0286] further provided that when q is 1, and R is Me, Cl, CN or F
at the 6 position of the benzothiazepine ring, or Br at position 7
of the benzothiazepine ring, then R.sub.2 is not H, Me,
--C(C.dbd.O)H, --C(C.dbd.O)Me, --C(C.dbd.O)Et, --C(C.dbd.O)Ph,
--S(.dbd.O).sub.2Me, or --S(.dbd.O).sub.2Et;
[0287] further provided that when q is 1, n is 0, and R is OH,
C.sub.1-C.sub.3 alkoxyl at the 7 position of the benzothiazepine
ring, then R.sub.2 is not H, --C(C.dbd.O)CH.dbd.CH.sub.2,
--C(C.dbd.O)CH.sub.2Br, --(CH.sub.2).sub.3-4-benzylpiperidine,
##STR00005##
[0288] further provided that when q is 0 and n is 0 or 2, then
R.sub.2 is not --C.dbd.ONHPh, --C.dbd.ONHCOCH.sub.2Cl,
--C.dbd.ONH.sub.2, --C.dbd.ONH(n-Bu), --C.dbd.S(NHPh),
--C.dbd.S(NHCOCH.sub.2Cl), --C.dbd.S(NH.sub.2), --C.dbd.SNH(n-Bu),
--CH.sub.2CH.sub.2N(Me).sub.2, --CH.sub.2CH.sub.2NH.sub.2 or
--C.dbd.OCHCl.sub.2;
[0289] further provided that when q is 2, each R is methoxy at
positions 7 and 8 of the benzothiazepine ring, and n is 0 or 2,
then R.sub.2 is not --C(C.dbd.O)Me;
[0290] further provided that when q is 0, R.sub.2 is not H;
[0291] further provided that when q is 0, and n is 0, then R.sub.2
is not methyl, benzotriazolylmethyl, 4-methoxybenzyl,
Ph-C.ident.C--CH.sub.2--, 4-chlorobenzyl, ethyl, pentyl,
--CH.sub.2P(O)(OCH.sub.2CH.sub.3).sub.2, Ph-CO--CH.sub.2CH.sub.2--,
C(.dbd.O)CH.dbd.CH.sub.2 and C(.dbd.O)CH.sub.2Br; and
[0292] further provided that when q is 1, R is CH.sub.3 at position
9 of the benzothiazepine ring, and n is 0, then R.sub.2 is not
methyl, benzotriazolylmethyl, pentyl,
--CH.sub.2P(O)(OCH.sub.2CH.sub.3).sub.2 or 4-methoxybenzyl.
[0293] In certain embodiments, the present invention provides
compounds of formula I-a, wherein each R is independently selected
from the group consisting of H, halogen, --OH, OMe, --NH.sub.2,
--NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1, or 2.
[0294] In other embodiments, the present invention provides
compounds of formula I-a, wherein R.sub.2 is selected from the
group consisting of --C.dbd.O(R.sub.5), --C.dbd.S(R.sub.6),
--SO.sub.2R.sub.7, --P(.dbd.O)R.sub.8R.sub.9, and
--(CH.sub.2).sub.m--R.sub.10.
[0295] In yet another embodiment, the present invention provides
compounds of formula I-b:
##STR00006##
wherein R' and R'' are independently selected from the group
consisting of H, halogen, --OH, --NH.sub.2, --NO.sub.2, --CN,
--CF.sub.3, --OCF.sub.3, --N.sub.3, --SO.sub.3H,
--S(.dbd.O).sub.2alkyl, --S(.dbd.O)alkyl,
--OS(.dbd.O).sub.2CF.sub.3, acyl, alkyl, alkoxyl, alkylamino,
alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino; and wherein each acyl, alkyl, alkoxyl,
alkylamino, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio may be
substituted or unsubstituted; R.sub.2 and n are as defined in
compounds of formula I-a above; and enantiomers, diastereomers,
tautomers, pharmaceutically acceptable salts, hydrates, solvates,
complexes and prodrugs thereof.
[0296] In certain embodiments, the present invention provides
compounds of formula I-b, wherein R' and R'' are independently
selected from the group consisting of H, halogen, --OH, OMe,
--NH.sub.2, --NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In
some cases, R' is H or OMe, and R'' is H.
[0297] In other embodiments, the present invention provides
compounds of formula I-b, wherein R.sub.2 is selected from the
group consisting of --C.dbd.O(R.sub.5), --C.dbd.S(R.sub.6),
--SO.sub.2R.sub.7, --P(.dbd.O)R.sub.8R.sub.9, and
--(CH.sub.2).sub.m--R.sub.10.
[0298] In yet another embodiment, the present invention provides
compounds formula of I-c:
##STR00007##
wherein each R, R.sub.7, q, and n is as defined in compounds of
formula I-a above; and enantiomers, diastereomers, tautomers,
pharmaceutically acceptable salts, hydrates, solvates, complexes
and prodrugs thereof.
[0299] In certain embodiments, the present invention provides
compounds of formula I-c, wherein each R is independently selected
from the group consisting of H, halogen, --OH, OMe, --NH.sub.2,
--NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1, or 2.
[0300] In other embodiments, the present invention provides
compounds of formula I-c, wherein R.sub.7 is selected from the
group consisting of --OH, --NR.sub.15R.sub.16, alkyl, alkenyl,
aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl; wherein each alkyl, akenyl, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be
substituted or unsubstituted.
[0301] In a further embodiment, the present invention provides
compounds of formula of I-d:
##STR00008##
wherein R' and R'' are independently selected from the group
consisting of H, halogen, --OH, --NH.sub.2, --NO.sub.2, --CN,
--CF.sub.3, --OCF.sub.3, --N.sub.3, --SO.sub.3H,
--S(.dbd.O).sub.2alkyl, --S(.dbd.O)alkyl,
--OS(.dbd.O).sub.2CF.sub.3, acyl, alkyl, alkoxyl, alkylamino,
alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino; and wherein each acyl, alkyl, alkoxyl,
alkylamino, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio may be
substituted or unsubstituted; R.sub.7 and n are as defined in
compounds of formula I-a above; and enantiomers, diastereomers,
tautomers, pharmaceutically acceptable salts, hydrates, solvates,
complexes and prodrugs thereof.
[0302] In certain embodiments, the present invention provides
compounds of formula I-d, wherein R' and R'' are independently
selected from the group consisting of H, halogen, --OH, OMe,
--NH.sub.2, --NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In
some cases, R' is H or OMe, and R'' is H.
[0303] In other embodiments, the present invention provides
compounds of formula I-d, wherein R.sub.7 is selected from the
group consisting of --OH, --NR.sub.15R.sub.16, alkyl, alkenyl,
aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl; wherein each alkyl, akenyl, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be
substituted or unsubstituted.
[0304] In one embodiment, the present invention provides compounds
of formula of I-e:
##STR00009##
wherein each R, R.sub.5, q and n is as defined compounds of formula
I-a above; and enantiomers, diastereomers, tautomers,
pharmaceutically acceptable salts, hydrates, solvates, complexes
and prodrugs thereof.
[0305] In certain embodiments, the present invention provides
compounds of formula I-e, wherein each R is independently selected
from the group consisting of H, halogen, --OH, OMe, --NH.sub.2,
--NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1, or 2.
[0306] In other embodiments, the present invention provides
compounds of formula I-e, wherein R.sub.5 is selected from the
group consisting of --NR.sub.15R.sub.16, --NHOH, --OR.sub.15,
--CH.sub.2X, alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkyl,
alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl may be substituted or unsubstituted.
[0307] In some embodiments, the present invention provides
compounds of formula I-e, wherein R.sub.5 is an alkyl substituted
by at least one labeling group, such as a fluorescent, a
bioluminescent, a chemiluminescent, a colorimetric and a
radioactive labeling group. A fluorescent labeling group can be
selected from bodipy, dansyl, fluorescein, rhodamine, Texas red,
cyanine dyes, pyrene, coumarins, Cascade Blue.TM., Pacific Blue,
Marina Blue, Oregon Green, 4',6-Diamidino-2-phenylindole (DAPI),
indopyra dyes, lucifer yellow, propidium iodide, porphyrins,
arginine, and variants and derivatives thereof.
[0308] In another embodiment, the present invention provides
compounds of formula of I-f:
##STR00010##
wherein R' and R'' are independently selected from the group
consisting of H, halogen, --OH, --NH.sub.2, --NO.sub.2, --CN,
--CF.sub.3, --OCF.sub.3, --N.sub.3, --SO.sub.3H,
--S(.dbd.O).sub.2alkyl, --S(.dbd.O)alkyl,
--OS(.dbd.O).sub.2CF.sub.3, acyl, alkyl, alkoxyl, alkylamino,
alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino; and wherein each acyl, alkyl, alkoxyl,
alkylamino, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio may be
substituted or unsubstituted; R.sub.5 and n are as defined in
compounds of formula I-a above; and enantiomers, diastereomers,
tautomers, pharmaceutically acceptable salts, hydrates, solvates,
complexes and prodrugs thereof.
[0309] In certain embodiments, the present invention provides
compounds of formula I-f, wherein R' and R'' are independently
selected from the group consisting of H, halogen, --OH, OMe,
--NH.sub.2, --NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In
some cases, R' is H or OMe, and R'' is H.
[0310] In other embodiments, the present invention provides
compounds of formula I-f, wherein R.sub.5 is selected from the
group consisting of --NR.sub.15R.sub.16, --NHOH, --OR.sub.15,
--CH.sub.2X, alkyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkyl,
alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl may be substituted or unsubstituted.
[0311] In yet another embodiment, the present invention provides
compounds of formula of I-g:
##STR00011##
wherein W is S or O; each R, R.sub.15, R.sub.16, q, and n is as
defined in compounds of formula I-a above; and enantiomers,
diastereomers, tautomers, pharmaceutically acceptable salts,
hydrates, solvates, complexes and prodrugs thereof.
[0312] In certain embodiments, the present invention provides
compounds of formula I-g, wherein each R is independently selected
from the group consisting of H, halogen, --OH, OMe, --NH.sub.2,
--NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1, or 2.
[0313] In other embodiments, the present invention provides
compounds of formula I-g, wherein R.sub.15 and R.sub.16
independently are selected from the group consisting of H, OH,
NH.sub.2, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, and heterocyclylalkyl; wherein each alkyl,
alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl may be substituted; and optionally R.sub.15 and
R.sub.16 together with the N to which they are bonded may form a
heterocycle which may be substituted.
[0314] In some embodiments, the present invention provides
compounds of formula I-g, wherein W is O or S.
[0315] In yet another embodiment, the present invention provides
compounds of formula of I-h:
##STR00012##
wherein W is S or O; wherein R' and R'' are independently selected
from the group consisting of H, halogen, --OH, --NH.sub.2,
--NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3, --SO.sub.3H,
--S(.dbd.O).sub.2alkyl, --S(.dbd.O)alkyl,
--OS(.dbd.O).sub.2CF.sub.3, acyl, alkyl, alkoxyl, alkylamino,
alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino; and wherein each acyl, alkyl, alkoxyl,
alkylamino, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio may be
substituted or unsubstituted; R.sub.15, R.sub.16 and n are as
defined in compounds of formula I-a above; and enantiomers,
diastereomers, tautomers, pharmaceutically acceptable salts,
hydrates, solvates, complexes and prodrugs thereof.
[0316] In certain embodiments, the present invention provides
compounds of formula I-h, wherein R' and R'' are independently
selected from the group consisting of H, halogen, --OH, OMe,
--NH.sub.2, --NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In
some cases, R' is H or OMe, and R'' is H.
[0317] In other embodiments, the present invention provides
compounds of formula I-h, wherein R.sub.15 and R.sub.16
independently are selected from the group consisting of H, OH,
NH.sub.2, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, and heterocyclylalkyl; wherein each alkyl,
alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl may be substituted; and optionally R.sub.15 and
R.sub.16 together with the N to which they are bonded may form a
heterocycle which may be substituted.
[0318] In some embodiments, the present invention provides
compounds of formula I-g, wherein W is O or S.
[0319] In a further embodiment, the present invention provides
compounds of formula of I-i:
##STR00013##
wherein R.sub.17 is selected from the group consisting of
--NR.sub.15R.sub.16, --NHNR.sub.15R.sub.16, --NHOH, --OR.sub.15,
--CH.sub.2X, alkenyl, aryl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, and heterocyclylalkyl; wherein each alkenyl, aryl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl
may be substituted or unsubstituted; each R, q, and n is as defined
in compounds of formula I-a above; and enantiomers, diastereomers,
tautomers, pharmaceutically acceptable salts, hydrates, solvates,
complexes and prodrugs thereof.
[0320] In certain embodiments, the present invention provides
compounds of formula I-i, wherein each R is independently selected
from the group consisting of H, halogen, --OH, OMe, --NH.sub.2,
--NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1, or 2.
[0321] In other embodiments, the present invention provides
compounds of formula I-i, wherein R.sub.17 is --NR.sub.15R.sub.16,
and --OR.sub.15. In certain other embodiments, R.sub.17 is --OH,
--OMe, --NEt, --NHEt, --NHPh, --NH.sub.2, or
--NHCH.sub.2pyridyl.
[0322] In one embodiment, the present invention provides compounds
of formula of I-j:
##STR00014##
wherein R' and R'' are independently selected from the group
consisting of H, halogen, --OH, --NH.sub.2, --NO.sub.2, --CN,
--CF.sub.3, --OCF.sub.3, --N.sub.3, --SO.sub.3H, --S(O).sub.2alkyl,
--S(.dbd.O)alkyl, --OS(.dbd.O).sub.2CF.sub.3, acyl, alkyl, alkoxyl,
alkylamino, alkylthio, cycloalkyl, aryl, heterocyclyl,
heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl,
(hetero-)arylthio, and (hetero-)arylamino; and wherein each acyl,
alkyl, alkoxyl, alkylamino, cycloalkyl, aryl, heterocyclyl,
heterocyclylalkyl, alkenyl, alkynyl, (hetero-)aryl,
(hetero-)arylthio may be substituted or unsubstituted; R.sub.17 is
selected from the group consisting of --NR.sub.15R.sub.16,
--NHNR.sub.15R.sub.16, --NHOH, --OR.sub.15, --CH.sub.2X, alkenyl,
aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl; wherein each alkenyl, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be
substituted or unsubstituted; n is as defined in compounds of
formula I-a; and enantiomers, diastereomers, tautomers,
pharmaceutically acceptable salts, hydrates, solvates, complexes
and prodrugs thereof.
[0323] In certain embodiments, the present invention provides
compounds of formula I-j, wherein R' and R'' are independently
selected from the group consisting of H, halogen, --OH, OMe,
--NH.sub.2, --NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In
some cases, R' is H or OMe, and R'' is H.
[0324] In other embodiments, the present invention provides
compounds of formula I-j, wherein R.sub.17 is --NR.sub.15R.sub.16
or --OR.sub.15. In certain other embodiments, R.sub.17 is --OH,
--OMe, --NEt, --NHEt, --NHPh, --NH.sub.2, or
--NHCH.sub.2pyridyl.
[0325] In another embodiment, the present invention provides
compounds of formula I-k or I-k-1:
##STR00015##
wherein R, R' and R'' are independently selected from the group
consisting of H, halogen, --OH, --NH.sub.2, --NO.sub.2, --CN,
--CF.sub.3, --OCF.sub.3, --N.sub.3, --SO.sub.3H,
--S(.dbd.O).sub.2alkyl, --S(.dbd.O)alkyl,
--OS(.dbd.O).sub.2CF.sub.3, acyl, alkyl, alkoxyl, alkylamino,
alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino; and wherein each acyl, alkyl, alkoxyl,
alkylamino, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio may be
substituted or unsubstituted; R.sub.18 is selected from the group
consisting of --NR.sub.15R.sub.16, --C(.dbd.O)NR.sub.15R.sub.16,
--(C.dbd.O)OR.sub.15, --OR.sub.15, alkyl, aryl, cycloalkyl,
heterocyclyl, and at one labeling group; wherein each alkyl, aryl,
cycloalkyl, and heterocyclyl may be substituted or unsubstituted;
wherein q is 0, 1, 2, 3, or 4; p is 1, 2, 3, 4, 5, 6, 7, 8 9, or
10; and n is 0, 1, or 2; and enantiomers, diastereomers, tautomers,
pharmaceutically acceptable salts, hydrates, solvates, complexes
and prodrugs thereof
[0326] In certain embodiments, the present invention provides
compounds of formula I-k, wherein each R is independently selected
from the group consisting of H, halogen, --OH, OMe, --NH.sub.2,
--NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In
some cases, R is H or is OMe at position 7 of the benzothiazepine
ring.
[0327] In certain embodiments, the present invention provides
compounds of formula I-k-1, wherein R' and R'' are independently
selected from the group consisting of H, halogen, --OH, OMe,
--NH.sub.2, --NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In
some cases, R' is H or OMe, and R'' is H.
[0328] In other embodiments, the present invention provides
compounds of formula I-k or I-k-1, wherein R.sub.18 is selected
from the group consisting of --NR.sub.15R.sub.16,
--(C.dbd.O)OR.sub.15, --OR.sub.15, alkyl, aryl, and at one labeling
group; and wherein each alkyl and aryl may be substituted or
unsubstituted. In some cases, m is 1, and R.sub.18 is Ph,
C(.dbd.O)OMe, C(.dbd.O)OH, aminoalkyl, NH.sub.2, NHOH, or NHCbz. In
other cases, m is 0, and R.sub.18 is C.sub.1-C.sub.4 alkyl, such as
Me, Et, propyl, and butyl. In yet other cases, m is 2, and R.sub.18
is pyrrolidine, piperidine, piperazine, or morpholine. In some
embodiments, m is 3, 4, 5, 5, 7, or 8, and R.sub.18 is a
fluorescent labeling group selected from bodipy, dansyl,
fluorescein, rhodamine, Texas red, cyanine dyes, pyrene, coumarins,
Cascade Blue.TM., Pacific Blue, Marina Blue, Oregon Green,
4',6-Diamidino-2-phenylindole (DAPI), indopyra dyes, lucifer
yellow, propidium iodide, porphyrins, arginine, and variants and
derivatives thereof
[0329] In yet another embodiment, the present invention provides
compounds of formula of I-l or I-l-1:
##STR00016##
wherein R, R' and R'' are independently selected from the group
consisting of H, halogen, --OH, --NH.sub.2, --NO.sub.2, --CN,
--CF.sub.3, --OCF.sub.3, --N.sub.3, --SO.sub.3H,
--S(.dbd.O).sub.2alkyl, --S(.dbd.O)alkyl,
--OS(.dbd.O).sub.2CF.sub.3, acyl, alkyl, alkoxyl, alkylamino,
alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino; and wherein each acyl, alkyl, alkoxyl,
alkylamino, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio may be
substituted or unsubstituted; R.sub.6 and n and q are as defined in
compounds of formula I-a; and enantiomers, diastereomers,
tautomers, pharmaceutically acceptable salts, hydrates, solvates,
complexes and prodrugs thereof.
[0330] In certain embodiments, the present invention provides
compounds of formula I-l, wherein each R is independently selected
from the group consisting of H, halogen, --OH, OMe, --NH.sub.2,
--NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In
some cases, R is H or is OMe at position 7 of the benzothiazepine
ring.
[0331] In certain embodiments, the present invention provides
compounds of formula I-l-1, wherein R' and R'' are independently
selected from the group consisting of H, halogen, --OH, OMe,
--NH.sub.2, --NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In
some cases, R' is H or OMe, and R'' is H.
[0332] In other embodiments, the present invention provides
compounds of formula I-l or I-l-1, wherein R.sub.6 is selected from
the group consisting of --NR.sub.15R.sub.16, --NHNR.sub.15R.sub.16,
--OR.sub.15, --NHOH, --CH.sub.2X, acyl, alkenyl, alkyl, aryl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl;
wherein each acyl, alkenyl, alkyl, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be
substituted or unsubstituted. In some cases, R.sub.6 is
--NR.sub.15R.sub.16 such as --NHPh, pyrrolidine, piperidine,
piperazine, morpholine, and the like. In some other cases, R.sub.6
is alkoxyl, such as --O-tBu.
[0333] In a further embodiment, the present invention provides
compounds of formula I-m or I-m-1:
##STR00017##
wherein R' and R'' are independently selected from the group
consisting of H, halogen, --OH, --NH.sub.2, --NO.sub.2, --CN,
--CF.sub.3, --OCF.sub.3, --N.sub.3, --SO.sub.3H,
--S(.dbd.O).sub.2alkyl, --S(.dbd.O)alkyl,
--OS(.dbd.O).sub.2CF.sub.3, acyl, alkyl, alkoxyl, alkylamino,
alkylthio, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino; and wherein each acyl, alkyl, alkoxyl,
alkylamino, cycloalkyl, aryl, heterocyclyl, heterocyclylalkyl,
alkenyl, alkynyl, (hetero-)aryl, (hetero-)arylthio may be
substituted or unsubstituted; wherein R.sub.8, R.sub.9 1 and n are
as defined in compounds of formula I-a above; and enantiomers,
diastereomers, tautomers, pharmaceutically acceptable salts,
hydrates, solvates, complexes and prodrugs thereof.
[0334] In certain embodiments, the present invention provides
compounds of formula I-m, wherein each R is independently selected
from the group consisting of H, halogen, --OH, OMe, --NH.sub.2,
--NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In
some cases, R is H or is OMe at position 7 of the benzothiazepine
ring.
[0335] In certain embodiments, the present invention provides
compounds of formula I-m-1, wherein R' and R'' are independently
selected from the group consisting of H, halogen, --OH, OMe,
--NH.sub.2, --NO.sub.2, --CN, --CF.sub.3, --OCF.sub.3, --N.sub.3,
--S(.dbd.O).sub.2C.sub.1-C.sub.4alkyl,
--S(.dbd.O)C.sub.1-C.sub.4alkyl, --S--C.sub.1-C.sub.4alkyl,
--OS(.dbd.O).sub.2CF.sub.3, Ph, --NHCH.sub.2Ph, --C(C.dbd.O)Me,
--OC(.dbd.O)Me, morpholinyl and propenyl; and n is 0, 1 or 2. In
some cases, R' is H or OMe, and R'' is H.
[0336] In other embodiments, the present invention provides
compounds of formula I-m or I-m-1, wherein R.sub.8 and R.sub.9 are
independently alkyl, aryl, --OH, alkoxyl, or alkylamino. In some
cases, R.sub.8 is C.sub.1-C.sub.4alkyl such as Me, Et, propyl and
butyl; and R.sub.9 is aryl such as phenyl.
[0337] In other embodiments, the present invention provides
compounds of formula I-n,
##STR00018##
wherein:
[0338] R.sub.d is CH.sub.2, or NR.sub.a; and
[0339] R.sub.a is H, alkoxy (for example but not limited to
methoxy), --(C.sub.1-C.sub.6 alkyl)-aryl, wherein the aryl is a
bisubstituted phenyl or a benzo[1,3]dioxo-5-yl group, or a Boc
group. In one embodiment, R.sub.a is H.
[0340] Representative compounds of Formula I-n include without
limitation S101, S102, S103, and S114.
[0341] In certain other embodiments, the invention provides
compounds of Formula I-o:
##STR00019##
wherein:
[0342] R.sub.e is --(C.sub.1-C.sub.6 alkyl)-phenyl,
--(C.sub.1-C.sub.6 alkyl)-C(O)R.sub.b, or substituted or
unsubstituted --C.sub.1-C.sub.6 alkyl; and
[0343] R.sub.b is --OH or --O--(C.sub.1-C.sub.6 alkyl), and
[0344] wherein the phenyl or substituted alkyl is substituted with
one or more of halogen, hydroxyl, --C.sub.1-C.sub.6 alkyl,
--O--(C.sub.1-C.sub.6 alkyl), --NH.sub.2, --NH(C.sub.1-C.sub.6
alkyl), --N(C.sub.1-C.sub.6 alkyl).sub.2, cyano, or dioxolane.
[0345] Representative compounds of Formula I-o include without
limitation S107, S110, S111, S120, and S121.
[0346] In certain other embodiments, the invention provides
compounds of Formula I-p:
##STR00020##
wherein:
[0347] R.sub.c is --(C.sub.1-C.sub.6 alkyl)-NH.sub.2,
--(C.sub.1-C.sub.6 alkyl)-OR.sub.f, wherein R.sub.f is H or
--C(O)--(C.sub.1-C.sub.6)alkyl, or --(C.sub.1-C.sub.6
alkyl)-NHR.sub.g wherein Rg is carboxybenzyl. Representative
compounds of Formula I-n include without limitation S109, S122, and
S123.
[0348] In non-limiting examples, Formulae I-a, I-b, I-e, I-f, I-g,
I-h, I-n are represented by compounds S101, S102, S103. In a
non-limiting example, Formulae I-a, I-b, I-e, I-f, I-i, I-j are
represented by compound S104. In a non-limiting example, Formulae
I-a, I-b, I-o are represented by S107. In a non-limiting example,
Formulae I-a, I-b, I-e, I-f are represented by S108. In a
non-limiting example, Formulae I-a, I-b, I-e, I-f, I-p are
represented by S109. In a non-limiting example, Formulae I-a, I-b,
I-k, I-k-1, I-o are represented by S110. In a non-limiting example,
Formulae I-a, I-b, I-k, I-k-1 I-o are represented by S111. In a
non-limiting example, Formulae I-a, I-b, I-c, I-d are represented
by S112. In a non-limiting example, Formulae I-a, I-b are
represented by S113. In a non-limiting example, Formulae I-a, I-b,
I-e, I-f, I-g, I-h are represented by S114. In a non-limiting
example, Formulae I-a, I-b, I-g, I-h, I-l and I-l-1 are represented
by S115. In a non-limiting example, Formulae I-a, I-b, I-g, I-h,
are represented by S116. In a non-limiting example, Formulae I-a,
I-b, I-e, I-f are represented by S117. In a non-limiting example,
Formulae I-a, I-b, I-e, I-f are represented by S118. In a
non-limiting example, I-a, I-b are represented by S119. In a
non-limiting example, Formulae I-a, I-b, I-k, I-k-1, I-o are
represented by S120. In a non-limiting example, Formulae I-a, I-b,
I-k, I-k-1 I-o, I-p are represented by S121. In a non-limiting
example, Formulae I-a, I-b, I-e, I-f, I-p are represented by S122.
In a non-limiting example, Formulae I-a, I-b, I-e, I-f, I-p are
represented by S123.
[0349] The compounds of Formula I, I-a, I-b, I-c, I-d, I-e, I-f,
I-g, I-h, I-i, I-j, I-k, I-k-1, I-l, I-l-1, I-m, I-m-1, I-n, I-o,
and I-p, and Formula II can be used in methods that treat or
prevent disorders and diseases associated with the RyR
receptors.
[0350] Examples of such compounds include, without limitation, S1,
S2, S3, S4, S5, S6, S7, S9, S11, S12, S13, S14, S19, S20, S22, S23,
S24, S25, S26, S27, S36, S37, S38, S40, S43, S44, S45, S46, S47,
S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60,
S61, S62, S63, S64, S66, S67, S68, S69, S70, S71, S72, S73, S74,
S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87,
S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, S100,
S101, S102, S103, 5104, 5105, 5107, S108, S109, S110, S111, S112,
S113, S114, S115, S116, S117, S118, S119, S120, S121, S122, and
S123, as herein defined. In certain embodiments, the compounds are
isolated and substantially pure.
[0351] In a certain embodiment of the methods the compound is not
S4. In another embodiment, the compound is not S7. In another
embodiment, the compound is not S8. In another embodiment, the
compound is not S10. In another embodiment, the compound is not
S20. In another embodiment, the compound is not S24. In another
embodiment, the compound is not S25. In another embodiment, the
compound is not S26. In another embodiment, the compound is not
S27. In another embodiment, the compound is not S36. In another
embodiment, the compound is not JTV-519.
[0352] Certain RyCal compounds of the invention have the following
structures:
##STR00021## ##STR00022## ##STR00023## ##STR00024## ##STR00025##
##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030##
##STR00031## ##STR00032## ##STR00033## ##STR00034##
##STR00035##
[0353] In one embodiment of the present invention, for compounds of
Formula I, if R.sub.2 is C.dbd.O(R.sub.5) or SO.sub.2R.sub.7, then
R is at positions 2, 3, or 5 on the benzene ring (i.e., positions
6, 7 or 9 of the benzothiazepine ring).
[0354] In another embodiment of the invention, for compounds of
Formula I, if R.sub.2 is C.dbd.O(R.sub.5) or SO.sub.2R.sub.7, then
each R is independently selected from the group consisting of H,
halogen, --OH, --NH.sub.2, --NO.sub.2, --CN, --N.sub.3,
--SO.sub.3H, acyl, alkyl, alkylamino, cycloalkyl, heterocyclyl,
heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino,
cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl,
(hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino may be
substituted with one or more radicals independently selected from
the group consisting of halogen, N, O, --S--, --CN, --N.sub.3,
--SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl,
(hetero-)cycloalkyl, and (hetero-)cyclyl.
[0355] In another embodiment of the invention, for compounds of
Formula I, if R.sub.2 is C.dbd.O(R.sub.5) or SO.sub.2R.sub.7, then
there are at least two R groups attached to the benzene ring.
Furthermore, there are at least two R groups attached to the
benzene ring, and both R groups are attached at positions 2, 3, or
5 on the benzene ring. Still furthermore, each R is independently
selected from the group consisting of H, halogen, --OH, --NH.sub.2,
--NO.sub.2, --CN, --N.sub.3, --SO.sub.3H, acyl, alkyl, alkylamino,
cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl,
(hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein
each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl,
heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-) arylthio, and
(hetero-)arylamino may be substituted with one or more radicals
independently selected from the group consisting of halogen, N, O,
--S--, --CN, --N.sub.3, --SH, nitro, oxo, acyl, alkyl, alkoxyl,
alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and
(hetero-)cyclyl.
[0356] In another embodiment of the invention, for compounds of
Formula I, if R.sub.2 is C.dbd.O(R.sub.5), then R.sub.5 is selected
from the group consisting of --NR.sub.16, NHNHR.sub.16, NHOH,
--OR.sub.15, CONH.sub.2NHR.sub.16, CONR.sub.16, CH.sub.2X, acyl,
aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and
heterocyclylalkyl; wherein each acyl, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be
substituted with one or more radicals independently selected from
the group consisting of halogen, N, O, --S--, --CN, --N.sub.3,
nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl,
(hetero-)cycloalkyl, and (hetero-)cyclyl.
[0357] In another embodiment, the present invention provides use of
compounds of Formula II:
##STR00036##
wherein R.dbd.OR', SR', N(R').sub.2, alkyl, or halide and R'=alkyl,
aryl, or H, and wherein R can be at position 2, 3, 4, or 5 (i.e.,
positions 6, 7, 8 or 9 of the benzothiazepine ring). Formula II is
discussed also in co-pending application Ser. No. 10/680,988, the
disclosure of which is incorporated herein in its entirety by
reference.
Routes of Activity
[0358] The compounds of the invention reduce the open probability
of RyR by increasing the affinity of FKBP12 (calstabin1) and
FKBP12.6 (calstabin2) for, respectively PKA-phosphorylated RyR1 and
PKA-phosphorylated RyR2. Moreover, the compounds of Formula I
normalize gating of mutant RyR channels, including CPVT-associated
mutant RyR2 channels, by increasing FKBP12 (calstabin1) and FKBP
12.6 (calstabin2) binding affinity. Therefore, the compounds of the
invention prevent disorders and conditions involving modulation of
the RyR receptors, particularly the RyR1 and RyR2 receptors.
Examples of such disorders and conditions include, without
limitation, cardiac disorders and diseases, skeletal muscular
disorders and diseases, cognitive disorders and diseases, malignant
hyperthermia, diabetes, and sudden infant death syndrome. Cardiac
disorder and diseases include, but are not limited to, irregular
heartbeat disorders and diseases; exercise-induced irregular
heartbeat disorders and diseases; sudden cardiac death;
exercise-induced sudden cardiac death; congestive heart failure;
chronic obstructive pulmonary disease; and high blood pressure.
Irregular heartbeat disorders and diseases include and
exercise-induced irregular heartbeat disorders and diseases
include, but are not limited to, atrial and ventricular arrhythmia;
atrial and ventricular fibrillation; atrial and ventricular
tachyarrhythmia; atrial and ventricular tachycardia;
catecholaminergic polymorphic ventricular tachycardia (CPVT); and
exercise-induced variants thereof. Skeletal muscular disorder and
diseases include, but are not limited to, skeletal muscle fatigue,
exercise-induced skeletal muscle fatigue, muscular dystrophy,
bladder disorders, and incontinence. Cognitive disorders and
diseases include, but are not limited to, Alzheimer's Disease,
forms of memory loss, and age-dependent memory loss. The compounds
of the invention treat these disorders and conditions by increasing
FKBP12 (calstabin1)-RyR1 binding affinity and increasing FKBP12.6
(calstabin2)-RyR2 binding affinity.
[0359] In accordance with the foregoing, the present invention
provides a method for limiting or preventing a decrease in the
level of RyR-bound FKBP (calstabin) in cells of a subject. As used
herein, "RyR" includes RyR1, RyR2, and RyR3. Additionally, FKBP
includes both FKBP12 (calstabin1) and FKBP12.6 (calstabin2).
"RyR-bound FKBP" therefore refers to RyR1-bound FKBP12
(calstabin1), RyR2-bound FKBP12.6 (calstabin2), and RyR3-bound
FKBP12 (calstabin1).
[0360] As used herein, "RyR" also includes an "RyR protein" and an
"RyR analogue." An "RyR analogue" is a functional variant of the
RyR protein, having RyR biological activity, that has 60% or
greater amino-acid-sequence homology with the RyR protein. The RyR
of the present invention are unphosphorylated, phosphorylated
(e.g., by PKA), or hyperphosphorylated (e.g., by PKA). As further
used herein, the term "RyR biological activity" refers to the
activity of a protein or peptide that demonstrates an ability to
associate physically with, or bind with, FKBP12 (calstabin1) in the
case of RyR1 and RyR3, and FKBP12.6 (calstabin2) in the case of
RyR2 (i.e., binding of approximately two fold or, approximately
five fold, above the background binding of a negative control),
under the conditions of the assays described herein.
[0361] As used herein, "FKBP" includes both an "FKBP protein" and
an "FKBP analogue," whether it be FKBP12 (calstabin1) or FKBP12.6
(calstabin2). Unless otherwise indicated herein, "protein" shall
include a protein, protein domain, polypeptide, or peptide, and any
fragment thereof. An "FKBP analogue" is a functional variant of the
FKBP protein, having FKBP biological activity, that has 60% or
greater amino-acid-sequence homology with the FKBP protein, whether
it be FKBP12 (calstabin1) or FKBP12.6 (calstabin2). As further used
herein, the term "FKBP biological activity" refers to the activity
of a protein or peptide that demonstrates an ability to associate
physically with, or bind with, unphosphorylated or
non-hyperphosphorylated RyR2 (i.e., binding of approximately two
fold, or approximately five fold, above the background binding of a
negative control), under the conditions of the assays described
herein.
[0362] FKBP binds to the RyR channel, one molecule per RyR subunit.
Accordingly, as used herein, the term "RyR-bound FKBP" includes a
molecule of an FKBP12 (calstabin1) protein that is bound to an RyR1
protein subunit or a tetramer of FKBP12 that is in association with
a tetramer of RyR1, a molecule of FKBP12.6 (calstabin2) protein
that is bound to an RyR2 protein subunit or a tetramer of FKBP12.6
that is in association with a tetramer of RyR2, and a molecule of
an FKBP12 (calstabin1) protein that is bound to an RyR3 protein
subunit or a tetramer of FKBP12 that is in association with a
tetramer of RyR3. Therefore, "RyR-bound FKBP" refers to "RyR1-bound
FKBP12," "RyR2-bound FKBP 12.6," and "RyR3-bound FKBP12."
[0363] In accordance with the method of the present invention, a
"decrease" or "disorder" in the level of RyR-bound FKBP in cells of
a subject refers to a detectable decrease, diminution or reduction
in the level of RyR-bound FKBP in cells of the subject. Such a
decrease is limited or prevented in cells of a subject when the
decrease is in any way halted, hindered, impeded, obstructed or
reduced by the administration of compounds of the invention, such
that the level of RyR-bound FKBP in cells of the subject is higher
than it would otherwise be in the absence of the administered
compound.
[0364] The level of RyR-bound FKBP in a subject is detected by
standard assays and techniques, including those readily determined
from the known art (e.g., immunological techniques, hybridization
analysis, immunoprecipitation, Western-blot analysis, fluorescence
imaging techniques and/or radiation detection, etc.), as well as
any assays and detection methods disclosed herein. For example,
protein is isolated and purified from cells of a subject using
standard methods known in the art, including, without limitation,
extraction from the cells (e.g., with a detergent that solubilizes
the protein) where necessary, followed by affinity purification on
a column, chromatography (e.g., FTLC and HPLC), immunoprecipitation
(with an antibody), and precipitation (e.g., with isopropanol and a
reagent such as Trizol). Isolation and purification of the protein
is followed by electrophoresis (e.g., on an SDS-polyacrylamide
gel). A decrease in the level of RyR-bound FKBP in a subject, or
the limiting or prevention thereof, is determined by comparing the
amount of RyR-bound FKBP detected prior to the administration of a
compound of the invention (in accordance with methods described
below) with the amount detected a suitable time after
administration of the compound.
[0365] A decrease in the level of RyR-bound FKBP in cells of a
subject is limited or prevented, for example, by inhibiting
dissociation of FKBP and RyR in cells of the subject; by increasing
binding between FKBP and RyR in cells of the subject; or by
stabilizing the RyR-FKBP complex in cells of a subject. As used
herein, the term "inhibiting dissociation" includes blocking,
decreasing, inhibiting, limiting or preventing the physical
dissociation or separation of an FKBP subunit from an RyR molecule
in cells of the subject, and blocking, decreasing, inhibiting,
limiting or preventing the physical dissociation or separation of
an RyR molecule from an FKBP subunit in cells of the subject. As
further used herein, the term "increasing binding" includes
enhancing, increasing, or improving the ability of phosphorylated
RyR to associate physically with FKBP (e.g., binding of
approximately two fold or, approximately five fold, above the
background binding of a negative control) in cells of the subject
and enhancing, increasing or improving the ability of FKBP to
associate physically with phosphorylated RyR (e.g., binding of
approximately two fold, or, approximately five fold, above the
background binding of a negative control) in cells of the subject.
Additionally, a decrease in the level of RyR-bound FKBP in cells of
a subject is limited or prevented by directly decreasing the level
of phosphorylated RyR in cells of the subject or by indirectly
decreasing the level of phosphorylated RyR in the cells (e.g., by
targeting an enzyme (such as PKA) or another endogenous molecule
that regulates or modulates the functions or levels of
phosphorylated RyR in the cells). In one embodiment, the level of
phosphorylated RyR in the cells is decreased by at least 10% in the
method of the present invention. In another embodiment, the level
of phosphorylated RyR is decreased by at least 20%.
[0366] The subject of the present invention are in vitro and in
vivo systems, including, without limitation, isolated or cultured
cells or tissues, non-cell in vitro assay systems and an animal
(e.g., an amphibian, a bird, a fish, a mammal, a marsupial, a
human, a domestic animal (such as a cat, dog, monkey, horse, mouse
or rat) or a commercial animal (such as a cow or pig)).
[0367] The cells of a subject include striated muscle cells. A
striated muscle is a muscle in which the repeating units
(sarcomeres) of the contractile myofibrils are arranged in registry
throughout the cell, resulting in transverse or oblique striations
that are observed at the level of a light microscope. Examples of
striated muscle cells include, without limitation, voluntary
(skeletal) muscle cells and cardiac muscle cells. In one
embodiment, the cell used in the method of the present invention is
a human cardiac muscle cell. As used herein, the term "cardiac
muscle cell" includes cardiac muscle fibers, such as those found in
the myocardium of the heart. Cardiac muscle fibers are composed of
chains of contiguous heart-muscle cells, or cardiomyocytes, joined
end to end at intercalated disks. These disks possess two kinds of
cell junctions: expanded desmosomes extending along their
transverse portions, and gap junctions, the largest of which lie
along their longitudinal portions.
[0368] A decrease in the level of RyR-bound FKBP is limited or
prevented in cells of a subject by administering the compounds of
the invention to the subject; this would also permit contact
between cells of the subject and the compounds of the invention.
The compounds of the invention are modulators of calcium-ion
channels. In addition to regulating Ca.sup.2+ levels in myocardial
cells, the compounds of the invention modulate the Na.sup.+ current
and the inward-rectifier K.sup.+ current in cells, such as guinea
pig ventricular cells, and inhibits the delayed-rectifier K.sup.+
current in cells, such as guinea pig atrial cells.
Pharmaceutical Composition
[0369] The compounds of the invention are formulated into
pharmaceutical compositions for administration to human subjects in
a biologically compatible form suitable for administration in vivo.
According to another aspect, the present invention provides a
pharmaceutical composition comprising compounds of the invention in
admixture with a pharmaceutically acceptable diluent and/or
carrier. The pharmaceutically-acceptable carrier must be
"acceptable" in the sense of being compatible with the other
ingredients of the composition and not deleterious to the recipient
thereof. The pharmaceutically-acceptable carrier employed herein is
selected from various organic or inorganic materials that are used
as materials for pharmaceutical formulations and which are
incorporated as analgesic agents, buffers, binders, disintegrants,
diluents, emulsifiers, excipients, extenders, glidants,
solubilizers, stabilizers, suspending agents, tonicity agents,
vehicles and viscosity-increasing agents. If necessary,
pharmaceutical additives, such as antioxidants, aromatics,
colorants, flavor-improving agents, preservatives, and sweeteners,
are also added. Examples of acceptable pharmaceutical carriers
include carboxymethyl cellulose, crystalline cellulose, glycerin,
gum arabic, lactose, magnesium stearate, methyl cellulose, powders,
saline, sodium alginate, sucrose, starch, talc and water, among
others.
[0370] The pharmaceutical formulations of the present invention are
prepared by methods well-known in the pharmaceutical arts. For
example, the compounds of the invention are brought into
association with a carrier and/or diluent, as a suspension or
solution. Optionally, one or more accessory ingredients (e.g.,
buffers, flavoring agents, surface active agents, and the like)
also are added. The choice of carrier is determined by the
solubility and chemical nature of the compounds, chosen route of
administration and standard pharmaceutical practice.
[0371] The compounds of the invention are administered to a subject
by contacting target cells (e.g., cardiac muscle cells) in vivo in
the subject with the compounds. The compounds of the invention are
contacted with (e.g., introduced into) cells of the subject using
known techniques utilized for the introduction and administration
of proteins, nucleic acids and other drugs. Examples of methods for
contacting the cells with (i.e., treating the cells with) the
compounds of the invention include, without limitation, absorption,
electroporation, immersion, injection, introduction, liposome
delivery, transfection, transfusion, vectors and other
drug-delivery vehicles and methods. When the target cells are
localized to a particular portion of a subject, it is desirable to
introduce the compounds for the invention directly to the cells, by
injection or by some other means (e.g., by introducing the
compounds into the blood or another body fluid). The target cells
are contained in tissue of a subject and are detected by standard
detection methods readily determined from the known art, examples
of which include, without limitation, immunological techniques
(e.g., immunohistochemical staining), fluorescence imaging
techniques, and microscopic techniques.
[0372] Additionally, the compounds of the present invention are
administered to a human or animal subject by known procedures
including, without limitation, oral administration, sublingual or
buccal administration, parenteral administration, transdermal
administration, via inhalation or intranasally, vaginally,
rectally, and intramuscularly. The compounds of the invention are
administered parenterally, by epifascial, intracapsular,
intracranial, intracutaneous, intrathecal, intramuscular,
intraorbital, intraperitoneal, intraspinal, intrasternal,
intravascular, intravenous, parenchymatous, subcutaneous or
sublingual injection, or by way of catheter. In one embodiment, the
agent is administered to the subject by way of delivery to the
subject's muscles including, but not limited to, the subject's
cardiac muscles. In an embodiment, the agent is administered to the
subject by way of targeted delivery to cardiac muscle cells via a
catheter inserted into the subject's heart. In other embodiments,
the agent is administered via a subcutaneous pump.
[0373] For oral administration, a formulation of the compounds of
the invention is presented as capsules, tablets, powders, granules
or as a suspension or solution. The formulation has conventional
additives, such as lactose, mannitol, corn starch or potato starch.
The formulation also is presented with binders, such as crystalline
cellulose, cellulose derivatives, acacia, corn starch or gelatins.
Additionally, the formulation is presented with disintegrators,
such as corn starch, potato starch or sodium
carboxymethylcellulose. The formulation also is presented with
dibasic calcium phosphate anhydrous or sodium starch glycolate.
Finally, the formulation is presented with lubricants, such as talc
or magnesium stearate.
[0374] For parenteral administration (i.e., administration by
injection through a route other than the alimentary canal), the
compounds of the invention are combined with a sterile aqueous
solution that is isotonic with the blood of the subject. Such a
formulation is prepared by dissolving a solid active ingredient in
water containing physiologically-compatible substances, such as
sodium chloride, glycine and the like, and having a buffered pH
compatible with physiological conditions, so as to produce an
aqueous solution, then rendering said solution sterile. The
formulation is presented in unit or multi-dose containers, such as
sealed ampoules or vials. The formulation is delivered by any mode
of injection, including, without limitation, epifascial,
intracapsular, intracranial, intracutaneous, intrathecal,
intramuscular, intraorbital, intraperitoneal, intraspinal,
intrasternal, intravascular, intravenous, parenchymatous,
subcutaneous, or sublingual or by way of catheter into the
subject's heart.
[0375] For transdermal administration, the compounds of the
invention are combined with skin penetration enhancers, such as
propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic
acid, N-methylpyrrolidone and the like, which increase the
permeability of the skin to the compounds of the invention and
permit the compounds to penetrate through the skin and into the
bloodstream. The compounds of the invention/enhancer composition
also are further combined with a polymeric substance, such as
ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate,
polyvinyl pyrrolidone, and the like, to provide the composition in
gel form, which are dissolved in a solvent, such as methylene
chloride, evaporated to the desired viscosity and then applied to
backing material to provide a patch.
[0376] In some embodiments, the composition is in unit dose form
such as a tablet, capsule or single-dose vial. Suitable unit doses,
i.e., therapeutically effective amounts, can be determined during
clinical trials designed appropriately for each of the conditions
for which administration of a chosen compound is indicated and
will, of course, vary depending on the desired clinical endpoint.
The present invention also provides articles of manufacture for
treating and preventing disorders, such as cardiac disorders, in a
subject. The articles of manufacture comprise a pharmaceutical
composition of one or more of the compounds of the invention as
described herein. The articles of manufacture are packaged with
indications for various disorders that the pharmaceutical
compositions are capable of treating and/or preventing. For
example, the articles of manufacture comprise a unit dose of a
compound disclosed herein that is capable of treating or preventing
a muscular disorder, and an indication that the unit dose is
capable of treating or preventing a certain disorder, for example
an arrhythmia.
[0377] In accordance with a method of the present invention, the
compounds of the invention are administered to the subject (or are
contacted with cells of the subject) in an amount effective to
limit or prevent a decrease in the level of RyR-bound FKBP in the
subject, particularly in cells of the subject. This amount is
readily determined by the skilled artisan, based upon known
procedures, including analysis of titration curves established in
vivo and methods and assays disclosed herein. A suitable amount of
the compounds of the invention effective to limit or prevent a
decrease in the level of RyR-bound FKBP in the subject ranges from
about 5 mg/kg/day to about 20 mg/kg/day, and/or is an amount
sufficient to achieve plasma levels ranging from about 300 ng/ml to
about 1000 ng/ml. In an embodiment, the amount of compounds from
the invention ranges from about 10 mg/kg/day to about 20 mg/kg/day.
In another embodiment, from about 0.01 mg/kg/day to about 10
mg/kg/day is administered. In another embodiment, from about 0.01
mg/kg/day to about 5 mg/kg/day is administered. In another
embodiment, from about 0.05 mg/kg/day to about 5 mg/kg/day is
administered. In another, preferred embodiment, from about 0.05
mg/kg/day to about 1 mg/kg/day is administered.
Uses
[0378] The present invention provides a new range of therapeutic
treatments for patients with various disorders involving modulation
of the RyR receptors, particularly skeletal muscular disorders
(RyR1), cardiac (RyR2) disorders, and cognitive (RyR3)
disorders.
[0379] In one embodiment of the present invention, the subject has
not yet developed a disorder, such as cardiac disorders (e.g.,
exercise-induced cardiac arrhythmia). In another embodiment of the
present invention, the subject is in need of treatment for a
disorder, including a cardiac disorder.
[0380] In one embodiment of the present invention, the subject has
not yet developed symptoms of muscle fatigue, (e.g.,
exercise-induced muscle fatigue). In another embodiment of the
present invention, the subject is in need of treatment for a
disorder associated with muscle fatigue, including skeletal
muscular disorders.
[0381] Various disorders that the compounds of the invention treat
or prevent include, but are not limited to, cardiac disorders and
diseases, skeletal muscular disorders and diseases, cognitive
disorders and diseases, malignant hyperthermia, diabetes, and
sudden infant death syndrome. Cardiac disorder and diseases
include, but are not limited to, irregular heartbeat disorders and
diseases; exercise-induced irregular heartbeat disorders and
diseases; sudden cardiac death; exercise-induced sudden cardiac
death; congestive heart failure; chronic obstructive pulmonary
disease; and high blood pressure. Irregular heartbeat disorders and
diseases include and exercise-induced irregular heartbeat disorders
and diseases include, but are not limited to, atrial and
ventricular arrhythmia; atrial and ventricular fibrillation; atrial
and ventricular tachyarrhythmia; atrial and ventricular
tachycardia; catecholaminergic polymorphic ventricular tachycardia
(CPVT); and exercise-induced variants thereof. Skeletal muscular
disorder and diseases include, but are not limited to, skeletal
muscle fatigue, exercise-induced skeletal muscle fatigue, muscular
dystrophy, bladder disorders, and incontinence. Cognitive disorders
and diseases include, but are not limited to, Alzheimer's Disease,
forms of memory loss, and age-dependent memory loss. One skilled in
the art will recognize still other diseases, including but not
limited to muscular and cardiac disorders, that the compounds of
the invention are useful to treat, in accordance with the
information provided herein.
[0382] The amount of compounds of the invention effective to limit
or prevent a decrease in the level of RyR2-bound FKBP 12.6 in the
subject is an amount effective to prevent exercise-induced cardiac
arrhythmia in the subject. Cardiac arrhythmia is a disturbance of
the electrical activity of the heart that manifests as an
abnormality in heart rate or heart rhythm. As used herein, an
amount of compounds of the invention "effective to prevent
exercise-induced cardiac arrhythmia" includes an amount of
compounds of Formula I, I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h,
I-i, I-j, I-k, I-k-1, I-l, I-l-1, I-m, I-m-1, I-n, I-o, I-p, and
II, effective to prevent the development of the clinical impairment
or symptoms of the exercise-induced cardiac arrhythmia (e.g.,
palpitations, fainting, ventricular fibrillation, ventricular
tachycardia and sudden cardiac death). The amount of the compounds
effective to prevent exercise-induced cardiac arrhythmia in a
subject will vary depending upon the particular factors of each
case, including the type of exercise-induced cardiac arrhythmia,
the subject's weight, the severity of the subject's condition, and
the mode of administration of the compounds. This amount is readily
determined by the skilled artisan, based upon known procedures,
including clinical trials, and methods disclosed herein. In one
embodiment, the amount of the compounds of the invention effective
to prevent the exercise-induced cardiac arrhythmia is an amount
effective to prevent exercise-induced sudden cardiac death in the
subject. In another embodiment, the compounds of the invention
prevent exercise-induced cardiac arrhythmia and exercise-induced
sudden cardiac death in the subject.
[0383] The amount of compounds of the invention effective to limit
or prevent a decrease in the level of RyR1-bound FKBP12 in the
subject is an amount effective to prevent muscle fatigue in the
subject. This amount is readily determined by the skilled artisan,
based upon known procedures, including clinical trials, and methods
disclosed herein.
[0384] Because of its ability to stabilize RyR-bound FKBP and
maintain and restore balance in the context of dynamic PKA
phosphorylation and dephosphorylation of RyR, the compounds of the
invention are also useful in treating a subject who has already
experienced clinical symptoms of these various disorders. For
example, if the symptoms of the disorder are observed in the
subject early enough, the compounds of the invention are effective
in limiting or preventing a further decrease in the level of
RyR-bound FKBP in the subject.
[0385] Additionally, the subject of the present invention is a
candidate for exercise-induced cardiac disorders, such as
exercise-induced cardiac arrhythmia. Exercise-induced cardiac
arrhythmia is a heart condition (e.g., a ventricular fibrillation
or ventricular tachycardia, including any that leads to sudden
cardiac death) that develops during/after a subject has undergone
physical exercise. A "candidate" for an exercise-induced cardiac
disorder is a subject who is known to be, or is believed to be, or
is suspected of being, at risk for developing a cardiac disorder
during/after physical exercise. Examples of candidates for
exercise-induced cardiac arrhythmia include, without limitation, an
animal/person known to have catecholaminergic polymorphic
ventricular tachycardia (CPVT); an animal/person suspected of
having CPVT; and an animal/person who is known to be, or is
believed to be, or is suspected of being at risk for developing
cardiac arrhythmia during/after physical exercise, and who is about
to exercise, is currently exercising or has just completed
exercise. As discussed above, CPVT is an inherited disorder in
individuals with structurally-normal hearts. It is characterized by
stress-induced ventricular tachycardia--a lethal arrhythmia that
causes sudden cardiac death. In subjects with CPVT, physical
exertion and/or stress induce bidirectional and/or polymorphic
ventricular tachycardias that lead to sudden cardiac death (SCD) in
the absence of detectable structural heart disease. Individuals
with CPVT have ventricular arrhythmias when subjected to exercise,
but do not develop arrhythmias at rest.
[0386] Also, the subject of the present invention can be a
candidate for muscle fatigue disorder, including but not limited to
any chronic disorder associated with muscle fatigue, or stress or
exercise-induced muscle fatigue.
[0387] Accordingly, in still another embodiment of the present
invention, the subject has been exercising, or is currently
exercising, and has developed an exercise-induced disorder. In this
case, the amount of the compounds of the invention effective to
limit or prevent a decrease in the level of RyR-bound FKBP in the
subject is an amount of compound effective to treat the
exercise-induced disorder in the subject. As used herein, an amount
of compounds of the invention "effective to treat an
exercise-induced disorder" includes an amount of compound of the
invention effective to alleviate or ameliorate the clinical
impairment or symptoms of the exercise-induced disorder
characterized by muscle fatigue or an amount of compound of the
invention effective to alleviate or ameliorate the clinical
impairment or symptoms of the exercise-induced disorder (e.g., in
the case of cardiac arrhythmia, palpitations, fainting, ventricular
fibrillation, ventricular tachycardia, and sudden cardiac death).
The amount of compounds of the invention effective to treat an
exercise-induced disorder in a subject will vary depending upon the
particular factors of each case, including the type of
exercise-induced disorder, the subject's weight, the severity of
the subject's condition, and the mode of administration of the
compounds of the invention. This amount is readily determined by
the skilled artisan, based upon known procedures, including
clinical trials, and methods disclosed herein. In an embodiment,
the compounds of the invention treat exercise-induced disorders in
the subject.
[0388] The present invention further provides a method for treating
exercise-induced disorders in a subject. The method comprises
administering the compounds of the invention to the subject in an
amount effective to treat the exercise-induced disorder in the
subject. A suitable amount of the compounds of the invention
effective to treat, for example, exercise-induced cardiac
arrhythmia in the subject ranges from about 5 mg/kg/day to about 20
mg/kg/day, and/or is an amount sufficient to achieve plasma levels
ranging from about 300 ng/ml to about 1000 ng/ml. The present
invention also provides a method for preventing an exercise-induced
disorder in a subject. The method comprises administering the
compounds of the invention to the subject in an amount effective to
prevent the exercise-induced disorder in the subject. A suitable
amount of the compounds of the invention effective to prevent the
exercise-induced disorder in the subject ranges from about 5
mg/kg/day to about 20 mg/kg/day, and/or is an amount sufficient to
achieve plasma levels ranging from about 300 ng/ml to about 1000
ng/ml. Additionally, the present invention provides a method for
preventing exercise-induced disorders in a subject. The method
comprises administering the compounds of the invention to the
subject in an amount effective to prevent an exercise-induced
disorder in the subject. A suitable amount of the compounds of the
invention effective to prevent an exercise-induced disorder in the
subject ranges from about 5 mg/kg/day to about 20 mg/kg/day, and/or
is an amount sufficient to achieve plasma levels ranging from about
300 ng/ml to about 1000 ng/ml.
[0389] Additionally, the compounds prevent irregular heartbeat
disorders in subjects with heterozygous defects in the FKBP 12.6
gene.
[0390] The compounds of the invention can be used alone, in
combination with each other, or in combination with other agents
that have cardiovascular activity including, but not limited to,
diuretics, anticoagulants, antiplatelet agents, antiarrhythmics,
inotropic agents, chronotropic agents, .alpha. and .beta. blockers,
angiotensin inhibitors and vasodilators. Further, such combinations
of the compounds of the present invention and other cardiovascular
agents are administered separately or in conjunction. In addition,
the administration of one element of the combination is prior to,
concurrent to or subsequent to the administration of other
agent(s).
[0391] In various embodiments of the above-described methods, the
exercise-induced cardiac arrhythmia in the subject is associated
with VT. In some embodiments, the VT is CPVT. In other embodiments
of these methods, the subject is a candidate for exercise-induced
cardiac arrhythmia, including candidates for exercise-induced
sudden cardiac death.
[0392] In view of the foregoing methods, the present invention also
provides use of the compounds of the invention in a method for
limiting or preventing a decrease in the level of RyR-bound FKBP in
a subject who is a candidate for a disorder. The present invention
also provides use of the compounds of the invention in a method for
treating or preventing a muscular disorder in a subject.
Furthermore, the present invention provides use of the compounds of
the invention in a method for treating or preventing
exercise-induced muscular disorders in a subject.
[0393] Accordingly, the present invention further provides a method
for assaying the effects of the compounds of the invention in
preventing disorders and diseases associated with the RyR
receptors. The method comprises the steps of: (a) obtaining or
generating a culture of cells containing RyR; (b) contacting the
cells with one or more of the compounds of the invention; (c)
exposing the cells to one or more conditions known to increase
phosphorylation of RyR in cells; and (d) determining if the one or
more compounds of the invention limits or prevents a decrease in
the level of RyR-bound FKBP in the cells. As used herein, a cell
"containing RyR" is a cell in which RyR, including RyR1, RyR2, and
RyR3, or a derivative or homologue thereof, is naturally expressed
or naturally occurs. Conditions known to increase phosphorylation
of RyR in cells include, without limitation, PKA.
[0394] In the method of the present invention, cells are contacted
with one or more of the compounds of the invention by any of the
standard methods of effecting contact between drugs/agents and
cells, including any modes of introduction and administration
described herein. The level of RyR-bound FKBP in the cell is
measured or detected by known procedures, including any of the
methods, molecular procedures and assays known to one of skill in
the art or described herein. In one embodiment of the present
invention, the one or more compounds of the invention limits or
prevents a decrease in the level of RyR-bound FKBP in the
cells.
[0395] RyR, including RyR1, RyR2, and RyR3, has been implicated in
a number of biological events in cells. For example, it has been
shown that RyR2 channels play an important role in EC coupling and
contractility in cardiac muscle cells. Therefore, it is clear that
preventive drugs designed to limit or prevent a decrease in the
level of RyR-bound FKBP in cells, particularly RyR2-bound FKPB12.6
in cardiac muscle cells, are useful in the regulation of a number
of RyR-associated biological events, including EC coupling and
contractility. Thus, the one or more compounds of the invention are
evaluated for effect on EC coupling and contractility in cells,
particularly cardiac muscle cells, and therefore, usefulness for
preventing exercise-induced sudden cardiac death.
[0396] Accordingly, the method of the present invention further
comprises the steps of contacting one or more compounds of the
invention with a culture of cells containing RyR; and determining
if the one or more compounds has an effect on an RyR-associated
biological event in the cells. As used herein, a "RyR-associated
biological event" includes a biochemical or physiological process
in which RyR levels or activity have been implicated. As disclosed
herein, examples of RyR-associated biological events include,
without limitation, EC coupling and contractility in cardiac muscle
cells. According to this method of the present invention, the one
or more compounds are contacted with one or more cells (such as
cardiac muscle cells) in vitro. For example, a culture of the cells
is incubated with a preparation containing the one or more
compounds of the invention. The compounds' effect on a
RyR-associated biological event then is assessed by any biological
assays or methods known in the art, including immunoblotting,
single-channel recordings and any others disclosed herein.
[0397] The present invention is further directed to one or more
compounds of the invention, identified by the above-described
identification method, as well as a pharmaceutical composition
comprising the compound and a pharmaceutically acceptable carrier
and/or diluent. The compounds are useful for preventing
exercise-induced sudden cardiac death in a subject, and for
treating or preventing other RyR-associated conditions. As used
herein, a "RyR-associated condition" is a condition, disease, or
disorder in which RyR level or activity has been implicated, and
includes an RyR-associated biological event. The RyR-associated
condition is treated or prevented in the subject by administering
to the subject an amount of the compound effective to treat or
prevent the RyR-associated condition in the subject. This amount is
readily determined by one skilled in the art. In one embodiment,
the present invention provides a method for preventing
exercise-induced sudden cardiac death in a subject, by
administering the one or more compounds of the invention to the
subject in an amount effective to prevent the exercise-induced
sudden cardiac death in the subject.
[0398] The present invention also provides an in vivo method for
assaying the effectiveness of the compounds of the invention in
preventing disorders and diseases associated with the RyR
receptors. The method comprises the steps of: (a) obtaining or
generating an animal containing RyR; (b) administering one or more
of the compounds of the invention to the animal; (c) exposing the
animal to one or more conditions known to increase phosphorylation
of RyR in cells; and (d) determining the extent the compound limits
or prevents a decrease in the level of RyR-bound FKBP in the
animal. The method further comprises the steps of: (e)
administering one or more of the compounds of the invention to an
animal containing RyR; and (f) determining the extent of the effect
of the compound on a RyR-associated biological event in the animal.
Also provided is a pharmaceutical composition comprising this
compound; and a method for preventing exercise-induced sudden
cardiac death in a subject, by administering this compound to the
subject in an amount effective to prevent the exercise-induced
sudden cardiac death in the subject.
[0399] It has been demonstrated that compounds which block PKA
activation would be expected to reduce the activation of the RyR
channel, resulting in less release of calcium into the cell.
Compounds that bind to the RyR channel at the FKBP binding site,
but do not come off the channel when the channel is phosphorylated
by PKA, would also be expected to decrease the activity of the
channel in response to PKA activation or other triggers that
activate the RyR channel. Such compounds would also result in less
calcium release into the cell.
[0400] By way of example, the diagnostic assays screen for the
release of calcium into cells via the RyR channel, using
calcium-sensitive fluorescent dyes (e.g., Fluo-3, Fura-2, and the
like). Cells are loaded with the fluorescent dye of choice, then
stimulated with RyR activators to determine the reduction of the
calcium-dependent fluorescent signal (Brillantes, et al.,
Stabilization of calcium release channel (ryanodine receptor)
function by FK506-binding protein. Cell, 77:513-23, 1994; Gillo, et
al., Calcium entry during induced differentiation in murine
erythroleukemia cells. Blood, 81:783-92, 1993; Jayaraman, et al.,
Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine
phosphorylation. Science, 272:1492-94, 1996). Calcium-dependent
fluorescent signals are monitored with a photomultiplier tube, and
analyzed with appropriate software. This assay can easily be
automated to screen the compounds of the invention using multiwell
dishes.
[0401] To demonstrate that the compounds of inhibit the
PKA-dependent activation of RyR-mediated intracellular calcium
release, an assay involves the expression of recombinant RyR
channels in a heterologous expression system, such as Sf9, HEK293,
or CHO cells. RyR could also be co-expressed with beta-adrenergic
receptors. This would permit assessment of the effect of compounds
of the invention on RyR activation, in response to addition of
beta-adrenergic receptor agonists.
[0402] The level of PKA phosphorylation of RyR2 which correlates
with the degree of heart failure also is assayed and then used to
determine the efficacy of the one or more compounds of the
invention to block the PKA phosphorylation of the RyR2 channel.
Such an assay is based on the use of antibodies that are specific
for the RyR2 protein. For example, the RyR2-channel protein is
immunoprecipitated and then back-phosphorylated with PKA and
[gamma.sup.32P]-ATP. The amount of radioactive [.sup.32P] label
that is transferred to the RyR2 protein then is measured using a
phosphorimager (Marx, et al., PKA phosphorylation dissociates
FKBP12.6 from the calcium release channel (ryanodine receptor):
defective regulation in failing hearts. Cell, 101:365-76,
2000).
[0403] Another assay of the compounds of the invention involves use
of a phosphoepitope-specific antibody that detects RyR1 that is PKA
phosphorylated on Ser 2843 or RyR2 that is PKA phosphorylated on
Ser 2809. Immunoblotting with such an antibody can be used to
assess efficacy of these compounds for therapy for heart failure
and cardiac arrhythmias. Additionally, RyR2 S2809A and RyR2 S2809D
knock-in mice are used to assess efficacy of therapy for heart
failure and cardiac arrhythmias. Such mice further provide evidence
that PKA hyperphosphorylation of RyR2 is a contributing factor in
heart failure and cardiac arrhythmias by showing that the RyR2
S2809A mutation inhibits heart failure and arrhythmias, and that
the RyR2 S2809D mutation worsens heart failure and arrhythmias.
[0404] Therefore, in a specific embodiment, the present invention
provides a method of treating heart failure, atrial fibrillation or
exercise-induced cardiac arrhythmia, comprising administering to an
animal in need thereof, a therapeutically effective amount of a
compound selected from the compounds of the invention.
[0405] Intracellular Ca.sup.2+ leak is proposed as a principal
mediator of depressed muscle performance and dystrophic muscle
remodeling. Muscular dystrophies are heterogeneous hereditary
diseases characterized by weakness and progressive muscle wasting.
Of all forms of muscular dystrophies involving the
dystrophin-associated protein complex (referred to as
dystrophinopathies), Duchenne muscular dystrophy (DMD) is one of
the most frequent genetic diseases (X-linked; 1 in 3,500 boys) with
death usually occurring before age 30 by respiratory and/or cardiac
failure in high numbers of patients. Becker muscular dystrophy
(BMD) represents a milder form of the disease associated with a
reduction in the amount or expression of a truncated form of the
dystrophin protein whereas Duchenne patients have been
characterized by complete absence or very low levels of dystrophin.
Duchenne and Becker's muscular dystrophy (DMD/BMD) are caused by
mutations in the gene encoding the 427-kDa cytoskeletal protein
dystrophin. However, with increasing age in BMD cardiac symptoms
are more common than in DMD patients and do not correlate with
skeletal muscle symptoms. Since genetic screening will not
eliminative DMD due to a high incidence of sporadic cases, an
effective therapy is highly desirable. DMD/BMD have been
consistently associated with disturbed intracellular calcium
metabolism. Because alterations of intracellular Ca.sup.2+
concentrations in DMD myofibers are believed to represent a central
pathogenic mechanism, development of a therapeutic intervention
that prevents intracellular Ca.sup.2+ abnormalities as a cause of
skeletal muscle degeneration is highly desirable.
[0406] It is well established that lack of dystrophin expression is
the primary genetic defect in DMD and BMD. However, the key
mechanism leading to progressive muscle damage is largely unknown.
It has been suggested that elevations of intracellular Ca.sup.2+
concentrations ([Ca.sup.2+].sub.i) under resting conditions
directly contributed to toxic muscle cell (myofiber) damage and
concurrent activation of Ca.sup.2+-dependent proteases. Since
calpain activity is increased in necrotic muscle fibers of mdx mice
and calpain dysfunction contributes to limb-girdle muscular
dystrophy, preventing activation of calcium-dependent proteases by
inhibiting intracellular Ca.sup.2+ elevations represents a strategy
to prevent muscle wasting in DMD. Significant differences in
[Ca.sup.2+].sub.i between normal and dystrophic muscles have been
reported in myotubes and animal models including the
dystrophin-deficient mdx mouse. Intracellular Ca.sup.2+ elevations
are prevented by administration of a pharmaceutical composition
comprising a compound of the invention.
[0407] The present invention also provides a method of diagnosis of
a disease or disorder in a subject, said method comprising:
obtaining a cell or tissue sample from the subject; obtaining DNA
from the cell or tissue; comparing the DNA from the cell or tissue
with a control DNA encoding RyR to determine whether a mutation is
present in the DNA from the cell or tissue, the presence of a
mutation indicating a disease or disorder. In one embodiment, the
mutation is a RyR2 mutation on chromosome 1q42-q43. In another
embodiment, the mutation is one or more CPTV mutations. In another
embodiment, the mutation may be a mutation that is present in the
DNA encoding RyR2 of a SIDS subject. The diagnostic method is used
to detect the presence of a disease or disorder in an adult, a
child or a fetus. The disease and disorders include, but are not
limited to, cardiac disorders and diseases, skeletal muscular
disorders and diseases, cognitive disorders and diseases, malignant
hyperthermia, diabetes, and sudden infant death syndrome. Cardiac
disorder and diseases include, but are not limited to, irregular
heartbeat disorders and diseases; exercise-induced irregular
heartbeat disorders and diseases; sudden cardiac death;
exercise-induced sudden cardiac death; congestive heart failure;
chronic obstructive pulmonary disease; and high blood pressure.
Irregular heartbeat disorders and diseases include and
exercise-induced irregular heartbeat disorders and diseases
include, but are not limited to, atrial and ventricular arrhythmia;
atrial and ventricular fibrillation; atrial and ventricular
tachyarrhythmia; atrial and ventricular tachycardia;
catecholaminergic polymorphic ventricular tachycardia (CPVT); and
exercise-induced variants thereof. Skeletal muscular disorder and
diseases include, but are not limited to, skeletal muscle fatigue,
exercise-induced skeletal muscle fatigue, muscular dystrophy,
bladder disorders, and incontinence. Cognitive disorders and
diseases include, but are not limited to, Alzheimer's Disease,
forms of memory loss, and age-dependent memory loss.
[0408] The present invention further provides a method of diagnosis
of disorders and diseases in a subject, said method comprising:
obtaining cells or tissue sample from the subject; incubating the
cells or tissue sample with the compound of the invention under
conditions which increase phosphorylation of RyR in cells;
determining (a) whether RyR bound to calstabin (i.e. RyR1 bound to
calstabin1, RyR2 bound to calstabin2, or RyR3 bound to calstabin1)
is increased in the cells or tissue compared to RyR bound to
calstabin in control cells or tissues said control cells or tissues
lacking mutant RyR calcium channels, or (b) whether a decrease in
release of calcium occurs in RyR channels compared to a lack of
decrease in release of calcium in the control cells; an increase in
RyR-bound calstabin in (a) indicating a disorder or disease in the
subject or a decrease in release of calcium in RyR channels in (b)
compared to the control cells indicating a cardiac disease or
disorder in the subject. The diagnostic method is used to detect
the presence of a disease or disorder in an adult, a child or a
fetus. The disease and disorders include, but are not limited to,
cardiac disorders and diseases, skeletal muscular disorders and
diseases, cognitive disorders and diseases, malignant hyperthermia,
diabetes, and sudden infant death syndrome. Cardiac disorder and
diseases include, but are not limited to, irregular heartbeat
disorders and diseases; exercise-induced irregular heartbeat
disorders and diseases; sudden cardiac death; exercise-induced
sudden cardiac death; congestive heart failure; chronic obstructive
pulmonary disease; and high blood pressure. Irregular heartbeat
disorders and diseases include and exercise-induced irregular
heartbeat disorders and diseases include, but are not limited to,
atrial and ventricular arrhythmia; atrial and ventricular
fibrillation; atrial and ventricular tachyarrhythmia; atrial and
ventricular tachycardia; catecholaminergic polymorphic ventricular
tachycardia (CPVT); and exercise-induced variants thereof. Skeletal
muscular disorder and diseases include, but are not limited to,
skeletal muscle fatigue, exercise-induced skeletal muscle fatigue,
muscular dystrophy, bladder disorders, and incontinence. Cognitive
disorders and diseases include, but are not limited to, Alzheimer's
Disease, forms of memory loss, and age-dependent memory loss.
[0409] The present invention further provides a method of diagnosis
of a cardiac disorder or disease in a subject, said method
comprising: obtaining cardiac cells or tissue sample from the
subject; incubating the cardiac cells or tissue sample with the
compound of the invention, under conditions which increase
phosphorylation of RyR2 in cells; determining (a) whether RyR2
bound to calstabin2 is increased in the cells or tissue compared to
RyR2 bound to calstabin2 in control cells or tissues said control
cells or tissues lacking mutant RyR2 calcium channels, or (b)
whether a decrease in release of calcium occurs in RyR2 channels
compared to a lack of decrease in release of calcium in the control
cells; an increase in RyR2-bound calstabin2 in (a) indicating a
disorder or disease in the subject or a decrease in release of
calcium in RyR2 channels in (b) compared to the control cells
indicating a cardiac disease or disorder in the subject. The
provided method is used to diagnose CPTV. The provided method also
is used to diagnose sudden infant death syndrome (SIDS). The
provided method additionally is used to diagnose cardiac irregular
heartbeat disorders and diseases; exercise-induced irregular
heartbeat disorders and diseases; sudden cardiac death;
exercise-induced sudden cardiac death; congestive heart failure;
chronic obstructive pulmonary disease; and high blood pressure.
Irregular heartbeat disorders and diseases include and
exercise-induced irregular heartbeat disorders and diseases
include, but are not limited to, atrial and ventricular arrhythmia;
atrial and ventricular fibrillation; atrial and ventricular
tachyarrhythmia; atrial and ventricular tachycardia;
catecholaminergic polymorphic ventricular tachycardia (CPVT); and
exercise-induced variants thereof.
[0410] In addition to the above-mentioned therapeutic uses, the
compounds of the invention are also useful in diagnostic assays,
screening assays and as research tools.
Methods of Synthesis
[0411] The present invention provides, in a further aspect,
processes for the preparation of a compound of invention, and
salts, solvates, hydrates, complexes, and prodrugs thereof, and
pharmaceutically acceptable salts of such prodrugs. These processes
are disclosed in the applications that are incorporate by reference
in the first paragraph of this document, as well as in U.S. Pat.
No. 7,704,409 and U.S. application Ser. No. 10/680,988 and Ser. No.
12/480,396, the content of each of which is also expressly
incorporated herein by reference thereto.
[0412] It should be noted that the compounds used as starting
materials for, or generated as intermediates in, the synthesis of
the compounds of the invention, may themselves have structures
encompassed by the formulae of the invention, and/or may themselves
be active agents useful in the methods and compositions of the
present invention. Such starting materials and intermediates may be
useful for, inter alia, treating or preventing various disorders
and diseases associated with RyR receptors such as muscular and
cardiac disorders, treating or preventing a leak in a RyR2 receptor
in a subject, or modulating the binding of RyR and FKBP in a
subject. The present invention encompasses any of the starting
materials or intermediates disclosed herein that have structures
encompassed by Formula I, I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h,
I-i, I-j, I-k, I-k-1, I-l, I-l-1, I-m, I-m-1, I-n, I-o, I-p or II,
and/or which are useful as active agents in the methods and
compositions of the present invention. For example, in one
embodiment the compound S68, which is useful as a starting material
for the synthesis of compounds S69-S75, may be used for, inter
alia, treating or preventing various disorders and diseases
associated with RyR receptors, treating or preventing a leak in a
RyR2 receptor, or modulating the binding of RyR and FKBP in a
subject.
[0413] In another embodiment, the compound S26, which is useful in
the synthesis of many of the compounds described herein (including
S3, S4, S5, S7, S9, S11, S12, S13, S14 and other compounds) may be
used for, inter alia, treating or preventing various disorders and
diseases associated with RyR receptors, treating or preventing a
leak in a RyR2 receptor, or modulating the binding of RyR and FKBP
in a subject.
[0414] Similarly, in another embodiment, the compound S25 (see U.S.
Pat. No. 7,718,644) may also be used for, inter alia, treating or
preventing various disorders and diseases associated with RyR
receptors, treating or preventing a leak in a RyR2 receptor, or
modulating the binding of RyR and FKBP in a subject.
[0415] The compounds of the present invention are prepared in
different forms, such as salts, hydrates, solvates, complexes,
prodrugs or salts of prodrugs and the invention includes all
variant forms of the compounds.
[0416] The present invention further provides a composition,
comprising radio labeled compounds of the invention. Labeling of
the compounds of the invention is accomplished using one of a
variety of different radioactive labels known in the art. The
radioactive label of the present invention is, for example, a
radioisotope. The radioisotope is any isotope that emits detectable
radiation including, without limitation, .sup.35S, .sup.125I,
.sup.3H, or .sup.14C. Radioactivity emitted by the radioisotope can
be detected by techniques well known in the art. For example, gamma
emission from the radioisotope is detected using gamma imaging
techniques, particularly scintigraphic imaging.
[0417] By way of non-limiting example, radio-labeled compounds of
the invention are prepared as follows. A compound of the invention
is demethylated at the phenyl ring using BBr.sub.3. The resulting
phenol compound then is re-methylated with a radio-labeled
methylating agent (such as .sup.3H-dimethyl sulfate) in the
presence of a base (such as NaH) to provide .sup.3H-labeled
compounds.
[0418] Using forced swimming as an efficient protocol to increase
skeletal muscle aerobic capacity in mice, the composition and
phosphorylation status of the skeletal RyR1 channel complex have
been investigated. Unexpectedly, after 3 weeks of 90 mins swimming
twice daily, C57Bl6 wild-type mice showed significantly increased
RyR1 phosphorylation by PKA while Ca.sup.2+-calmodulin kinase II
(CaMKII) phosphorylation was not changed indicating specificity of
the stress pathway RyR1 protein expression was stable, however,
RyR1 channels were depleted of the stabilizing subunit calstabin1
(FKBP12). It has been shown that RyR1 hyperphosphorylation and
calstabin1 depletion are consistent with leaky RyR1 channels that
cause intracellular SR Ca.sup.2+ leak.
[0419] RyR1 channels are PKA hyperphosphorylated and depleted of
the stabilizing calstabin1 subunit after 3 weeks of 90 mins
swimming twice daily. The immunoprecipitated RyR1 macromolecular
channel complex shows increased PKA phosphorylation at Ser-2844
(corresponding to human RyR1-Ser-2843) whereas CaMKII
phosphorylation at Ser-2849 (corresponding to human RyR1-Ser-2848)
is unchanged. Concomitant with increased RyR1-Ser-2844 PKA
hyperphosphorylation, calstabin1 is depleted from the channel
complex. Normalization of phosphorylation and calstabin1 content to
four subunits of the tetrameric channel complex shows a significant
in increase in PKA phosphorylation and depletion of the stabilizing
calstabin1 subunit.
[0420] The compounds of the present invention are prepared in
different forms, such as salts, hydrates, solvates, complexes,
pro-drugs or salts of prodrugs and the invention includes all
variant forms of the compounds.
[0421] The term "compound(s) of the invention" as used herein means
a compound of Formula I, I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h,
I-i, I-j, I-k, I-k-1, I-l, I-l-1, I-m, I-m-1, I-n, I-o, I-p and II,
and salts, hydrates, prodrugs and solvates thereof.
[0422] A "pharmaceutical composition" refers to a mixture of one or
more of the compounds described herein, or pharmaceutically
acceptable salts, hydrates or pro-drugs thereof, with other
chemical components, such as physiologically acceptable carriers
and excipients. The purpose of a pharmaceutical composition is to
facilitate administration of a compound to an organism.
[0423] A "prodrug" refers to an agent which is converted into the
parent drug in vivo. Pro-drugs are often useful because, in some
situations, they are easier to administer than the parent drug.
They are bioavailable, for instance, by oral administration whereas
the parent drug is not. The prodrug also has improved solubility in
pharmaceutical compositions over the parent drug. For example, the
compound carries protective groups which are split off by
hydrolysis in body fluids, e.g., in the bloodstream, thus releasing
active compound or is oxidized or reduced in body fluids to release
the compound.
[0424] A compound of the present invention also can be formulated
as a pharmaceutically acceptable salt, e.g., acid addition salt,
and complexes thereof. The preparation of such salts can facilitate
the pharmacological use by altering the physical characteristics of
the agent without preventing its physiological effect. Examples of
useful alterations in physical properties include, but are not
limited to, lowering the melting point to facilitate transmucosal
administration and increasing the solubility to facilitate
administering higher concentrations of the drug.
[0425] The compounds of the present invention form hydrates or
solvates, which are included in the scope of the claims. When the
compounds of the present invention exist as regioisomers,
configurational isomers, conformers or diasteroisomeric forms all
such forms and various mixtures thereof are included in the scope
of Formula I, I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j,
I-k, I-k-1, I-l, I-l-1, I-m, I-m-1, I-n, I-o, I-p and II. It is
possible to isolate individual isomers using known separation and
purification methods, if desired. For example, when a compound of
the present invention is a racemate, the racemate can be separated
into the (S)-compound and (R)-compound by optical resolution.
Individual optical isomers and mixtures thereof are included in the
scope of Formula I, I-a, I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i,
I-j, I-k, I-k-1, I-l, I-l-1, I-m, I-m-1, I-n, I-o, I-p and II.
[0426] The terms "animal," "subject" and "patient" as used herein
include all members of the animal kingdom including, but not
limited to, mammals, animals (e.g., cats, dogs, horses, etc.) and
humans.
[0427] The present invention further provides a composition,
comprising radio labeled compounds of Formula I, I-a, I-b, I-c,
I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-k-1, I-l, I-l-1, I-m,
I-m-1, I-n, I-o, and I-p. Labeling of the compounds is accomplished
using one of a variety of different radioactive labels known in the
art. The radioactive label of the present invention is, for
example, a radioisotope. The radioisotope is any isotope that emits
detectable radiation including, without limitation, .sup.35s,
.sup.125I, .sup.3H, or .sup.14C. Radioactivity emitted by the
radioisotope can be detected by techniques well known in the art.
For example, gamma emission from the radioisotope is detected using
gamma imaging techniques, particularly scintigraphic imaging.
[0428] By way of example, radio-labeled compounds of the invention
are prepared as follows. A compound of the invention may be
demethylated at the phenyl ring using BBr.sub.3. The resulting
phenol compound then is re-methylated with a radio-labeled
methylating agent (such as .sup.3H-dimethyl sulfate) in the
presence of a base (such as NaH) to provide .sup.3H-labeled
compounds.
[0429] In accordance with the method of the present invention, the
decrease in the level of RyR-bound FKBP is limited or prevented in
the subject by decreasing the level of phosphorylated RyR in the
subject. In one embodiment, the amount of the agent effective to
limit or prevent a decrease in the level of RyR2-bound FKBP 12.6 in
the subject is an amount of the agent effective to treat or prevent
heart failure, atrial fibrillation and/or exercise-induced cardiac
arrhythmia in the subject. In another embodiment, the amount of the
agent effective to limit or prevent a decrease in the level of
RyR2-bound FKBP 12.6 in the subject is an amount of the agent
effective to prevent exercise-induced sudden cardiac death in the
subject.
[0430] In view of the foregoing, the present invention further
provides a method for treating or preventing exercise-induced
cardiac arrhythmia in a subject, comprising administering to the
subject a 1,4-benzothiazepine compound, as disclosed herein, in an
amount effective to treat or prevent exercise-induced cardiac
arrhythmia in the subject. Similarly, the present invention
provides a method for preventing exercise-induced sudden cardiac
death in a subject, comprising administering to the subject a
1,4-benzothiazepine compound, as disclosed herein, in an amount
effective to prevent exercise-induced sudden cardiac death in the
subject. Additionally, the present invention provides a method for
treating or preventing atrial fibrillation or heart failure in a
subject, comprising administering to the subject a compound, as
disclosed herein, in an amount effective to treat or prevent the
atrial fibrillation or heart failure in the subject. In each of
these methods, the compound is selected from the group of compounds
consisting of compounds of the formula:
##STR00037##
wherein, n is 0, 1, or 2; R is located at one or more positions on
the benzene ring; each R is independently selected from the group
consisting of H, halogen, --OH, --NH.sub.2, --NO.sub.2, --CN,
--N.sub.3, --SO.sub.3H, acyl, alkyl, alkoxyl, alkylamino,
cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl,
(hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein
each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl,
heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino may be substituted with one or more radicals
independently selected from the group consisting of halogen, N, O,
--S--, --CN, --N.sub.3, --SH, nitro, oxo, acyl, alkyl, alkoxyl,
alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and
(hetero-)cyclyl; R.sub.1 is selected from the group consisting of
H, oxo, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein
each alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be
substituted with one or more radicals independently selected from
the group consisting of halogen, N, O, --S--, --CN, --N.sub.3,
--SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl,
(hetero-)cycloalkyl, and (hetero-)cyclyl; R.sub.2 is selected from
the group consisting of H, --C.dbd.O(R.sub.5), --C.dbd.S(R.sub.6),
--SO.sub.2R.sub.7, --POR.sub.8R.sub.9, --(CH.sub.2)m-R.sub.10,
alkyl, aryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and
heterocyclyl; wherein each alkyl, aryl, heteroaryl, cycloalkyl,
cycloalkylalkyl, and heterocyclyl may be substituted with one or
more radicals independently selected from the group consisting of
halogen, N, O, --S--, --CN, --N.sub.3, nitro, oxo, acyl, alkyl,
alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and
(hetero-)cyclyl; R.sub.3 is selected from the group consisting of
H, CO.sub.2Y, CONY, acyl, alkyl, alkenyl, aryl, cycloalkyl, and
heterocyclyl; wherein each acyl, alkyl, alkenyl, aryl, cycloalkyl,
and heterocyclyl may be substituted with one or more radicals
independently selected from the group consisting of halogen, N, O,
--S--, --CN, --N3, --SH, nitro, oxo, acyl, alkyl, alkoxyl,
alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and
(hetero-)cyclyl; and wherein Y is selected from the group
consisting of H, alkyl, aryl, cycloalkyl, and heterocyclyl; R.sub.4
is selected from the group consisting of H, alkyl, alkenyl, aryl,
cycloalkyl, and heterocyclyl; wherein each alkyl, alkenyl, aryl,
cycloalkyl, and heterocyclyl may be substituted with one or more
radicals independently selected from the group consisting of
halogen, N, O, --S--, --CN, --N.sub.3, --SH, nitro, oxo, acyl,
alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and
(hetero-)cyclyl; R.sub.5 is selected from the group consisting of
--NR.sub.16, NHNHR.sub.16, NHOH, --OR.sub.15, CONH.sub.2NHR.sub.16,
CO.sub.2R.sub.15, CONR.sub.16, CH.sub.2X, acyl, alkenyl, aryl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl;
wherein each acyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, and heterocyclylalkyl may be substituted with one or
more radicals independently selected from the group consisting of
halogen, N, O, --S--, --CN, --N.sub.3, nitro, oxo, acyl, alkyl,
alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and
(hetero-)cyclyl; R.sub.6 is selected from the group consisting of
--OR.sub.15, NHNR.sub.16, NHOH, --NR.sub.16, CH.sub.2X, acyl,
alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl,
and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl
may be substituted with one or more radicals independently selected
from the group consisting of halogen, N, O, --S--, --CN, --N.sub.3,
nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl,
(hetero-)cycloalkyl, and (hetero-)cyclyl; R.sub.7 is selected from
the group consisting of --OR.sub.15, --NR.sub.16, NHNHR.sub.16,
NHOH, CH.sub.2X, alkyl, aryl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, and heterocyclylalkyl; wherein each alkyl, aryl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl
may be substituted with one or more radicals independently selected
from the group consisting of halogen, N, O, --S--, --CN, --N.sub.3,
nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl,
(hetero-)cycloalkyl, and (hetero-)cyclyl; R.sub.8 and R.sub.9
independently are selected from the group consisting of OH, acyl,
alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each
acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be
substituted with one or more radicals independently selected from
the group consisting of halogen, N, O, --S--, --CN, --N.sub.3,
nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl,
(hetero-)cycloalkyl, and (hetero-)cyclyl; R.sub.10 is selected from
the group consisting of NH.sub.2, OH, --SO.sub.2R.sub.11,
--NHSO.sub.2R.sub.11, C.dbd.O(R.sub.12), NHC.dbd.O(R.sub.12),
--OC.dbd.O(R.sub.12), and --POR.sub.13R.sub.14; R.sub.11, R.sub.12,
R.sub.13, and R.sub.14 independently are selected from the group
consisting of H, OH, NH.sub.2, NHNH.sub.2, NHOH, acyl, alkenyl,
alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl,
alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl,
heterocyclyl, and heterocyclylalkyl may be substituted with one or
more radicals independently selected from the group consisting of
halogen, --N--, --O--, --S--, --CN, --N.sub.3, nitro, oxo, acyl,
alkenyl, alkoxyl, alkyl, alkylamino, amino, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, and hydroxyl; X
is selected from the group consisting of halogen, CN,
CO.sub.2R.sub.15, CONR.sub.16, --NR.sub.16, --OR.sub.15,
--SO.sub.2R.sub.7, and --POR.sub.8R.sub.9; and R.sub.15 and
R.sub.16 independently are selected from the group consisting of H,
acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each
acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be
substituted with one or more radicals independently selected from
the group consisting of halogen, --N--, --O--, --S--, --CN,
--N.sub.3, nitro, oxo, acyl, alkenyl, alkoxyl, alkyl, alkylamino,
amino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl,
heterocyclylalkyl, and hydroxyl, and wherein each substituted acyl,
alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl,
cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl radical may
itself be substituted with one or more radicals independently
selected from the group consisting of halogen, --N--, --O--, --S--,
--CN, --N.sub.3, nitro, oxo, acyl, alkenyl, alkoxyl, alkyl,
alkylamino, amino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl,
heterocyclylalkyl, and hydroxy; and salts, hydrates, solvates,
complexes, and prodrugs thereof.
[0431] In an embodiment of the present invention, if R.sub.2 is
C.dbd.O(R.sub.5) or SO.sub.2R.sub.7, then R is at positions 2, 3,
or 5 on the benzene ring.
[0432] In another embodiment of the invention, if R.sub.2 is
C.dbd.O(R.sub.5) or SO.sub.2R.sub.7, then each R is independently
selected from the group consisting of H, halogen, --OH, --NH.sub.2,
--NO.sub.2, --CN, --N.sub.3, --SO.sub.3H, acyl, alkyl, alkylamino,
cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl,
(hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein
each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl,
heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino may be substituted with one or more radicals
independently selected from the group consisting of halogen, N, O,
--S--, --CN, --N.sub.3, --SH, nitro, oxo, acyl, alkyl, alkoxyl,
alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and
(hetero-)cyclyl.
[0433] In another embodiment of the invention, if R.sub.2 is
C.dbd.O(R.sub.5) or SO.sub.2R.sub.7, then there are at least two R
groups attached to the benzene ring. Furthermore, there are at
least two R groups attached to the benzene ring, and both R groups
are attached at positions 2, 3, or 5 on the benzene ring. Still
further, each R is independently selected from the group consisting
of H, halogen, --OH, --NH.sub.2, --NO.sub.2, --CN, --N.sub.3,
--SO.sub.3H, acyl, alkyl, alkylamino, cycloalkyl, heterocyclyl,
heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and
(hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino,
cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl,
(hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino may be
substituted with one or more radicals independently selected from
the group consisting of halogen, N, O, --S--, --CN, --N.sub.3,
--SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl,
(hetero-)cycloalkyl, and (hetero-)cyclyl.
[0434] In another embodiment of the invention, if R.sub.2 is
C.dbd.O(R.sub.5), then R.sub.5 is selected from the group
consisting of --NR.sub.16, NHNHR.sub.16, NHOH, --OR.sub.15,
CONH.sub.2NHR.sub.16, CONR.sub.16, CH.sub.2X, acyl, aryl,
cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl;
wherein each acyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl,
and heterocyclylalkyl may be substituted with one or more radicals
independently selected from the group consisting of halogen, N, O,
--S--, --CN, --N.sub.3, nitro, oxo, acyl, alkyl, alkoxyl,
alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and
(hetero-)cyclyl.
Efficacy Demonstrations
[0435] As demonstrated by FIG. 1, embodiments A, B, C, and D, S36
is more potent at increasing the binding of FKBP12.6 and RyR2 than
JTV-519 and does not block the L-type Ca2+ channel (I.sub.Ca,L) or
HERG K.sup.+ channel (I.sub.Kr). In embodiment A, PKA
phosphorylated RyR2 is generated as follows: cardiac SR membrane
preparations (5 .mu.l, 50 .mu.g) are added to a total of 100 .mu.l
of kinase buffer (8 mM MgCl.sub.2, 10 mM EGTA, 50 mM Tris-PIPES, pH
6.8) containing 100 .mu.M MgATP and 40 units of PKA, and incubated
at room temperature. Samples are centrifuged at 95,000 g for 10 min
and the pellets are washed three times in 0.2 ml imidazole buffer.
The final pellets are pooled and resuspended in imidazole buffer
(final concentration.apprxeq.10 .mu.g/.mu.l). To test for the
FKBP12.6 rebinding efficiency of JTV-519, PKA phosphorylated
cardiac SR (50 mg) is incubated for 30 minutes at room temperature
with the test compounds and 250 nM FKBP12.6 in 10 mM imidizol
buffer, pH 7.0. Samples then are centrifuged at 100,000 g for 10
minutes and pellets washed 3 times with imidizol buffer. After
washing, proteins are size-fractionated on 15% PAGE. Immunoblots
are developed using an anti-FKBP antibody (1:3,000 dilution). The
amount of rebinding is quantified using densitometry of Western
blots and is compared to the amount of FKBP associated with RyR in
non-phosphorylated SR. EC.sub.50's for the compounds are determined
by generating FKBP binding data using concentrations of compounds
ranging from 0.5-1000 nM. In embodiment B, currents through L-type
Ca.sup.2+ channels in isolated mouse cardiomyocytes are recorded
using whole-cell patch clamp recording conditions with Ba.sup.2+ as
the charge carrier. The extracellular solution contains (in mM):
N-methyl-D-glucamine, 125; BaCl.sub.2, 20; CsCl, 5; MgCl.sub.2, 1;
HEPES, 10; glucose, 5; pH 7.4 (HCl). The intracellular solution
contains (in mM): CsCl, 60; CaCl.sub.2, 1; EGTA, 11; MgCl.sub.2, 1;
K.sub.2ATP, 5; HEPES, 10; aspartic acid, 50; pH 7.4 (CsOH). Under
these conditions, it is expected that the measured current was
carried by Ba.sup.2+ primarily through L-type calcium channels
which is referred to as I.sub.Ca,L. Drugs are applied by a local
solution changer and reach the cell membrane within 1 s. The
effects of nifedipine and S36 are tested with 20 ms long
voltage-clamp steps to +10 or +20 mV (peak of current-voltage
relation for each individual cell) from holding potentials of -80
mV or -40 mV. In embodiment C, the voltage-dependence of L-type
Ca.sup.2+ current blocked by JTV-519 (1 .mu.M) and S36 (l .mu.M)
are measured and presented.
[0436] As demonstrated by FIG. 2, embodiments A, B, C, and D, S36
prevents exercise-induced sudden cardiac death at lower plasma
levels compared with JTV-519. In embodiment A are shown
representative ECGs of an untreated FKBP12.6.sup.-/- mouse and
JTV-519-treated FKBP12.6.sup.-/- and FKBP12.6.sup.-/- mice. Mice
are treated with 0.5 mg JTV-519/per kilogram of body weight per
hour for 7 days with an implanted osmotic mini-pump. JTV-519 has no
effect on resting heart rate or other ECG parameters such as heart
rate (HR). In embodiment B are shown sustained polymorphic
ventricular tachycardia recorded by telemetry in an untreated
FKBP12.6.sup.-/- mouse (upper tracing) subjected to exercise
testing, immediately followed by injection with 0.5 mg epinephrine
per kilogram of body weight. Representative telemetry ECG recording
of a JTV-519-treated FKBP12.6.sup.-/- mouse following the same
protocol is shown in the bottom tracing. In embodiment C are shown
numbers of mice with cardiac death (left), sustained VTs (>10
beats, middle), and nonsustained VTs (3 to 10 arrhythmogenic beats,
right) in experimental groups of mice subjected to exercise testing
and injection with 0.5 mg/kg epinephrine. In embodiment D, the
dose-dependence of pharmacological effects of JTV-519 and S36 is
shown. Plasma levels of 1 .mu.M JTV-519 prevent cardiac arrhythmias
and sudden cardiac death in FKBP12.6.sup.-/- mice. Plasma levels of
1 .mu.M and 0.02 .mu.M S36 also prevent cardiac arrhythmias and
sudden cardiac death in FKBP12.6.sup.-/- mice.
[0437] FIG. 3 shows the results of treated mice that are subjected
to permanent ligation of the left anterior descending coronary
artery resulting in myocardial infarction.
[0438] As demonstrated by FIG. 4, S36 improves cardiac function in
chronic heart failure post-myocardial infarction. Wild-type mice
are subjected to permanent ligation of the left anterior descending
coronary artery resulting in myocardial infarction. Seven days
following myocardial infartion, mice are treated with S36 (plasma
concentration 200 nM) or placebo. Heart weight to body weight
(HW/BW) ratios and pressure-volume loops quantifications (dP/dt,
slope of the maximum derivative of change in systolic pressure over
time) show reverse remodeling and improved cardiac contractility in
S36-treated mice compared with placebo.
[0439] FIG. 5 is a summary graph of EC50 values of JTV-519 and
compounds S1-S67 disclosed herein. The FKBP12.6 rebinding assay
described above is used to determine the amount of FKBP12.6 binding
to PKA-phosphorylated RyR2 at various concentrations (0.5-1000 nM)
of the compounds shown. EC.sub.50 values are calculated using
Michaelis-Menten curve fitting.
[0440] FIG. 6, embodiments A, B, and C, show CPVT-associated
RyR2-P2328S channel function and structure. In embodiment A are
shown representative single-channel current traces of RyR2-P2328S
and RyR2-WT while embodiment B shows RyR2-P2328S. Embodiment C
shows immunoblot analysis of calstabin-2 binding of
RyR2-P2328S.
[0441] As demonstrated by FIG. 7, embodiments A and B, treatment
with JTV-519 reduces PKA phosphorylation of RyR2 in mice with heart
failure. Equivalent amounts of RyR2 are immunoprecipitated with an
antibody against RyR2 (top blot). Representative immunoblots
(embodiment A) and bar graphs (embodiment B) show the amount of PKA
phosphorylated RyR2 at Ser-2808 bound to RyR2 in wild-type and
calstabin2(FKBP12.6).sup.-/- mice. Treatment with JTV-519 (0.5
mg/kg/h) for 28 days post-myocardial infarction reduces
PKA-phosphorylation of RyR2, presumably due to reverse cardiac
remodeling, in wildtype but not calstabin-2 (FKBP12.6).sup.-/-
mice.
[0442] As demonstrated by FIG. 8, embodiments A and B, mice in
which cardiac RyR2 cannot be PKA phosphorylated (RyR2-S2808A
knockin mice) have improved cardiac function following myocardial
infarction. Shown in embodiment A is the quantification of M-mode
echocardiograms showing improved ejection fraction in RyR2-S2808A
knockin mice compared with wildtype 28 days following permanent
coronary artery ligation. Shown in embodiments B and C are
pressure-volume loop quantifications showing (embodiment B)
improved cardiac contractility and decreased cardiac dilation
(embodiment C) in RyR2-S2808A knockin mice compared with wildtype
following myocardial infarction.
[0443] FIG. 9, embodiments A and B, demonstrate that mdx skeletal
muscle has normal levels of RyR1 PKA phosphorylation, but depleted
levels of calstabin1. The immunoblots in embodiment A show that mdx
type mice have depleted levels of calstabin1 compared to a control
(wild-type) mouse. The summary bar graphs of embodiment B show that
the mdx mouse, nevertheless, has an equivalent level of
PKA-phosphorylation. Therefore, it is concluded that calstabin1
depletion is a defect that is consistent with the intracellular
Ca.sup.2+ leak observed in skeletal muscle cells from mdx mice and
myofibers from human mutation carriers. Intracellular SR Ca.sup.2+
leak is likely to contribute to myofiber death and wasting of
muscle mass by toxic intracellular Ca.sup.2+ overload and
activation of proteases.
[0444] FIG. 10, embodiments A, B, and C, demonstrates that SR
Ca.sup.2+ leak at the subcellular level in skeletal muscles of
animals with heart failure is detectable. Life quality and
prognosis in heart failure (HF) patients is severely decreased due
to skeletal muscle dysfunction (e.g., shortness of breath due to
diaphragmatic weakness, and exercise intolerance due to limb
skeletal muscle fatigue) in addition to depressed cardiac function.
Dysregulation of intracellular SR Ca.sup.2+ release is a pathogenic
mechanism underlying skeletal muscle dysfunction in HF. HF in
animals causes significantly accelerates intrinsic skeletal muscle
fatigue.
[0445] Embodiments A and B of FIG. 10 are AF/F fluorescence line
scan images of representative examples of Ca.sup.2+ sparks in
myofibers from sham and postmyocardial infarction (PMI) rats and
corresponding Ca.sup.2+ spark time course. Embodiment C shows the
relative distribution of the spatio-temporal properties of the
Ca.sup.2+ sparks. Charts indicate 25, 50, 75 percentiles, the
horizontal lines indicate the range from 1-99% of the distribution.
Sham, open symbols (n=137, three animals); postmyocardial
infarction (PMI), gray symbols (n=82, two animals). *, P<0.05.
FDHM, full duration at 50% peak amplitude; FWHM, full width at 50%
peak amplitude.
[0446] FIG. 11, embodiment A, B, C, and D demonstrates that
Ser-2843 is the unique PKA phosphorylation site in skeletal RyR1
channels. (A) Representative single channel traces of wild-type
RyR1, (B) effect of exogenous PKA phosphorylation of RyR1 (wt
RyR1-P), (C) PKA does not affect RyR1-52843A that contains a
non-functional PKA phosphorylation site. Since PKA does not
increase RyR1-52843A activity, Ser-2843 appears to constitute the
unique PKA phosphorylation site in RyR1 channels in skeletal
muscle. Accordingly, (D) constitutively phosphorylated RyR1-52843D
mimics exogenous PKA phosphorylation shown in (B) confirming that
Ser-2843 is the unique PKA phosphorylation site in skeletal RyR1
channels. RyR1 single channel recordings in planar lipid bilayers
show activity of the channels at 150 nM [Ca.sup.2+].sub.cis
(cytosolic side) with 1 mM ATP. Recordings were at 0 mV, closed
state of the channels as indicated by `c`, and channel openings are
upward deflections. All point amplitude histograms are shown on the
right. Open probability (P.sub.o) and mean closed (Tc) and open
(To) dwell times are indicated above each channel tracing.
[0447] FIG. 12, embodiments A and B, demonstrates the depletion of
stabilizing calstabin1 and PKA hyperphosphorylation of RyR1
channels from sustained exercise. Aerobic exercise can be defined
as a form of physical exercise that increases the heart rate and
enhances oxygen intake to improve performance. Examples of aerobic
exercise are running, cycling, and swimming. During the study of
FIG. 12, mice were challenged by aerobic exercise (forced swimming)
for 90 mins twice daily. The animals were accustomed to swimming in
preliminary training sessions: day -3 twice 30 mins, day -2 twice
45 mins, day -1 twice 60 mins, day 0 and following twice 90 mins.
Mice were then exercised for 1, 7, or 21 additional, consecutive
days for 90 mins twice daily. Between swimming sessions separated
by a 4 hour rest period the mice are kept warm and given food and
water. An adjustable-current water pool was used to exercise mice
by swimming. An acrylic pool (90 cm long.times.45 cm wide.times.45
cm deep) filled with water to a depth of 25 cm was used. A current
in the pool was generated with a pump. The current speed during the
swimming session was at a constant speed of 1 l/min flow rate. The
water temperature was maintained at 34.degree. C. with an electric
heater. Age- and weight-matched mice were used to exclude
differences in buoyancy from body fat.
[0448] Using forced swimming as an efficient protocol to increase
skeletal muscle aerobic capacity in mice, the composition and
phosphorylation status of the skeletal RyR1 channel complex have
been investigated. Unexpectedly, after 3 weeks of 90 mins swimming
twice daily, C57Bl6 wild-type mice showed significantly increased
RyR1 phosphorylation by PKA while Ca.sup.2+-calmodulin kinase II
(CaMKII) phosphorylation was not changed indicating specificity of
the stress pathway RyR1 protein expression was stable, however,
RyR1 channels were depleted of the stabilizing subunit calstabin1
(FKBP12). It has been shown that RyR1 hyperphosphorylation and
calstabin1 depletion are consistent with leaky RyR1 channels that
cause intracellular SR Ca.sup.2+ leak.
[0449] RyR1 channels are PKA hyperphosphorylated and depleted of
the stabilizing calstabin1 subunit after 3 weeks of 90 mins
swimming twice daily. As seen in Embodiment A, the
immunoprecipitated RyR1 macromolecular channel complex shows
increased PKA phosphorylation at Ser-2844 (corresponding to human
RyR1-Ser-2843) whereas CaMKII phosphorylation at Ser-2849
(corresponding to human RyR1-Ser-2848) is unchanged. Concomitant
with increased RyR1-Ser-2844 PKA hyperphosphorylation, calstabin1
is depleted from the channel complex. As seen in embodiment B,
normalization of phosphorylation and calstabin1 content to four
subunits of the tetrameric channel complex shows a significant in
increase in PKA phosphorylation and depletion of the stabilizing
calstabin1 subunit. Control, non-exercised mice; swim, mice
exercised 90 mins twice daily for 3 weeks (preliminary data).
P<0.05.
[0450] FIG. 13, embodiments A and B, demonstrate that PKA
phosphorylation increases for increasing durations of sustained
exercise. To investigate the influence of the duration of sustained
exercise on the RyR1 Ca.sup.2+ release channel defect, mice were
exposed to swimming for 1, 7, or 21 days followed by immediate
sacrifice. Longer exposure to sustained exercise results in a
significant increase of RyR1 PKA hyperphosphorylation beginning at
7 days and saturating at 21 days.
[0451] In FIG. 13, embodiment A, the immunoprecipitated RyR1
channel complex shows significantly and above physiologic levels
increased PKA phosphorylation at Ser-2844 (corresponding to human
RyR1-Ser-2843) after 7 days of swimming exercise. In FIG. 13,
embodiment B, inormalization of RyR2-Ser-2844 phosphorylation
within the tetrameric channel complex documents a significant
increase in PKA phosphorylation. *, P<0.05; **, P<0.005.
[0452] In summary, the data of FIG. 13 shows that sustained
exercise results in significantly increased RyR1 phosphorylation by
protein kinase A (PKA) which contributes to depletion of the
stabilizing calstabin1 subunit from the channel complex as the
cause of a gain-of-function defect.
[0453] FIG. 14 provides data showing that showing that chronically
increased sympathetic stimulation of skeletal muscles, results in
RyR1-dependent intracellular Ca.sup.2+ leak and significantly
increased muscle fatigue. As shown in FIG. 14 for mice and rats
with heart failure from myocardial infarction, chronic RyR1 PKA
hyperphosphorylation results in increased muscle fatigue.
[0454] In embodiment A, it can be seen that heart failure skeletal
muscle fatigues earlier than control. Rat soleus muscle (n=5
control, n=8 HF) was mounted in a tissue bath to assess contractile
function. Representative fatigue time tracing is shown for control
and HF skeletal muscles. Bar graph shows mean (.+-.S.D.) time to
40% fatigue. *, P<0.05. In embodiment B, it can be seen that
heart failure skeletal muscle achieved maximal tetanic force more
slowly than control skeletal muscles. Tetanic force was induced by
high-frequency field stimulation. Bar graph shows tetanic 50%
contraction time. **, P<0.01. Embodiment C demonstrates the
correlation between time to fatigue and RyR1 PKA phosphorylation
(r=0.88) in rat skeletal muscle from sham and heart failure
animals. Muscle function and RyR1 PKA phosphorylation were assessed
using contralateral soleus muscles from each animal.
[0455] In summary, FIG. 14 provides data showing that sustained
exercise causes RyR1 PKA hyperphosphorylation and calstabin1
depletion, and FIG. 14 shows that the identical defect occurs in
disease forms with increased sympathetic activity causing
intracellular SR Ca.sup.2+ leak and significantly accelerated
skeletal muscle fatigue.
[0456] An additional problem during sustained exercise and stress
is skeletal muscle degeneration further contributing to decreased
skeletal muscle performance. To assess structural changes during
sustained exercise, histologic changes in the fast-twitch muscles
of mice exposed to 3 weeks of exercise by swimming have been
characterized. Results are shown in FIG. 15. Cross-sections of the
mouse M. extensor digitorum longus (EDL) showed histologic changes
consistent with myofiber degeneration from intracellular Ca.sup.2+
overload from defective RyR1 channels. Therefore sustained exercise
for 90 mins twice daily triggers a dystrophic phenotype in EDL
muscles of normal C57Bl6 mice.
[0457] Trichrome stain shows packed myofibers of similar
cross-sectional dimension in non-exercised control (WT) mice
(left). Three weeks swimming results in myofiber degeneration and
interstitial collagen deposits with irregular fiber sizes.
Hematoxylin-eosin (H&E) stain indicates nuclear changes and
myofiber death. These changes are consistent with dystrophic
remodeling.
[0458] The rapid delayed rectifier potassium channel (I(Kr)) is
important for repolarization of the cardiac action potential. HERG
is the pore-forming subunit of the I(Kr) channel. Suppression of
I(Kr) function, for example as a side-effect of a drug or the
result of a mutation in hERG, can lead to long-QT (LQT) syndrome,
which is associated with increased risk of life-threatening
arrhythmias. The compounds of the present invention exhibit a lower
level of hERG blocking activity than JTV-519, as demonstrated in
FIGS. 16-35. Thus, the compounds of the present invention are
expected to be less toxic and/or exhibit fewer side effects than
JTV-519.
[0459] FIGS. 16 to 19 illustrate the effect of the compound ARM036
(also referred to as S36) and ARM036-Na (a sodium salt of ARM036)
on hERG currents.
[0460] FIG. 16 shows a typical hERG voltage-clamp current recording
before (control) and after application of ARM036 at 100 .mu.M. The
voltage pulse protocol used to activate the hERG currents is
illustrated below the current trace. It can be seen that, following
activation by the conditioning prepulse (to +20 mV), partial
repolarization (-50 mV test pulse) of the membrane evoked a large,
slowly decaying outward tail current. Application of ARM036
minimally reduced the outward tail current in a concentration- and
time-dependent manner.
[0461] FIG. 17 shows a typical time course of the effect of ARM036
at 100 mM on the amplitude of the hERG channel current.
[0462] FIG. 18 is a graph showing the concentration-dependence of
the effect of ARM036 on the hERG current. Table 1 provides the
numerical data that is illustrated graphically in FIG. 18. Because
the highest concentration of ARM036 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM036.
TABLE-US-00001 TABLE 1 Concentration (.mu.M) Mean SD SEM N 10 0.7%
0.3% 0.2% 3 100 0.9% 0.7% 0.4% 3
[0463] FIG. 19 is a graph showing the concentration-dependence of
the effect of ARM036-Na on the hERG current. Table 2 provides the
numerical data that is illustrated graphically in FIG. 18. Because
the highest concentration of ARM036-Na tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM036-NA.
TABLE-US-00002 TABLE 2 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM036-Na ND 0.01 0.2% 0.2% 0.2% 2
0.0% 0.3% 0.1 5.0% 7.1% 5.0% 2 10.0% 0.0% 1 5.5% 4.4% 3.1% 2 2.4%
8.6% 10 6.7% 2.2% 1.6% 2 5.1% 8.2%
[0464] FIG. 20 is a graph showing the concentration-dependence of
the effect of ARM047 on the hERG current. Table 3 provides the
numerical data that is illustrated graphically in FIG. 20. The
IC.sub.50 value for ARM047 block of the hERG current was 2.496
.mu.M.
TABLE-US-00003 TABLE 3 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM047 2.495 0.01 2.7% 1.4% 1.0% 2
1.7% 3.7% 0.1 9.7% 4.5% 3.2% 2 6.5% 12.9% 1 29.6% 8.6% 5.0% 3 30.8%
20.4% 37.5% 10 78.0% 3.6% 2.1% 3 82.1% 75.9% 75.9%
[0465] FIG. 21 is a graph showing the concentration-dependence of
the effect of ARM048 on the hERG current. Table 4 provides the
numerical data that is illustrated graphically in FIG. 21. Because
the highest concentration of ARM048 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM048.
TABLE-US-00004 TABLE 4 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM048 ND 0.01 1.3% 1.1% 0.8% 2
0.5% 2.0% 0.1 3.6% 0.9% 0.8% 2 4.2% 2.9% 1 4.1% 0.8% 0.6% 2 4.7%
3.5% 10 14.0% 1.7% 1.2% 2 15.2% 12.8%
[0466] FIG. 22 is a graph showing the concentration-dependence of
the effect of ARM050 on the hERG current. Table 5 provides the
numerical data that is illustrated graphically in FIG. 22. Because
the highest concentration of ARM050 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM050.
TABLE-US-00005 TABLE 5 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM050 ND 0.01 1.8% 0.9% 0.7% 2
1.1% 2.4% 0.1 3.7% 1.4% 1.0% 2 2.7% 4.7% 1 6.1% 1.4% 1.0% 2 7.1%
5.1% 10 24.2% 4.0% 2.9% 2 27.0% 21.3%
[0467] FIG. 23 is a graph showing the concentration-dependence of
the effect of ARM057 on the hERG current. Table 6 provides the
numerical data that is illustrated graphically in FIG. 23. Because
the highest concentration of ARM057 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM057.
TABLE-US-00006 TABLE 6 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM057* ND 0.01 0.2% 0.2% 0.2% 2
0.3% 0.0% 0.1 7.3% 4.5% 3.2% 2 4.1% 10.4% 1 2.7% 3.7% 2.6% 2 5.3%
0.1% 10 13.6% 6.7% 4.8% 2 18.3% 8.8%
[0468] FIG. 24 is a graph showing the concentration-dependence of
the effect of ARM064 on the hERG current. Table 7 provides the
numerical data that is illustrated graphically in FIG. 24. Because
the highest concentration of ARM064 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM064.
TABLE-US-00007 TABLE 7 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM064 ND 0.01 0.2% 1.1% 0.8% 2
-0.6% 1.0% 0.1 9.0% 1.3% 0.9% 2 9.9% 8.1% 1 9.6% 5.1% 3.6% 2 13.2%
6.0% 10 30.3% 2.5% 1.7% 2 32.0% 28.5%
[0469] FIG. 25 is a graph showing the concentration-dependence of
the effect of ARM074 on the hERG current. Table 8 provides the
numerical data that is illustrated graphically in FIG. 25. Because
the highest concentration of ARM050 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM074.
TABLE-US-00008 TABLE 8 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM074 ND 0.01 1.9% 1.6% 1.1% 2
0.6% 3.0% 0.1 4.8% 1.6% 1.1% 2 5.9% 3.7% 1 1.3% 1.6% 1.1% 2 0.2%
2.4% 5.6% 10 9.5% 0.2% 0.2% 2 9.3% 9.6% 14.0%
[0470] FIG. 26 is a graph showing the concentration-dependence of
the effect of ARM075 on the hERG current. Table 9 provides the
numerical data that is illustrated graphically in FIG. 26. Because
the highest concentration of ARM075 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM075.
TABLE-US-00009 TABLE 9 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM075 ND 0.01 1.4% 0.4% 0.3% 2
1.7% 1.1% 0.1 3.7% 1.6% 1.1% 2 2.6% 4.8% 1 3.9% 0.3% 0.2% 2 3.7%
4.1% 10 16.0% 1.8% 1.3% 2 14.7% 17.2%
[0471] FIG. 27 is a graph showing the concentration-dependence of
the effect of ARM076 on the hERG current. Table 10 provides the
numerical data that is illustrated graphically in FIG. 27. Because
the highest concentration of ARM076 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM076.
TABLE-US-00010 TABLE 10 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM076 ND 0.01 0.4% 0.5% 0.4% 2
0.0% 0.7% 0.1 2.5% 2.2% 1.6% 2 4.0% 0.9% 1 3.1% 1.5% 1.1% 2 2.0%
4.1% 10 11.2% 1.6% 1.2% 2 10.0% 12.3%
[0472] FIG. 28 is a graph showing the concentration-dependence of
the effect of ARM077 on the hERG current. Table 11 provides the
numerical data that is illustrated graphically in FIG. 28. Because
the highest concentration of ARM077 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM077.
TABLE-US-00011 TABLE 11 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM077 ND 0.01 1.3% 0.2% 0.2% 2
1.4% 1.1% 0.1 7.5% 5.5% 3.9% 2 11.4% 3.6% 1 3.6% 0.6% 0.4% 2 3.1%
4.0% 10 4.1% 4.5% 3.2% 2 0.9% 7.2%
[0473] FIG. 29 is a graph showing the concentration-dependence of
the effect of ARM101 on the hERG current. Table 12 provides the
numerical data that is illustrated graphically in FIG. 29. Because
the highest concentration of ARM101 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM101.
TABLE-US-00012 TABLE 12 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM101 4.406 0.01 3.4% 1.3% 0.9% 2
4.3% 2.5% 0.1 6.4% 1.8% 1.3% 2 5.1% 7.6% 1 23.0% 5.7% 4.1% 2 18.9%
27.0% 10 65.8% 1.3% 0.9% 2 66.7% 64.9%
[0474] FIG. 30 is a graph showing the concentration-dependence of
the effect of ARM102 on the hERG current. Table 13 provides the
numerical data that is illustrated graphically in FIG. 30. Because
the highest concentration of ARM102 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM102.
TABLE-US-00013 TABLE 13 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM102* ND 0.01 2.5% 2.8% 2.0% 2
0.5% 4.4% 0.1 4.2% 3.3% 2.4% 2 1.8% 6.5% 1 5.7% 6.9% 4.9% 2 0.8%
10.5% 10 47.3% 1.5% 1.1% 2 46.2% 48.3%
[0475] FIG. 31 is a graph showing the concentration-dependence of
the effect of ARM103 on the hERG current. Table 14 provides the
numerical data that is illustrated graphically in FIG. 31. Because
the highest concentration of ARM103 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM103.
TABLE-US-00014 TABLE 14 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM103* ND 0.1 6.8% 7.5% 5.3% 2
12.1% 1.5% 0.1 3.3% 1.3% 0.9% 2 4.2% 2.4% 1 7.3% 3.3% 2.3% 2 5.0%
9.6% 10 29.2% 1.1% 0.8% 2 28.4% 29.9%
[0476] FIG. 32 is a graph showing the concentration-dependence of
the effect of ARM104 on the hERG current. Table 15 provides the
numerical data that is illustrated graphically in FIG. 32. Because
the highest concentration of ARM104 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM104.
TABLE-US-00015 TABLE 15 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM104 ND 0.01 1.6% 0.5% 0.4% 2
1.2% 1.9% 0.1 2.5% 2.0% 1.4% 2 1.1% 3.9% 1 4.6% 2.1% 1.5% 2 3.1%
6.0% 10 7.4% 1.3% 0.9% 2 8.3% 6.5%
[0477] FIG. 33 is a graph showing the concentration-dependence of
the effect of ARM106 on the hERG current. Table 16 provides the
numerical data that is illustrated graphically in FIG. 33. Because
the highest concentration of ARM106 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM106.
TABLE-US-00016 TABLE 16 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM106 ND 0.01 1.3% 0.6% 0.4% 2
0.9% 1.7% 0.1 7.2% 3.5% 2.5% 2 9.6% 4.7% 1 6.1% 3.7% 2.7% 2 8.7%
3.4% 10 15.9% 4.0% 2.8% 2 18.7% 13.1%
[0478] FIG. 34 is a graph showing the concentration-dependence of
the effect of ARM107 on the hERG current. Table 17 provides the
numerical data that is illustrated graphically in FIG. 34. Because
the highest concentration of ARM107 tested resulted in less than
50% current inhibition, it was not possible to determine an
IC.sub.50 value for ARM107.
TABLE-US-00017 TABLE 17 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM107 ND 0.01 2.9% 1.6% 1.2% 2
1.7% 4.0% 0.1 4.0% 1.2% 0.9% 2 3.1% 4.8% 1 6.2% 5.9% 4.2% 2 2.0%
10.4% 10 32.1% 11.1% 7.9% 2 24.2% 39.9%
[0479] FIG. 35A is a graph showing the concentration-dependence of
the effect of S26 on the hERG current. Table 18 provides the
numerical data that is illustrated graphically in FIG. 35A. The
IC.sub.50 value for S26 was 7.029 .mu.M.
TABLE-US-00018 TABLE 18 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) S26 7.029 0.01 8.9% 0.3% 0.2% 2
9.1% 8.7% 0.1 10.5% 2.6% 1.9% 2 12.3% 8.6% 1 12.3% 1.3% 1.0% 2
11.3% 13.2% 10 58.3% 2.6% 1.8% 2 56.4% 60.1%
[0480] FIG. 35B is a graph showing the concentration-dependence of
the effect of JTV-519 (referred to in the figure as "ARM00X") on
the hERG current. Table 19 provides the numerical data that is
illustrated graphically in FIG. 35B. The IC.sub.50 value for
JTV-519 was 0.463 .mu.M.
TABLE-US-00019 TABLE 19 Mean % % % IC.sub.50 Conc. hERG Standard
Standard Individual Data Test Article ID (.mu.M) (.mu.M) Inhibition
Deviation Error n (% Inhibition) ARM0XX 0.463 0.01 5.0% 0.3% 0.2% 2
5.2% 4.8% 0.1 18.1% 11.4% 8.1% 2 10.0% 26.1% 1 68.4% 19.1% 13.5% 2
81.9% 54.9% 10 92.8% 5.8% 4.1% 2 96.9% 88.7%
[0481] The antiarrhythmic drug E-4031, a known blocker of hERG
currents, was used as a positive control. E-4031 blocked the hERG
current with an IC.sub.50 of 0.5 .mu.M (n=6).
[0482] In summary, the compounds of the present invention exhibit
reduced hERG blocking activity as compared to JTV-519. Thus, the
compounds of the invention are expected to be less toxic and/or
exhibit fewer side effects than JTV-519.
[0483] Table 20 below provides EC.sub.50 values for compounds
S1-S107. These EC.sub.50 data were obtained using thee FKBP 12.6
rebinding assay described above to determine the amount of FKBP12.6
binding to PKA-phosphorylated RyR2 at various concentrations
(0.5-1000 nM) of the compounds shown in Table 20. The EC.sub.50
values are calculated using Michaelis-Menten curve fitting.
TABLE-US-00020 TABLE 20 Compound No. EC50 (nM) 1 150 2 211 3 4 102
5 208 6 252 7 55 9 205 11 181 12 197 13 174 14 182 19 265 20 22 355
23 268 25 40 26 40 27 ca. 50 36 15 37 38 44 40 100 43 80 44 121 45
80 46 150 47 20 48 100 49 81 50 40 51 175 52 143 53 200 54 77 55
111 56 95 57 73 58 55 59 102 60 68 61 95 62 45 63 52 64 44 66 110
67 89 68 ca. 100 74 220 75 150 76 25 77 60 101 105 102 135 104 111
107 190
High-Throughput Screening Method
[0484] In addition to the compounds disclosed herein, other
compounds can be discovered that are capable of modulating calcium
ion channel activity, in particular those channels related to the
RyR series of calcium ion channels. Provided herein is a
highly-efficient assay for high-throughput screening of other
compounds that are capable of modulating calcium ion channel
activity.
[0485] By way of example, and as shown in Example 5 below, a
highly-efficient assay for high-throughput screening for small
molecules is developed by immobilizing FKBP, either FKBP12 or
FKBP12.6 (e.g., wild-type FKBP12.6 or a fusion protein, such as
GST-FKBP12.6) onto a 96-well plate coated with glutathione, using
standard procedures. PKA-phosphorylated ryanodine receptor (RyR),
specifically RyR1 or RyR3 in the case of FKBP12 and RyR2 in the
case of FKBP12.6, is loaded onto the FKBP-coated plate, and
incubated with compounds at various concentrations (10-100 nM) for
30 min. Thereafter, the plate is washed to remove the unbound RyR,
and then incubated with anti-RyR antibody (e.g., for 30 min). The
plate is washed again to remove unbound anti-RyR antibody, and then
treated with florescent-labeled secondary antibody. The plate is
read by an automatic fluorescent plate reader for binding
activity.
[0486] Alternatively, RyR is PKA-phosphorylated in the presence of
.sup.32P-ATP. Radioactive PKA-phosphorylated RyR is loaded onto an
FKBP-coated, 96-well plate, in the presence of JTV-519 analogues
and other compounds at various concentrations (10-100 nM) for 30
min. The plate is washed to remove the unbound radiolabeled RyR,
and then read by an automatic plate reader. PKA-phosphorylated RyR
also is coated to the plate, and incubated with .sup.32S-labeled
FKBP in the presence of the compounds.
[0487] The present invention is described in the following
Examples, which are set forth to aid in the understanding of the
invention and should not be construed to limit in any way the scope
of the invention as defined in the claims which follow
thereafter.
EXAMPLES
Example 1
RyR2 PKA Phosphorylation and FKBP12.6 Binding
[0488] Cardiac SR membranes are prepared, as previously described
(Marx, et al., PKA phosphorylation dissociates FKBP12.6 from the
calcium release channel (ryanodine receptor): defective regulation
in failing hearts. Cell, 101:365-76, 2000; Kaftan, et al., Effects
of rapamycin on ryanodine receptor/Ca.sup.(2+)-release channels
from cardiac muscle. Circ. Res., 78:990-97, 1996).
.sup.35S-labelled FKBP12.6 was generated using the TNT.TM. Quick
Coupled Transcription/Translation system from Promega (Madison,
Wis.). [.sup.3H] ryanodine binding is used to quantify RyR2 levels.
100 .mu.g of microsomes are diluted in 100 .mu.l of 10-mM imidazole
buffer (pH 6.8), incubated with 250-nM (final concentration)
[.sup.35S]-FKBP12.6 at 37.degree. C. for 60 min, then quenched with
500 .mu.l of ice-cold imidazole buffer. Samples are centrifuged at
100,000 g for 10 min and washed three times in imidazole buffer.
The amount of bound [.sup.35S]-FKBP12.6 is determined by liquid
scintillation counting of the pellet.
Example 2
Immunoblots
[0489] Immunoblotting of microsomes (50 .mu.g) is performed as
described, with anti-FKBP12/12.6 (1:1,000), anti-RyR-5029 (1:3,000)
(Jayaraman, et al., FK506 binding protein associated with the
calcium release channel (ryanodine receptor). J. Biol. Chem.,
267:9474-77, 1992), or anti-phosphoRyR2-P2809 (1:5,000) for 1 h at
room temperature (Reiken, et al., Beta-blockers restore calcium
release channel function and improve cardiac muscle performance in
human heart failure. Circulation, 107:2459-66, 2003). The
P2809-phosphoepitope-specific anti-RyR2 antibody is an
affinity-purified polyclonal rabbit antibody, custom-made by Zymed
Laboratories (San Francisco, Calif.) using the peptide,
CRTRRI-(pS)-QTSQ, which corresponds to RyR2 PKA-phosphorylated at
Ser.sup.2809. After incubation with HRP-labeled anti-rabbit IgG
(1:5,000 dilution; Transduction Laboratories, Lexington, Ky.), the
blots are developed using ECL (Amersham Pharmacia, Piscataway,
N.J.).
Example 3
Single-Channel Recordings
[0490] Single-channel recordings of native RyR2 from mouse hearts,
or recombinant RyR2, are acquired under voltage-clamp conditions at
0 mV, as previously described (Marx, et al., PKA phosphorylation
dissociates FKBP12.6 from the calcium release channel (ryanodine
receptor): defective regulation in failing hearts. Cell,
101:365-76, 2000). Symmetric solutions used for channel recordings
are: trans compartment--HEPES, 250 mmol/L; Ba(OH).sub.2, 53 mmol/L
(in some experiments, Ba(OH).sub.2 is replaced by Ca(OH).sub.2); pH
7.35; and cis compartment--HEPES, 250 mmol/L; Tris-base, 125
mmol/L; EGTA, 1.0 mmol/L; and CaCl.sub.2, 0.5 mmol/L; pH 7.35.
Unless otherwise indicated, single-channels recordings are made in
the presence of 150-nM [Ca.sup.2+] and 1.0-mM [Mg.sup.2+] in the
cis compartment. Ryanodine (5 mM) is applied to the cis compartment
to confirm identity of all channels. Data is analyzed from
digitized current recordings using Fetchan software (Axon
Instruments, Union City, Calif.). All data is expressed as
mean.+-.SE. The unpaired Student's t-testis used for statistical
comparison of mean values between experiments. A value of p<0.05
is considered statistically significant.
Example 4
High-Throughput Screening Method
[0491] Assays for screening biologically-active small molecules
have been developed. These assays are based on rebinding of FKBP12
protein to RyR.
[0492] A highly-efficient assay for high-throughput screening for
small molecules is developed by immobilization of FKBP12.6
(GST-fusion protein) onto a 96-well plate coated with glutathione.
PKA-phosphorylated ryanodine receptor type 2 (RyR2) is loaded onto
the FKBP12.6-coated plate, and incubated with JTV-519 analogues at
various concentrations (10-100 nM) for 30 min. Thereafter, the
plate is washed to remove the unbound RyR2, and then incubated with
anti-RyR2 antibody for 30 min. The plate is again washed to remove
unbound anti-RyR2 antibody, and then treated with
florescent-labeled secondary antibody. The plate is read by an
automatic fluorescent plate reader for binding activity.
[0493] In an alternative assay, RyR2 is PKA-phosphorylated in the
presence of 32P-ATP. Radioactive PKA-phosphorylated RyR2 is loaded
onto an FKBP12.6-coated, 96-well plate, in the presence of JTV-519
analogues at various concentrations (10-100 nM) for 30 min. The
plate is washed to remove the unbound radiolabeled RyR2, and then
read by an automatic plate reader.
Example 5
Effect of ARM036 Compounds on hERG Currents
[0494] The effects of the compounds of the invention on hERG
currents were studied using cultured human embryonic kidney 293
(HEK 293) cells which had been stably tranfected with hERG cDNA.
HEK 293 cells do not express endogenous hERG. HEK293 cells were
transfected with a plasmid containing the hERG cDNA and a neomycin
resistance gene. Stable transfectants were selected by culturing
the cells in the presence of G418. The selection pressure was
maintained by continued culture in the presence of G418. Cells were
cultures in Dulbecco's Modified Eagle Medium/Nutreint Mizture F-12
(D-MEM/F-12) supplemented with 10% fetal bovin serum, 199 U/ml
penicillin G sodium, 10 .mu.g/mL streptomycin sulfate and 500
.mu.g/mL G418. Cells for use in electrophysiology were cultured in
35 mm dishes.
[0495] Electrophysiological recordings (using the whole-cell patch
clamp method) were performed at room temperature (18.degree.
C.-24.degree. C.). Each cell acted as its own control. The effect
of ARM0036 was evaluated at two concentrations: 10 and 100 .mu.M.
Each concentration was tested in at least three cells (n.gtoreq.3).
90 nM Cisapride (commercially available from TOCRIS Bioscience) was
used as a positive control for hERG blockade. For recording, cells
were transferred to the recording chamber and superfused with
vehicle control solution. The patch pipette solution for whole cell
recording contained 130 mM potassium aspartate, 5 mM MgCl.sub.2, 5
mM EGTA, 4 mM ATP and 10 mM HEPES. The pH was adjusted to 7.2 with
KOH. The pipette solution was prepared in batches, aliquoted, and
stored frozen. A fresh aliquot was thawed and used each day. Patch
pipettes were made from glass capillary tubing using a P-97
micropipette puller (Sutter Instruments, Novato, Calif.). A
commercial patch clamp amplifier was used for whole cell
recordings. Before digitization, current records were low-pass
filtered at one-fifth of the sampling frequency.
[0496] Onset and steady state block of hERG current was measured
using a pulse pattern with fixed amplitudes (conditioning prepulse:
+20 mV for 2 seconds; test pulse: -50 mV for 2 seconds) repeated at
10 second intervals, from a holding potential of -80 mV. Peak tail
current was measured during the 2 second step to -50 mV. A steady
state was maintained for at least 30 seconds before applying the
test compound or the positive control. Peak tail current was
monitored until a new steady state was achieved. Test compound
concentrations were applied cumulatively in ascending order without
washout between applications.
[0497] Data acquisition and analysis was performed using the suite
of pCLAMP (Vre. 8.2) programs (Axon Instruments, Union City,
Calif.). Steady state was defined by the limiting constant rate of
change with time (linear time dependence). The steady state before
and after application of the test or control compounds was used to
calculate the percentage of current inhibited at each
concentration. Concentration-response data were fit to an equation
of the form:
% Block={1-1/[Test]/IC.sub.50)N]}.times.100
[0498] where [Test] is the concentration of the test compound,
IC.sub.50 (inhibitory concentration 50) is the concentration of the
test compound producing half-maximal inhibition, N is the Hill
coefficient, and % Block is the percentage of hERG current
inhibited at each concentration of the test compound. Nonlinear
squares fits were solved with the Solver add-in for Excel 2000
(Microsoft, Redmond, Wash.). For some compounds it was not possible
to determine the IC.sub.50 because the highest concentration of the
test compound used did not block the hERG channel by 50% or
more.
Example 6
Effect of Various Compounds on hERG Currents
[0499] Multiple compounds of the invention were tested for their
effects on hERG currents. The compounds tested were: ARM036-Na,
ARM047, ARM048, ARM050, ARM057, ARM064, ARM074, ARM075, ARM076,
ARM077, ARM101, ARM102, ARM103, ARM104, ARM106, ARM107 and ARM26.
By way of comparison, the effect of JTV-519 (referred to in the
figures as "ARM00X") on hERG currents was also tested.
Electrophysiological recordings were made using the PatchXpress
7000A (Molecular Devices) automatic parallel patch clamp system.
Each compound was tested at 0.01, 0.1, 1 and 10 mM, with each
concentration tested in 2 cells (n>2). The duration of exposure
to each test concentration was 5 minutes. Other aspects of the
experimental protocols were essentially similar to those described
in Example 5. For some compounds it was not possible to determine
the IC.sub.50 because the highest concentration of the test
compound used did not block the hERG channel by 50% or more.
Example 7
Effect of S36
[0500] The RyCal compounds referred to as S36, S107 were
synthesized as described herein.
[0501] FIGS. 36-41 show some aspects of the molecular mechanisms
which lead to muscle fatigue and the effect of S36 on muscle
fatigue.
[0502] Drug Delivery:
[0503] Eight-week-old, wild-type, weight-matched, C57BL/6J
littermate mice were randomized to either S36 or vehicle treatment.
On day -2, osmotic infusion pumps (Alzet Model 2004, 200 .mu.l
total volume, 0.25 .mu.l/hr delivery, Durect, Cupertino, Calif.)
filled with either 200 ul of PBS or 200 .mu.l of S36 (10 .mu.g/ul
diluted in PBS) were implanted subcutaneously on the dorsal surface
of the mice by a horizontal incision just behind the neck. Mice
were allowed to recover for three days prior to the initiation of
exercise. Standard food and water were provided ad libitum.
[0504] Exercise Protocols:
[0505] Beginning on day 1, mice were exercised for three weeks by
swimming 5 days/week and by running on a treadmill an additional 1
day/week.
[0506] Swimming Model:
[0507] The daily swimming protocol consisted of swimming sessions
twice-daily separated by one-hour rest periods. After an initial
conditioning regimen lasting 5 days during which the swimming
sessions were increased in 10 minute increments from 40 minutes
each to 80 minutes each, the swimming sessions thereafter lasted 90
minutes each. A 30 cm wide by 30 cm long opaque acrylic tank was
filled with tap water to a depth of at least 20 cm. Water was
circulated and warmed to 32-34 degrees C. using a separate
reservoir with heating element, thermostat, and pump. Compressed
room air was bubbled from Tygon tubing with small needle holes
placed at the bottom of the tank to agitate the water surface. 4
mice, matched pair-wise with respect to treatment group, swam at
any one time in the tank. Littermates who did not exercise were
reserved as negative controls.
[0508] In order to track the swimming activity of each individually
identified mouse, a video tracking system was used (San Diego
Instruments, San Diego, Calif.), which includes Sony CCD video
recorder, DVD/Hard Drive, frame grabber card, and custom SMART 2.0
software with Social Behavior package capable of tracking up to 8
mice simultaneously under ideal conditions.) Mice were anesthetized
by using 1.5% isoflurane in O.sub.2, and small, 0.75 cm, Velcro
coins were sutured with 5-0 nylon suture to the scalp of each
mouse. Plastic 1 cm colored dots, glued to the hook side of the
Velcro, could be securely attached to the mouse and used for
multiple subject tracking under appropriate lighting conditions.
Each resultant mouse track in x,y, and time was analyzed and mean
velocities, and distance swam over 2, 5, and 10 minute intervals
was obtained.
[0509] Treadmill Running:
[0510] A Columbus Instruments (Columbus, Ohio) treadmill (Model:
Exer-6M Treadmill with Treadmill Shock Detection Unit) with 6 lanes
was used to run the mice. Mice were placed in their respective
lanes at the lowest speed (7 meters/min) with the shocking
apparatus turned off and allowed to adjust to the surroundings for
6 minutes. The forward half of the treadmill was covered with
aluminum foil to block out light. A desk lamp illuminated the
shocking area at the rear of the treadmill. After the adjustment
period, the electric current was turned on, and the number of
shocks delivered during the next two three minute intervals
(training period) were recorded. The shock counter was then reset,
the speed was increased to 10 m/min, and visits to the shocking
area and shocks delivered to each mouse were recorded at
three-minute intervals until the end of the experiment. At regular
intervals, the speed of the treadmill was ramped up from the
initial 10 m/min to as high as 36 m/min. The speed was increased no
more than 2 m/min every 6 minutes. Consistent treadmill speed
increases were used for all mice on a given day, but the protocol
increased in difficulty over the course of the 21 day experiment.
Task failure was defined when a mouse could not continue running to
avoid the shocking area and gave up or when the mouse had received
200 cumulative shocks. In nearly all cases, these two times were
very close to identical.
[0511] Muscle Isolation:
[0512] Following the 21.sup.st and 22.sup.nd day of exercise, mice
were swum a final time on a staggered schedule. Following 90
minutes of swimming, each mouse was immediately sacrificed by
carbon dioxide inhalation and cervical dislocation. Blood was
removed by intracardiac aspiration, spun down, and plasma was
eluted and frozen in liquid nitrogen. Both extensor digitorum
longus (EDL) muscles were exposed, moistened with Tyrode's
solution, and 4-0 silk sutures were tied to the proximal and distal
tendons and the muscles were dissected free. The muscles were
perfused with Tyrode's solution containing 2.0 mM CaCl.sub.2,
bubbled with 100% O.sub.2, warmed to 35 C, and hung on isometric
force transducers (F-30, Harvard Apparatus, Cambridge, Mass.).
After equilibration for 10 minutes at a resting tension of 1 cN and
a brief potentiation protocol, force-frequency relationships were
measured, with 60 second delays between 800 ms stimulations at
40-150 Hz. Fatigue was produced with a protocol of 50 Hz tetani
(each 600 ms long) every 2 seconds for 120 seconds. DMC v4.1.6
(Aurora Scientific, Canada) was used to stimulate and record muscle
responses, and DMA v3.2 (Aurora Scientific, Canada) was used to
analyze the resultant data.
[0513] Following stimulation, muscle length was determined at the
resting tension, and muscle dry weight was recorded. One EDL muscle
was frozen in isopentane (-80.degree. C.) for histology and the
other was frozen in liquid N.sub.2 for biochemistry.
[0514] In addition, both soleus muscles were dissected and likewise
one was frozen in isopentane for histology and one was frozen in
liquid N.sub.2 for biochemical analysis. The vastus lateralis,
heart, and diaphragm were also dissected from each animal and
frozen in liquid N.sub.2 for biochemical analysis.
[0515] Biochemistry:
[0516] RyR channels were immunoprecipitated from skeletal muscle
homogenates with anti-RyR antibody in 0.5 ml of buffer (50 mM Tris
HCl buffer, pH 7.4 0.9% NaCl 5.0 mM NaF 1.0 mM Na.sub.3VO.sub.4
0.5% Triton X-100+protease inhibitors) for 1 hour at 4.degree. C.
The samples were incubated with protein A Sepharose beads (Amersham
Pharmacia) at 4.degree. C. for 1 h, after which the beads were
washed three times with buffer. Proteins were separated on SDS PAGE
gels (6% for RyR2 and 15% for calstabin2) and transferred onto
nitrocellulose membranes overnight (SemiDry transfer blot,
Bio-Rad). After incubation with 5% nonfat milk to prevent
non-specific antibody binding and a wash in Tris-buffered saline
with 0.1% Tween-20, membranes were incubated for 1-2 h at room
temperature with primary antibodies anti-calstabin (1:1,000),
anti-RyR (5029; 1:5,000), or anti-phospho-RyR2-pSer2809 (1:5,000),
which detects PKA-phosphorylated mouse RyR1-pSer2844 and
RyR2-pSer2808. After three washes, membranes were incubated with
horseradish peroxidase-labeled anti-rabbit IgG (1:5,000,
Transduction Laboratories, Lexington, Ky.), and developed with an
enhanced chemiluminescent detection system (Amersham Pharmacia).
Band densities were quantified by using QUANTITY ONE software
(Bio-Rad).
Example 8
Effect of S107
[0517] FIGS. 42-55 show molecular mechanisms of muscle fatigue and
the effect of S107.
[0518] Drug Delivery:
[0519] Eight-week-old, weight-matched, C57BL/6J littermate mice
were randomized to dosing with either S107 or vehicle (H.sub.2O).
On day -3 of each trial, osmotic pumps (Alzet Model 2004, 200 ul
total volume, 0.25 ul/hr delivery, Durect, Cupertino, Calif.)
filled with either 200 ul of PBS or 200 ul of S107 (10 ug/ul
diluted in H.sub.2O) were implanted subcutaneously on the dorsal
surface of each mouse by a horizontal incision on the neck. Mice
were allowed to recover for three days prior to the initiation of
exercise. Standard food and water were provided ad libitum through
the experiment.
[0520] Chronic Exercise Model:
[0521] The daily swimming protocol consisted of twice-daily
swimming sessions separated by a one-hour rest period. After an
initial conditioning regimen lasting 5 days during which the
swimming sessions were increased in 10 minute increments from 40
minutes each to 80 minutes each, the swimming sessions thereafter
lasted 90 minutes each. A 30 cm wide by 30 cm long opaque acrylic
tank was filled with tap water to a depth of at least 20 cm. Water
was circulated and warmed to 32-34.degree. C. using a separate
reservoir with heating element, thermostat, and pump. 8 mice,
balanced pair-wise with respect to genotype and/or treatment group,
swam at any one time in the tank. Littermates who did not exercise
were reserved as sedentary controls.
[0522] In order to confirm that uniform exercise conditions were
achieved, pilot experiments were performed in which the motion of
each individually identified mouse was tracked with a video
tracking system (San Diego Instruments, San Diego, Calif.).
Individual recorded tracks over the full 90 minutes of each swim
were analyzed for distance swam, mean velocities over time, etc.
using the SMART 2.0 software with Social Behavior package (San
Diego Instruments). No significant differences in the degree of
exercise were noted.
[0523] Treadmill Performance:
[0524] A Columbus Instruments (Columbus, Ohio) treadmill (Model:
Exer-6M Treadmill with Treadmill Shock Detection Unit) with 6 lanes
was used to run the mice. Mice were placed in their respective
lanes with the shocking apparatus turned off and allowed to adjust
to the surroundings for 10 minutes. The forward half of the
treadmill was covered with aluminum foil to block out light and a
desk lamp illuminated the shocking area at the rear of the
treadmill. After the adjustment period, the treadmill was set to 10
meters/min, and the mice were trained to run with gentle prodding
for 6 minutes. The electric current was then turned on, and the
number of shocks delivered during the next two three minute
intervals (training period) were recorded. The shock counter was
then reset and visits to the shocking area and shocks delivered to
each mouse were recorded at three-minute intervals until the end of
the experiment. At regular intervals, the speed of the treadmill
was ramped up from the initial 10 m/min to 24 m/min. The speed was
increased no more than 2 m/min every 6 minutes. Task failure was
defined when a mouse could not continue running despite gentle
prodding.
[0525] Intact Muscle Preparation:
[0526] Immediately following a forced exercise session, each mouse
was sacrificed by carbon dioxide inhalation and cervical
dislocation. Blood was removed by intracardiac aspiration, spun
down, and plasma was eluted and frozen in liquid nitrogen. 4-0 silk
sutures were tied to the proximal and distal tendons of intact EDL
and soleus muscles and the muscles were dissected free and placed
in a modified Ringer's solution (140 mM NaCl, 5 mM KCl, 2.0 mM
CaCl.sub.2, 2 mM MgCl.sub.2, 10 mM HEPES, 10 mM glucose, pH 7.4)
bubbled with 100% O.sub.2. Muscles were hung vertically in 50 mL
Radnoti jacketed glass chambers with one tendon attached with 4-0
silk suture to an isometric force transducer (F30, Harvard
Apparatus, Cambridge, Mass.) and the other tendon attached by
suture to a stationary arm with built in platinum stimulating plate
electrodes. After perfusion with 35C Ringer's and equilibration for
10 minutes at a resting tension of 1 cN and a brief potentiation
protocol, force-frequency relationships were measured, with 60
second delays between 800 ms stimulations at 40-150 Hz. DMCv4.1.6
(Aurora Scientific, Canada) was used to stimulate and record muscle
responses, and DMA v3.2 (Aurora Scientific, Canada) was used to
analyze the resultant data. Following stimulation, muscle length
was determined at resting tension, and muscle dry weight was
recorded.
[0527] Confocal Microscopy:
[0528] Single flexor digitorum brevis (FDB) fibers were
enzymatically dissociated by standard methods (Reiken, Lacampagne
et al. 2003). Briefly, the muscle was dissected from the paw,
placed in a modified Ringer's solution, and stripped of all fascia.
Type 1 collagenase (2 mg/ml, Sigma) in Ringer's solution was
prepared fresh, and the muscle was digested for 2 hours at
37.degree. C. in a incubator shaking at 125 rpm. The muscle was
placed in fresh Ringer's and gently triturated. Single fibers were
collected and allowed to attach to glass coverslips coated in
laminin (Sigma L-2020). The cells were loaded with 2 .mu.M fluo4-AM
ester (Invitrogen) for 20 minutes, placed on the Zeiss Live 5
microscope stage and superfused for 15 minutes with Ringer's. The
fibers were paced at 1 Hz for 10 minutes prior to imaging baseline
fiber properties. Linescan images were continuously acquired at 1
ms scan rate during a tetanic sequence consisting of 300 ms
stimulation at 100 Hz with a 2 Hz train rate. Images were analyzed
in ImageJ, and an F/F0 ratio was calculated for each fiber.
[0529] Human Exercise Protocol:
[0530] Three weeks prior to test sessions, subjects reported to the
Human Performance Laboratory at Appalachian State University for
baseline measurements of cardiorespiratory fitness and body
composition. On three consecutive test session days, subjects ate a
standardized breakfast (7-8:00 am) and lunch (completed by 12:30
pm), and then reported to the ASU Human Performance Laboratory at
2:00 pm. Subjects exercised on exercise bikes at 70% VO.sub.2max
from 3:00-6:00 pm. Test sessions days were Monday, Tuesday, and
Wednesday afternoons. Oxygen consumption and other metabolic
parameters were measured using a metabolic cart (with a mouthpiece
and noseclip) every 30 minutes, and blood lactate and glucose (via
finger stick) every 60 minutes to verify that subjects were
adhering to the prescribed exercise workloads. Subjects ingested
0.5-1.0 liters water every hour of exercise while avoiding all
forms of ingested energy (e.g., bars, drinks). Resting control
subjects sat in the laboratory during the exercise test sessions.
Blood, urine, and saliva samples were collected 15-30 minutes
before exercise/sitting, and then within 5-10 minutes post-exercise
on each of the 3 test sessions. Muscle biopsy samples were obtained
15-30 minutes before exercise/sitting, and then within 5-10 minutes
post-exercise using a needle biopsy procedure on Days 1 and 3. Four
samples were taken (two from each thigh), about 2 inches apart.
Biopsies were snap frozen in liquid nitrogen and stored at
-80.degree. C.
[0531] Single Channel Recording and Data Acquisition:
[0532] SR vesicles from skeletal muscle of sedentary mice and mice
chronically exercised and treated either with vehicle or S107 were
prepared as described previously (Reiken, Lacampagne et al. 2003).
RyR1 channels were reconstituted by spontaneous fusion of
microsomes into the planar lipid bilayer (a mixture of
phosphatidylethanolamine and phosphatidylserine in a 3:1 ratio,
Avanti Polar Lipids). Planar lipid bilayers were formed across a
200 .mu.m aperture in a polysulfonate cup (Warner Instruments,
Inc.), which separated two bathing solutions (1 mM EGTA, 250/125 mM
HEPES/Tris, 50 mM KCl, 0.5 mM CaCl.sub.2, pH 7.35 as cis solution
and 53 mM Ba(OH).sub.2, 50 mM KCl, 250 mM HEPES, pH 7.35 as trans
solution). After incorporation, RyR1 channel activity was recorded
continuously for at least 10 minutes. The concentration of free
Ca.sup.2+ in the cis chamber was calculated with WinMaxC program
(version 2.50) (Bers, Patton et al. 1994). Single channel currents
were recorded at 0 mV using the Axopatch 200A patch-clamp amplifier
(Axon Instruments, USA) in gap-free mode, filtered at 1 kHz, and
digitized at 10 kHz. Data acquisition was performed using Digidata
1322A and Axoscope 9 software (Axon Instruments, USA). The
recordings were analyzed using Clampfit 10.1 (Molecular Devices,
USA) and Origin software (ver. 6.0, Microcal Software, Inc.,
USA).
[0533] Analysis of Ryanodine Receptor Complex:
[0534] 10 mg muscle samples were isotonically lysed. The ryanodine
receptor (RyR1) was immunoprecipitated by incubating 250 .mu.g of
homogenate with anti-RyR antibody (2 .mu.l 5029 Ab) in 0.5 ml of a
modified RIPA buffer (50 mM Tris-HCl pH 7.4, 0.9% NaCl, 5.0 mM NaF,
1.0 mM Na.sub.3VO.sub.4, 0.5% Triton-X100, and protease inhibitors)
for 1 hr at 4.degree. C. The samples were incubated with protein A
Sepharose beads (Amersham Pharmacia) at 4.degree. C. for 1 h, after
which the beads were washed three times with buffer. Proteins were
separated on SDS-PAGE gels (4-20% gradient) and transferred onto
nitrocellulose membranes overnight (SemiDry transfer blot,
Bio-Rad). After incubation with blocking solution (LICOR
Biosciences, Lincoln Nebr.) to prevent non-specific antibody
binding and a wash in Tris-buffered saline with 0.1% Tween-20,
membranes were incubated for 1-2 h at room temperature with primary
antibodies anti-calstabin (1:2500 in blocking buffer), anti-RyR
(5029, 1:5,000), or anti-phospho-RyR2-pSer2809 (1:5000), which
detects PKA-phosphorylated mouse RyR1-pSer2844 and RyR2-pSer2808,
anti-PDE4D3 (1:1000). After three washes, membranes were incubated
with infrared labeled secondary antibodies (1:10,000 dilution,
LICOR Biosystems). Band densities were quantified using the Odyssey
Infrared Imaging System (LICOR Biosciences).
[0535] Calpain and Creatine Kinase Assays:
[0536] Tissue calpain activities were measured using a calpain
activity assay kit (Calbiochem, San Diego, Calif.) (FIG. 42B). This
assay is based on the degradation of the fluorescent peptide
substrate Suc-LLVY-AMC (Calbiochem). Muscle homogenates were
diluted to a final concentration of 600 .mu.g/ml and the calpain
activity in the homogenate was determined as per manufactures
instructions. Plasma creatine kinase (CK) activity was assayed
using the regent kit from Pointe Scientific, Inc. (Canton, Mich.)
(FIG. 42A). Plasma samples (duplicates, 5 .mu.l each) were added to
200 .mu.l of CK reagent and the change in absorbance at 340 nM was
recorded over 4 minutes using a plate reader. The average
absorbance change per minute was used to determine the CPK levels
as per manufacturer's instructions.
[0537] Statistics:
[0538] Data are presented as mean.+-.SEM. Independent t-test with a
significance level of 0.05 was employed to test differences between
cal1-/- and WT, PDE4D-/- and WT, and Ex+veh and Ex+S107, except as
noted below. The distributions of treadmill failure data were found
in several cases to be asymmetric. As such, Wilcoxon rank sum tests
were used for all such data comparisons.
[0539] High Intensity Exercise Induces RyR1 PKA
Hyperphosphorylation, and Depletion of Calstabin1 and PDE4D3 from
the Channel Complex:
[0540] Exercise models in the mouse have been grouped into two
categories: 1) voluntary exercise including running wheels; and 2)
involuntary exercise, including swimming or forced treadmill runs
to exhaustion. In order to achieve high intensity exercise, a twice
daily swimming protocol was adapted to achieve uniform exercise in
mice over days to weeks. This mouse exercise protocol was not
designed to be either explicitly eccentric or isometric in
character, but rather a physiologic mix of eccentric and isometric
exercise. Following forced exercise, RyR1 was immunoprecipitated
out of whole muscle homogenates from hind limb muscles, and the
RyR1 channel complex was size-fractionated on SDS-PAGE and
immunoblotted for channel complex components. High-intensity
exercise in the mouse resulted in progressive phosphorylation of
RyR1 at the PKA site Ser2844 (RyR1-pS2844) that saturated by 14
days of twice daily swimming (90 min sessions, e.g. FIG. 43A).
[0541] In addition, the RyR1 macromolecular complex from extensor
digitorum longus (EDL) muscle underwent remodeling, including
depletion of calstabin1 and PDE4D3 from the channel by day 14
(FIGS. 43A and 43B). An identical pattern of biochemical changes
was seen in all other hind limb skeletal muscles isolated from the
same mice, including soleus, tibialis anterior, and gastrocnemius.
RyR1 PKA hyperphosphorylation and depletion of calstabin1 and
PDE4D3 were dependent on the intensity of the exercise, with only
relatively high intensity exercise resulting in significant channel
modifications (FIGS. 43C and 43D). Furthermore, the remodeling of
the RyR1 channel complex persisted after exercise and recovered
only partially following three days of rest after chronic exercise
(FIG. 51A). PKA hyperphosphorylation of RyR1 was not due to changes
in the amount of PKA and PP1 bound the RyR1 complex, as these
levels were not affected by exercise conditions (FIGS. 51A and
51B). Calstabin1 levels in whole muscle homogenate, measured by
immunoblot, were not altered during exercise (FIG. 51C). Thus,
intense daily exercise, over weeks causes remodeling of the RyR1
channel complex manifested by depletion of the phosphodiesterase
PDE4D3 from the channel, PKA hyperphosphorylation, and depletion of
the stabilizing subunit calstabin1 from the RyR1 channel
complex.
[0542] RyR1 channel defects occur during human exercise: To assess
whether the remodeling of the RyR1 channel macromolecular complex
observed in exercised mice is relevant to human physiology, human
thigh muscle biopsies were obtained from trained athletes before
and after exercise on Days 1 and 3 of a high-intensity exercise
protocol (cycling 3 hr/day at 57% of VO.sub.2max) (Nieman, Henson
et al. 2006). The human RyR1 macromolecular complex was
immunoprecipitated from muscle homogenate, size fractionated by
SDS-PAGE, and immunoblotted to assess RyR1 PKA phosphorylation and
levels of calstabin1 and PDE4D3 in the RyR1 complex. High intensity
exercise resulted in PKA hyperphosphorylation of RyR1 and
calstabin1 depletion compared to controls who did not exercise
(FIG. 44). Prior to exercise on Day 3, PKA phosphorylation of RyR1
in the trained cyclists was at or near resting levels and no
significant calstabin1 depletion from the RyR1 complex was
observed, however, PDE4D3 was stably depleted from the RyR1 complex
by the beginning of the third day of the high intensity exercise
(FIG. 44B). Thus, the same remodeling of the RyR1 channel complex
observed in chronically exercised mice occurs in highly trained
athletes subjected to intense exercise.
[0543] Muscle-Specific Calstabin1-/- Mice have a High-Intensity
Exercise Defect:
[0544] Muscle-specific deficiency of calstabin1 has been previously
shown to be associated with alterations in the force-frequency
relationships of isolated muscle preparations and a reduction in
Cav1.1 current (Tang, Ingalls et al. 2004). To determine if
calstabin1 binding to RyR1 has an effect on exercise performance,
we assessed treadmill run to exhaustion times in mice with
muscle-specific deficiency of calstabin1 (cal1-/-). There was a
significant defect in the high intensity exercise capacity of
cal1-/- mice compared to WT littermate controls (FIG. 45A). The
exercise defect was similar in both males and females (FIG. 45B),
and despite a small reduction in the body weights of the cal1-/-
mice (FIG. 45C), there was no correlation between failure time and
reduced body weight (FIG. 45D). The exercise defect was most
apparent in high intensity exercise (treadmill speeds equal to or
greater than 24 m/min) or in eccentric exercise such as 14 degree
downhill treadmill runs. 0/2 cal1-/- mice were able to complete a
30 min downhill treadmill exercise protocol while 2/2 WT
littermates were able to complete the protocol.
[0545] Twenty-four hours following a downhill exercise regimen (as
described herein), plasma creatine kinase (CPK), was elevated
consistent with increased muscle damage in the cal1-/- mice
compared to WT littermate controls (FIG. 45E). Following several
weeks of daily exercise training, the exercise capacity of WT mice
approached that of cal1-/- presumably because of the progressive
depletion of calstabin1 from the RyR1 complex that occurs with
chronic exercise in WT mice (FIG. 43) resulting in an
exercised-induced depletion of calstabin1 from the RyR1 channel
complex that is comparable to that observed prior to exercise in
the cal1-/- mice (FIG. 45F). Thus, muscle-specific calstabin1
deficient mice exhibit enhanced fatigue consistent with depletion
of calstabin1 from the RyR1 complex playing a role in muscle
fatigue.
[0546] PDE4D-/- Mice Exhibit an Exercise Defect:
[0547] Global deficiency of PDE4D results in an age-dependent
progressive cardiomyopathy (Lehnart, Wehrens et al. 2005). Prior to
the age of three months, however, PDE4D-/- mice exhibit no cardiac
defect as determined by echocardiogram or cardiac catheterization.
The exercise capacity of 2 month-old PDE4D-/- mice was compared
with their WT littermates. A significant reduction in exercise
capacity was observed (FIG. 46A) without any correlation with body
weight (FIG. 46B,C,D). Remarkably, CPK levels were increased,
consistent with muscle damage at rest, in PDE4D-/- mice relative to
WT littermate controls, and there was a significant increase in CPK
levels 24 hours following a single episode of eccentric exercise
consisting of thirty minutes of downhill treadmill running (FIG.
46E) in PDE4D-/- mice. PDE4D-/- mice exhibited an absence of RyR1
bound PDE4D3, a small increase in basal RyR1 PKA phosphorylation,
and a significant increase in calstabin1 depletion following only a
single day of mild exercise (FIG. 46F). These data show that PDE4D3
plays a significant role in the RyR1 complex by regulating the
extent of PKA phosphorylation of RyR1 and that depletion of PDE4D3
from the channel complex during exercise promotes PKA
phosphorylation of RyR1 which in turn contributes to muscle damage
and skeletal muscle fatigue during exercise.
[0548] Pharmacologic Rebinding of Calstabin1 to RyR1 Improves
Chronic Exercise Performance:
[0549] Having demonstrated that a deficiency of calstabin1 is
associated with an exercise defect and that chronic or high
intensity exercise can result in calstabin1 depletion from RyR1,
the effect on chronic or high intensity exercise performance of
pharmacologic rebinding of calstabin1 to RyR1 was determined.
1,4-benzothiazepine derivatives, RyCal compounds, were screened to
identify compounds with increased target activity, improved
specificity (absence of activity against other known ion channels)
and in vivo efficacy in terms of improved exercise capacity.
Compound S107 at a concentration of 500 nM was found not to affect
L-type calcium channel current or hERG potassium current.
[0550] Age and sex-matched WT mice were randomized to receive
osmotic pumps containing either S107 or vehicle. Dosing with S107
at 2.5 ug/hr or vehicle was initiated four days prior to the
beginning of a 21-day forced swimming exercise protocol. Exercise
capacity was assessed once a week by a level treadmill run to
exhaustion during the nocturnal cycle of the mouse. The mice were
not exercised on the same day as treadmill assessments. FIG. 47A
shows that calstabin1 rebinding with S107 had no acute effect on WT
exercise performance, but that over time the S107 treated WT mice
were relatively protected against a decline in treadmill exercise
capacity that occurred in vehicle treated mice (p<0.05 Wilcoxon
rank test, S107 vs. vehicle at Day 21). These studies are
complicated by the training effect of repeated exercise which leads
to improved performance due to enhanced musculature. Individual
treadmill failure times on Day 21 are shown in FIG. 47B. Isometric
force production was measured in EDL muscles in a tissue bath
during field stimulation.
[0551] EDL muscles from S107 treated mice showed increased force
production at stimulation frequencies greater than 80 Hz consistent
with a left-shift of the force-frequency relationship (FIG. 47C).
Drug treatment did not result in a change in body weight (FIG. 47D)
or muscle weight. In parallel chronic exercise trials with mice
deficient in calstabin1, S107 failed to improve treadmill
performance (FIG. 47E). The chronic exercise protocol resulted in
substantial PKA phosphorylation of RyR1 and calstabin1 depletion
from immunoprecipitated RyR1. Calstabin1 depletion from RyR1 was
nearly entirely reversed by S107 treatment (FIG. 47F). Taken
together these data show that preventing the SR Ca.sup.2+ leak due
to PKA hyperphosphorylated RyR1 channel with a drug that enhances
calstabin1 binding to the channel can protect against muscle
damage, muscle fatigue, enhance muscle function and improve
exercise performance during fatigue protocols.
[0552] Reduced Fatiguability in Calstabin1 Rebound FDB Muscle
Fibers:
[0553] Flexor digitorum brevis (FDB) muscle fibers were
enzymatically dissociated from mice following the chronic exercise
protocol and loaded with the calcium indicator fluo-4. Individual
muscle fibers were imaged on a Zeiss Live5 confocal microscope
during field stimulation at 1 Hz and during a fatiguing protocol
consisting of repeated 300 ms long 120 Hz tetani every 2 seconds
for 400 seconds. Representative F/F0 traces during the fatigue
protocol are shown for a FDB fiber isolated from a vehicle treated
mouse (FIG. 48A) and a S107 treated mouse (FIG. 48B). FDB fibers
from S107 treated mice exhibited a delayed decline in peak tetanic
calcium transients (FIG. 48C). It is known that muscle fibers with
slower kinetics of Ca.sup.2+ release and reuptake are less prone to
fatigue. The kinetics of Ca.sup.2+ release and reuptake during
single twitches at 1 Hz were assessed. The distribution of 50%
reuptake times (tau) showed no significant differences between
vehicle and S107 treatment (FIG. 52), indicating no shift in the
calcium reuptake kinetics of the FDB fibers. These data indicate
that treatment with S107 improves Ca.sup.2+ handling in muscle
fibers during fatigue protocols.
[0554] Chronic Exercise Results in Leaky RyR1 Channels which can be
Reversed by Calstabin1 Rebinding:
[0555] A critical issue is whether the biochemical changes in the
RyR1 macromolecular complex identified during exercise result in
changes in RyR1 channel activity. To address this directly, SR
microsomes were prepared from the hind limb muscle of sedentary
mice, mice chronically exercised and treated with vehicle, and mice
chronically exercised and treated with S107. Using standard
techniques, vesicles were fused to planar lipid bilayers and the
single channel activity of incorporated RyR1 channels was
continuously measured for at least 10 minutes at 90 nM
[Ca.sup.2+].sub.cis (FIG. 49A). In agreement with previously
published data (Meissner, 1994, Reiken, 2003), the activity of RyR1
from sedentary mice at resting calcium concentrations was very low
resulting in a small number of openings over long period of time
(in some experiments as long as 20 min of recording was necessary
to calculate an open probability for the channel). In contrast,
RyR1 channels from mice chronically exercised and treated with
vehicle displayed increased activity with significantly higher open
probabilities (p<0.005, Ex+veh, n=9 vs sedentary, n=9) due to an
increased frequency of openings (FIG. 49B). Administration of S107
caused a significant decrease of RyR1 open probability (p<0.005,
Ex+S107, n=12 vs Ex+veh, n=9) to a level comparable to that
observed in channels from sedentary mice. Neither chronic exercise
nor S107 had any effect on the duration of the channel dwell times
meaning that observed changes in open probability were due to
changes in the number of opening events (FIG. 49B). These data show
that RyR1 channels from exercised animals exhibit "leaky" channel
behavior (increased open probability) and that channels from
animals treated with S107 were not "leaky".
[0556] Reduced Calpain Activation in Muscle Tissue and Reduced
Muscle Tissue Damage Due to Calstabin1 Rebinding:
[0557] One possible mechanism by which calcium released by leaky
RyR1 channels impairs exercise performance is the activation of a
member of the calpain family of Ca.sup.2+-dependent neutral
proteases, which are known to be responsible for muscle damage in a
number of pathophysiological states. (Belcastro 1993; Berchtold,
Brinkmeier et al. 2000). Calpain activity in muscle homogenates was
assessed by means of the degradation of the synthetic calpain
substrate Suc-LLVY-AMC, which fluoresces upon cleavage by calpain.
Chronically exercised EDL muscle exhibited elevated calpain
activity compared to sedentary controls. Calpain activity was
significantly reduced in S107 treated and chronically exercised
mice (FIG. 50A). Evidence of a protection from muscle damage was
further provided by measurement of plasma CPK activity levels which
were elevated in the chronically exercised mice, but reduced close
to the levels observed in sedentary controls in the S107 treated
mice (FIG. 50B). Muscle histology showed evidence of muscle
hypertrophy with some inflammation and scattered loci of damaged
fibers in the chronically exercise mice, without evidence of
extensive necrosis in either treatment group.
Example 9
Muscular Dystrophy and Effects of S107
[0558] RyR1 calcium release channels become PKA hyperphosphorylated
and depleted of the stabilizing protein calstabin1 during exercise.
The compounds of the invention increase the binding affinity of
calstabin1 to PKA hyperphosphorylated RyR1. These compounds
(referred to as called "calcium channel stabilizers" or "rycal")
are 1,4-benzothiazepines and derivatives thereof. Treatment with
these compounds improves exercise performance of mice running on a
treadmill. A calcium leak via PKA hyperphoshorylated RyR1 channels
causes muscle damage due to activation of calcium-dependent
proteases and rycals prevent the calcium leak and inhibit muscle
damage during chronic exercise. Rycals can be used to improve
muscle fatigue in chronic diseases including heart failure, AIDS,
cancer, renal failure, and can also be used to treat muscular
dystrophies.
[0559] Duchenne muscular dystrophy (DMD) is an X-linked muscle
disease characterized by mutations in the dystrophin gene.
Increased calcium-activated calpain proteolysis in the sarcolemma
membrane is thought to be a primary mechanism in the
pathophysiology of DMD. The mdx mouse, carrying a stop codon inside
exon 23 of the dystrophin gene, provides a useful system to study
the effectiveness of different therapeutic strategies for the cure
of this disease.
[0560] A RyCal compound, S107, reduces calpain activity in mdx mice
during exercise. FIGS. 42, 53, 54, 55 provide data from these
studies. This indicates that RyCals may be useful for treating
muscle related diseases, including, but not limited to, muscular
dystrophies.
[0561] Animal Model:
[0562] Mdx mice (21 days old) were treated with S107 (0.125
mg/kg/h) or vehicle using implantable, osmotic pumps for 28 days.
After treatment, the mice were subjected to downhill (14.degree.
angel) treadmill running for 30 min at 18 m/min. Immediately after
the exercise, the animals were sacrificed and the skeletal muscles
were harvested.
[0563] Preparation of EDL Homogenates:
[0564] EDL muscles homogenates were prepared in 0.5 ml of
homogenization buffer (20 mM NaF, 10 mM Tris-maleate,
pH7.2+protease inhibitors). Cardiac sarcoplasmic reticulum (CSR)
fractions were obtained by centrifuging the homogenates at
50,000.times.g for 30 min. Homogenates were centrifuged at
4000.times.g for 20 min and the supernatants were centrifuged for
20 min at 10000.times.g. Aliquots of the homogenates were assayed
for protein concentration and stored at -80.degree. C.
[0565] Calpain Activity Assay:
[0566] Calpain activity of EDL homogenates (30 .mu.g) were measured
using a Calpain Activity Assay kit (Calbiochem). The assay utilizes
a synthetic calpain substrate, suc-LLVY-AMC. AMC is released upon
cleavage with calpain and is measured fluorometrically. Assays are
performed with both an activation and an inhibition buffer to
determine specific calpain activity in the sample.
[0567] Serum Creatine Kinase:
[0568] Serum (10 .mu.l) proteins were separated using 4-20% PAGE.
After transferring the proteins to nitrocellulose, the immunoblots
were developed using an anti-creatine kinase Antibody (Research
Diagnostics, 1:1000 dilution). Bands were quantified by
densitometry.
[0569] Immunoprecipitation of Ryanodine Receptor:
[0570] The ryanodine receptor (RyR1) was immunoprecipitated from
samples by incubating 250 .mu.g of EDL homogenate with anti-RyR
antibody (2 .mu.l 5029 Ab) in 0.5 ml of as modified RIPA buffer (50
mM Tris-HCl (pH 7.4), 0.9% NaCl, 5.0 mM NaF, 1.0 mM
Na.sub.3VO.sub.4, 0.5% Triton-X100, and protease inhibitors) 1 hr
at 4.degree. C. The samples were incubated with Protein A sepharose
beads (Ammersham Pharmacia Biotech, Piscatawy, N.J.) at 4.degree.
C. for 1 hour, after which, the beads were washed three times with
RIPA. Samples were heated to 95.degree. C. and size fractionated by
PAGE.
[0571] Western Analysis:
[0572] Samples (immunoprecipitates or 10 .mu.g CSR) were heated to
95.degree. C. and the proteins were size fractionated on 6% SDS
PAGE for RYR and 15% PAGE for calstabin. Immunoblots were developed
using antibodies against RyR (5029, 1:5000 dilution), PKA
phosphorylated RyR (1:10000), or Calstabin (1:2000). Dilutions are
made in 5% milk in TBS-T.
Example 10
Preparation of Tissue Lysates
[0573] Tissue lysates were prepared by homogenizing the tissue
(e.g., brain, cardiac, muscle) with Tissuemiser in 0.7 ml lysis
buffer (pH 7.4, 10 mM HEPES, 1 mM EDTA, 20 mM NaF, 2 mM Na.sub.3
VO.sub.4, 320 mM sucrose, and protease inhibitors) and centrifuged
for 15 min at 4,000.times.g at 4.degree. C. The supernatant was
then centrifuged for 15 min at 10,000.times.g at 4.degree. C. For
brain homogenates, the supernatant was centrifuged at
50,000.times.g at 4.degree. C. for 30 minutes, and the pellet
(microsomes) was resuspended in homogenization buffer which was
supplemented with 0.9% NaCl. For brain tissue, the resuspended
pellet was used for immunoprecipitation of RyR. For cardiac and
muscle tissue homogenates, the supernatant of the 10,000.times.g
spin was used for immunoprecipitation of the RyR. Protein
concentrations were measured by Bradford protein assay. The sample
was frozen at -80.degree. C. until use.
Example 11
Immunoprecipitation of Ryanodine Receptors (RyRs)
[0574] 100 .mu.g of microsomes were brought to a volume of 500
.mu.l with modified RIP A buffer (50 mM Tris-HCl (PH 7.4), 0.9%
NaCl, 5.0 mM NaF, 1.0 mM Na.sub.3VO.sub.4, 0.5% TritonX100, and
protease inhibitors). The ryanodine receptor was immunoprecipitated
by adding 21 of anti-RyR antibody (5209) and rotating the sample
for 1 hr at 4.degree. C. The sample was incubated with 40 .mu.l of
Protein A Sepharose beads and rotated for 1 hr at 4.degree. C.
After washing the beads with 500 .mu.l RIP A buffer three times,
the resulted pellet was resuspended in 15 .mu.l of 2X SDS sample
buffer and boiled for 5 min.
[0575] For Western Blot Analysis, proteins were size fractionated
on SDS-PAGE 4-20% gradient (BioRad). Immunoblots were developed
with anti-FKBP and anti-RyR antibody or anti-phosphorylated
FKBP.
Example 12
Effects of S107 on Spatial Learning and Cognitive Function
[0576] Experiments were performed to determine whether the
compounds described herein cross the blood brain barrier and
enhance binding of cal stab in to ryanodine receptors in the brain.
FIG. 56 shows the results of Western blots performed on RyR
immunoprecipitated from the tissue samples indicated (i.e. heart,
soleus muscle, mid-brain, and cerebellum). As illustrated in FIG.
56, the compound S107 crosses the blood brain barrier and restores
in vivo binding of calstabin to RyR in both the mid-brain and the
cerebellum, following depletion of calstabin from the RyR complex
by treatment of the mice with isoproterenol ("ISO") by chronic
infusion for 5 days. RyR was immunoprecipitated using an antibody
to RyR, and the presence of calstabin in the immunoprecipitates was
detected using an antibody to calstabin. The figure shows that the
compound S107 penetrates the brain and restores in vivo binding of
calstabin to RyR. Thus, S107 has calstabin rebinding activity in
the brain.
[0577] FIG. 57 provides a schematic representation of in vivo
experiments used to test the effect of S107 on cognitive function
in mice, using the Morris water maze system (described below in
FIG. 58). 16 wild type C57BL/6J 3-month-old mice, pairwise-matched
for sex, age, and body weight, were randomized to either S107
treatment (10 mg/ml; 0.25 .mu.l/hr subcutaneous osmotic pump) or
"vehicle" (25% DMSO in dH.sub.2O) treatment groups.
[0578] Two days after initiation of treatment, mice were subject to
an exercise regimen for 21 days, and effect of S107 treatment was
assayed by the weekly protocol as described below. Mice were
sacrificed after 21 days for performing biochemistry, calcium
imaging and ex vivo function studies.
[0579] FIG. 58 (A) provides a schematic representation of in vivo
experiments used to test the effect of S107 on learning in the
Morris water maze system. The layout of the water maze system
consists of a circular water tank divided into four quadrants
(labeled 1 thru 4 in FIG. 58, with four hidden platforms (labeled 5
to 8 in FIG. 58). The following protocol was followed: Day 1: mice
trained to find "hidden" platform with visible marker on platform
from random starting location. On days 2-4, the visible cue was
removed, and mice were repeatedly challenged to find hidden
platform at target 5 in quadrant 1. The time taken for each mouse
to reach the target, i.e. the "latency," was recorded. On day 5,
the previous day's protocol was repeated, then the hidden platform
was removed, and each mouse's movements recorded to quantify time
in various target regions. The protocol was repeated in week 2. The
bar graphs at the bottom of FIG. 3 show the latency to target(s)
(panel B) and mean velocity (cm/s) (panel C) for the vehicle and
S107 treated groups at the end of the 21-day testing period.
[0580] The platform was then removed, and the swimming pattern of
the mice was assessed at the end of the 21-day testing period. FIG.
59 shows a trend towards improved learning or increased persistence
in S107-treated mice as compared to vehicle. FIG. 60 provides
graphical data from the above experiments and shows a trend towards
altered behavior consistent with improved learning and persistence
in the S107-treated mice. p was approximately 0.2 vs. control (n=8
in both groups). The difference in permanence times between
S107-treated and vehicle-treated mice does not appear to be due to
swimming differences during the 2-minute probe learning assay.
[0581] FIG. 61 shows biochemical data for mice subjected to an
exercise regimen in the absence and presence of S107 at the end of
the 21-day testing period. Ryanodine receptor (types 1 and 2) was
immunoprecipitated from whole brain microsomes. Immunoprecipitates
were separated by 4-20% PAGE and analyzed for total RyR, PKA
phosphorylated RyR, and calstabin. The figure shows
exercised-induced RyR1 and RyR2 phosphorylation, accompanied with
reduction in calstabin 1 or 2 binding. Treatment with S107 restores
the binding of calstabin to RyR in exercised mice.
Example 13
Effects of Restraint Stress on PKA Phosphorylation at Different
Stress Periods
[0582] FIGS. 62-65 illustrate the effect of restraint stress on PKA
phosphorylation at different stress periods. Restraint Stress
Model: Chronic stress has been found to induce PKA phosphorylation
of ryanodine receptors (RyRs) in cardiac (RyR2) and skeletal (RyR1)
muscle cells. The effects of chronic stress on PKA phosphorylation
of RyRs in the brain, however, have not been explored. The
Restraint Stress Model is designed to investigate whether chronic
stress induces PKA phosphorylation of neuronal RyRs. As shown in
FIG. 62, twelve C57BL/6J wild type male mice were assigned to
different stress groups (n=2/group), generating 6 groups. Five of
the 6 groups were stressed and sacrificed at the end of each stress
period: 1, 5, 10, 14, and 21 days of stress (respectively 1D, 5D,
10D, 14D and 21D). The remaining group served as control (0D) that
was not restrained, and was sacrificed together with the 1D group.
Subjects in each stress group were restraint stressed in Plexiglas
restrainer tubes (10.times.21/2.times.31/4 cm) 2 hr in the morning
and 2 hr in the afternoon of each stress period. The two
nonstressed control subjects were handled in their home cage. At
the end of each stress period, subjects were sacrificed (sac) by
CO.sub.2 and their brains were immediately removed and frozen for
later immunoblot analysis. RyR2 was immunoprecipitated from whole
brain microsomes.
[0583] FIG. 63 shows the results of PKA phosphorylation of RyR2
channels in brain following restraint induced stress in mice. The
mice were restrained for time periods as indicated. Ryanodine
Receptor (type 2) was immunoprecipitated from whole brain
microsomes. Immunoprecipitates were separated by 4-20% PAGE and
analyzed for total RyR2, PKA Phosphorylated RyR2, and calstabin2.
FIG. 63 shows stress-induced RyR2 phosphorylation, accompanied with
reduction in calstabin 2 binding.
[0584] FIG. 64 is a bar graph summarizing the relative amounts of
PKA phosphorylation of RyR2 from FIG. 63. The relative
phosphorylation of RyR2 is represented using arbitrary units. A
one-way ANOVA shows that there was a significant difference between
groups [T(5,6)=27.58, P<0.0005]. Fisher's LSD post hoc test
reveals that 14 and 21 days of chronic restraint stress (CRS)
induced the highest PKA phosphorylation of RyR2 in the brain, where
***(P<0.001) and **(P<0.01) compared with nonstressed
controls (0 days). FIG. 65 is a bar graph summarizing the relative
amounts of calstabin2 bound to RyR2 from FIG. 63. A one-way ANOVA
also shows a group difference between the stress periods
[T(5,6)=5.91, P<0.037]. Fisher's LSD post hoc test reveals that
only the 21 days of CRS showed the lowest calstabin2 binding to the
RyR2 where *(P<0.05) compared with nonstressed controls (0
days).
[0585] All publications, references, patents and patent
applications cited herein are incorporated by reference in their
entirety to the same extent as if each individual application,
patent or patent application was specifically and individually
indicated to be incorporated by reference in its entirety.
[0586] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be
appreciated by one skilled in the art, from a reading of the
disclosure, that various changes in form and detail can be made
without departing from the true scope of the invention in the
appended claims.
* * * * *