U.S. patent application number 16/584071 was filed with the patent office on 2020-01-16 for use of fluorinated derivatives of 4-aminopyridine in therapeutics and medical imaging.
The applicant listed for this patent is The University of Chicago. Invention is credited to Daniel Appelbaum, Pedro Brugarolas, Chin-Tu Chen, Brian Popko.
Application Number | 20200017445 16/584071 |
Document ID | / |
Family ID | 52019393 |
Filed Date | 2020-01-16 |
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United States Patent
Application |
20200017445 |
Kind Code |
A1 |
Brugarolas; Pedro ; et
al. |
January 16, 2020 |
USE OF FLUORINATED DERIVATIVES OF 4-AMINOPYRIDINE IN THERAPEUTICS
AND MEDICAL IMAGING
Abstract
The present disclosure provides novel compounds, including
compounds that bind to potassium channels, methods for their
manufacture, and methods for their use, including their use to
diagnose and/or assess traumatic brain injury and use to treat
dymeylinating diseases, and/or in vivo imaging of the central
neverous system, and to diagnose and/or assess the progression of
MS or other diseases.
Inventors: |
Brugarolas; Pedro; (Chicago,
IL) ; Popko; Brian; (Chicago, IL) ; Appelbaum;
Daniel; (Chicago, IL) ; Chen; Chin-Tu;
(Chicago, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Chicago |
Chicago |
IL |
US |
|
|
Family ID: |
52019393 |
Appl. No.: |
16/584071 |
Filed: |
September 26, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15452179 |
Mar 7, 2017 |
10442767 |
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16584071 |
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14329597 |
Jul 11, 2014 |
9617215 |
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15452179 |
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13897035 |
May 17, 2013 |
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14329597 |
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PCT/US2013/041638 |
May 17, 2013 |
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13897035 |
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61648214 |
May 17, 2012 |
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61648214 |
May 17, 2012 |
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61845878 |
Jul 12, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07B 59/002 20130101;
A61K 51/0455 20130101; C07D 213/75 20130101; C07D 213/73
20130101 |
International
Class: |
C07D 213/73 20060101
C07D213/73; C07B 59/00 20060101 C07B059/00; C07D 213/75 20060101
C07D213/75; A61K 51/04 20060101 A61K051/04 |
Claims
1. An imaging method comprising administering to a subject a
compound of Formula (I): ##STR00018## wherein: R.sub.1, R.sub.2,
R.sub.3, and R.sub.4 are each independently selected from the group
consisting of H, (CH.sub.2).sub.nX, NH.sub.2, CH.sub.2OH,
OCH.sub.3, CF.sub.3, OCHF.sub.2, and OCF.sub.3; at least one of
R.sub.1, R.sub.3, and R.sub.4 is H; R.sub.5 is selected from the
group consisting of H, (CH.sub.2).sub.mX, C(CH.sub.3).sub.3, OH,
COOCF.sub.3, and COO(CH.sub.2).sub.mX; wherein n=0, 1, 2, 3, 4, or
5 and m=1, 2, 3, 4, or 5; wherein X represents a fluorine atom or
.sup.18F; wherein the compound contains at least one .sup.18F,
.sup.11C, .sup.13N, or .sup.15O isotope; and wherein the compound
is not [.sup.18F]3-fluoro-4-aminopyridine; or a pharmaceutical
acceptable salt thereof, or a deuterated version thereof; and
detecting the compound in the subject.
2. A method for diagnosing a demyelinating disease or evaluating
the progression of a demyelinating disease comprising administering
to a subject a compound of Formula (I): ##STR00019## wherein:
R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are each independently
selected from the group consisting of H, (CH.sub.2).sub.nX,
NH.sub.2, CH.sub.2OH, OCH.sub.3, CF.sub.3, OCHF.sub.2, and
OCF.sub.3; at least one of R.sub.1, R.sub.3, and R.sub.4 is H;
R.sub.5 is selected from the group consisting of H,
(CH.sub.2).sub.mX, C(CH.sub.3).sub.3, OH, COOCF.sub.3, and
COO(CH.sub.2).sub.mX; wherein n=0, 1, 2, 3, 4, or 5 and m=1, 2, 3,
4, or 5; wherein X represents a fluorine atom or .sup.18F; wherein
the compound contains at least one .sup.18F, .sup.11C, .sup.13N, or
.sup.15O isotope; and wherein the compound is not
[.sup.18F]3-fluoro-4-aminopyridine; or a pharmaceutical acceptable
salt thereof, or a deuterated version thereof; and detecting the
compound in the subject by a radiodiagnostic method.
3. The method of claim 2, wherein a dose is from about 0.005 to 50
mCi.
4. The method of claim 2, further comprising quantifying an amount
of the compound in the subject.
5. The method of claim 2, wherein the demyelinating disease is
multiple sclerosis, spinal cord compression, ischemia, acute
disseminated encephalomyelitis, optic neuromyelitis,
leukodystrophy, progressive multifocal leukoencephalopathy,
metabolic disorders, toxic exposure, congenital demyelinating
disease, peripheral neuropathy, encephalomyelitis, central pontine
myelolysis, Anti-MAG Disease, Guillain-Barre syndrome, chronic
inflammatory demyelinating polyneuropathy, or multifocal motor
neuropathy (MMN).
6. The method of claim 2, wherein the radiodiagnostic method is
Positron Emission Tomography (PET), PET-Time-Activity Curve (TAC),
PET-Magnetic Resonance Imaging (MRI), or PET/CT.
7. The method of claim 2, wherein a demyelinated region in the
subject is detected by detecting the compound.
8. The method of claim 2, wherein the compound binds to potassium
channels located at a demyelinated region in an axon in the
subject.
9. A method for diagnosing traumatic brain injury or evaluating the
progression of traumatic brain injury in a subject comprising
administering to the subject a compound of Formula (I):
##STR00020## wherein: R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are
each independently selected from the group consisting of H,
(CH.sub.2).sub.nX, NH.sub.2, CH.sub.2OH, OCH.sub.3, CF.sub.3,
OCHF.sub.2, and OCF.sub.3; at least one of R.sub.1, R.sub.3, and
R.sub.4 is H; R.sub.5 is selected from the group consisting of H,
(CH.sub.2).sub.mX, C(CH.sub.3).sub.3, OH, COOCF.sub.3, and
COO(CH.sub.2).sub.mX; wherein n=0, 1, 2, 3, 4, or 5 and m=1, 2, 3,
4, or 5; wherein X represents a fluorine atom or .sup.18F; wherein
the compound contains at least one .sup.18F, .sup.11C, .sup.13N, or
.sup.15O isotope; and wherein the compound is not
[.sup.18F]3-fluoro-4-aminopyridine; or a pharmaceutical acceptable
salt thereof, or a deuterated version thereof; and detecting the
compound in the subject by a radiodiagnostic method.
10. The method of claim 9, wherein the subject is at risk for
traumatic brain injury or a concussion.
11. The method of claim 9, wherein the imaging is affected by a
radiodiagnostic method.
12. The method of claim 11, wherein the radiodiagnostic method is
Positron Emission Tomography (PET), PET-Time-Activity Curve (TAC),
PET-Magnetic Resonance Imaging (MRI), or PET/CT.
13. The method of claim 9, further comprising quantifying the
amount of the compound in the subject.
14. The method of claim 9, wherein a demyelinated region in an axon
in the subject is detected by detecting the compound and an
increase in demyelination indicates traumatic brain injury.
15. The method of claim 9, wherein the compound blocks potassium
channels located at a demyelinated region in an axon in the
subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/452,179 filed Mar. 7, 2017, which is a
continuation of U.S. patent application Ser. No. 14/329,597 filed
Jul. 11, 2014, which is a continuation-in-part of U.S. patent
application Ser. No. 13/897,035 filed May 17, 2013 and PCT
Application PCT/US2013/041638 filed May 17, 2013, both of which
claim priority to U.S. Provisional Patent Application Ser. No.
61/648,214 filed May 17, 2012. U.S. patent application Ser. No.
14/329,597 also claims the benefit of priority to U.S. Provisional
Patent Application Ser. No. 61/845,878 filed Jul. 12, 2013. The
entire contents of each of the above-referenced disclosures are
specifically incorporated herein by reference without
disclaimer.
BACKGROUND OF THE INVENTION
I. Field of the Invention
[0002] The present invention relates generally to the fields of
biology, chemistry and medicine. More particularly, it concerns
derivatives of potassium channel inhibitors, including derivatives
of 4-aminopyridine, and methods of making and using thereof,
including for the treatment and medical imaging of
neurodegenerative conditions.
II. Description of Related Art
[0003] With nearly 400,000 people affected in the U.S. and 2.5
million worldwide, Multiple Sclerosis (MS) is the most common
neurodegenerative condition in young adults (Calabresi, 2007). The
progressive demyelination of neurons in the brain leads to diverse
neurological symptoms. Myelin is the multilayered membrane that
surrounds most axons of the central and peripheral nervous systems
and is essential for the propagation of rapid nerve impulses. In
people with MS, the myelin sheath that normal covers the axons is
lost and this leads to aberrant leakage of potassium ions from the
axon and improper impulse conduction.
[0004] One approach to treat MS or to mitigate the symptoms
associated with MS is to block potassium channels to reduce the
leakage of potassium ions, thus enhancing impulse conduction. In
January 2010, the FDA approved 4-aminopyridine (4-AP), as a therapy
for MS (Ampyra, Acorda Therapeutics, Inc., 2010). 4-AP is a
relatively selective blocker of K.sub.v1 family of K.sup.+ channels
(Wulff et al., 2009). By blocking K.sup.+ channels, impulse
conduction along the axon is partially restored and symptoms
ameliorate.
[0005] To develop new neuroprotective therapies for MS or other
neurodegenerative diseases, it is essential to have proper tools to
diagnose and assess disease progression.
[0006] According to CDC around 1.74 million people sustain a
traumatic brain injury in the U.S. each year. Most of these
injuries are mild (75%) and certain populations are at a higher
risk: men aged 0-4, 15-19 and over 60 as well as military personnel
and people engaged in contact sports. Recent studies have shown
that even mild TBIs can have serious consequences later in life.
TBI has been linked to depression, anxiety, substance abuse and
suicide. All these reasons make screening for TBI particularly
important.
[0007] Currently, the diagnosis of TBI is based on clinical
evaluation aided by Computed Tomography (CT) or Magnetic Resonance
Diffusion Tensor Imaging (MR-DTI). CT scans are very useful for
detecting mass lesions and fractures but do not allow visualization
of mild TBIs. More recently, MR-DTI has emerged as a sensitive
method to evaluate white matter integrity in TBI but it too can be
difficult to interpret.
[0008] During TBI, compression or stretching of the brain often
causes damage to axons and/or the myelin sheath. Oligodendrocytes,
the cells responsible for producing and maintaining myelin, have
also been shown to be sensitive to TBI (Flygt et al., 2013; Sharp
and Ham, 2011; Morey et al., 2012). Oligodendrocyte injury results
in axonal demyelination. In addition to facilitating raping nerve
conduaction velocities, myelin provides axonal protection, such
that demyelinated axons are prone to degeneration. Therefore,
TBI-induced damage to myelin and/or oligodendrocytes likely
contributes to the acute and long-term clinical manifestations of
TBI.
[0009] Axonal proteins are compartmentalized in myelinated axons,
with the voltage-gated sodium channels concentrated at the
unmyelinated node of Ranvier and the rectifying potassium (K+)
channels residing under the myelin sheath (Waxman and Ritchie,
1993). Following demyelination, like that which occurs in multiple
sclerosis (MS) and TBI, the K+ channels become exposed and leaky.
In 2010, the FDA approved 4-amino-pyridine (4-AP, Ampyra.RTM.) as a
drug to improve symptoms in people with MS. 4-AP is a K+ channel
blocker that binds to the exposed channels on demyelinated axons,
which reduces the aberrant efflux of K+ ions and enhances neuronal
conduction. Since 4-AP selectively targets K+ channels that have
become uncovered as a result of demyelination we propose to test
its usefulness as a tracer for demyelinated axons.
[0010] Not much is known about the role of axonal K+ channels and
the effects of 4-AP in TBI. However, there have been numerous
studies looking at the effects of 4-AP after Spinal Cord Injury
(SCI; Blight et al., 1989; Blight et al., 1991; Hayes et al., 1993;
Fehlings and Nashmi, 1996; Gruner and Yee, 1999). Similarly to TBI,
SCI is an injury to the CNS that occurs after a violent impact.
Depending on the location and severity of the injury the symptoms
can vary from partial loss of movement and sensation (incomplete
injury) to complete loss. In cases of incomplete injury, 4-AP has
been shown to enhance neuronal conduction through injured areas
both in animals and in humans (Blight et al., 1989; Blight et al.,
1991; Hayes et al., 1993). In addition, injured spinal cord areas
have been shown to have higher pharmacological sensitivity to 4-AP
(Fehlings and Nashmi, 1996), which agrees with our hypothesis that
K+ channels on demyelinated fibers are more accessible and suggest
the potential of using radioactive 4-AP to map injured areas. The
similarities between TBI and SCI in etiology and at the
histopathological level justify evaluating 4-AP based PET tracers
for TBI. In addition, if 4-AP is found to localize to injured areas
in TBI it could also be useful for restoring function/ameliorating
symptoms in TBI patients. Fluorine-18 is the preferred isotope for
PET imaging because its long half-life allows for off-site
production and commercialization. In addition, its low positron
energy gives higher resolution than for example carbon-11. We have
also shown that these fluorinated molecules have very similar
properties to 4-AP both in vitro and in vivo indicating that
fluorination does not disrupt its properties and therefore these
molecules could be used as surrogates of 4-AP.
[0011] Thus, there is a pressing need for new, accurate methods to
evaluate and diagnose TBI.
SUMMARY OF THE INVENTION
[0012] In some embodiments, there are provided compounds that bind
to potassium channels, methods for their manufacture, and methods
for their use. In a particular embodiment, the compounds may be
compounds of formula (I):
##STR00001##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are each
independently selected from the group consisting of H,
(CH.sub.2).sub.nX, CH.sub.2OCH.sub.2CH.sub.2X, NH.sub.2,
CH.sub.2OH, CF.sub.3, OCH.sub.3, OCH.sub.2F, OCHF.sub.2, OCF.sub.3
and R.sub.5 is selected from the group consisting of H,
(CH.sub.2).sub.mX, OH, COOCF.sub.3, COOC(CH.sub.3).sub.3, and
COO(CH.sub.2).sub.mX; wherein n=0, 1, 2, 3, 4, or 5 and m=2, 3, 4,
or 5; wherein X represents a fluorine atom or an isotope thereof;
as well as pharmaceutically acceptable salts, tautomers, or
deuterated versions thereof.
[0013] In one aspect, the isotope of fluorine is a radioactive
isotope. In a particular aspect, the fluorine isotope is .sup.18F.
In some aspects, any of C, N, O is optionally replaced by an
isotope thereof. An isotope of C, N, O may be any known C, N, O
isotope. In particular aspects, the isotope is a radioisotope. For
example, any of C, N, O may be optionally replaced by the isotope
.sup.11C, .sup.13N, .sup.15O, respectively.
[0014] In further embodiments, at least one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4 and R.sub.5 is not hydrogen. In still further
embodiments, at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4 and
R.sub.5 contains a fluorine atom or an isotope thereof. In certain
aspects, when R.sub.2 is NH.sub.2, CH.sub.2OH, a nonradioactive
fluorine, or CF.sub.3, at least one of R.sub.1, R.sub.3, R.sub.4,
and R.sub.5 is not hydrogen. In additional aspects, when R.sub.4 is
NH.sub.2, CH.sub.2OH, a nonradioactive fluorine, or CF.sub.3, at
least one of R.sub.1, R.sub.2, R.sub.4, and R.sub.5 is not
hydrogen.
[0015] In some embodiments, the compounds are not the following
compounds:
##STR00002##
[0016] In some embodiments, the compounds have the following
formulas:
##STR00003##
[0017] In certain embodiments, there are provided compounds of
formula (II):
##STR00004##
wherein M is (CH.sub.2).sub.nY and wherein n=0, 1, or 2, and Y is
fluorine or an isotope thereof, as well as pharmaceutical
acceptable salts, tautomers, or deuterated versions thereof.
[0018] In certain embodiments, M is CH.sub.2F, or
(CH.sub.2).sub.2F. In further embodiments, M is .sup.18F,
CH.sub.2.sup.18F, or (CH.sub.2).sub.2.sup.18F. In some aspects, any
of C, N, O is optionally replaced by an isotope thereof. An isotope
of C, N, O may be any known C, N, O isotope. In particular aspects,
the isotope is a radioisotope. For example, any of C, N, O may be
optionally replaced by the isotope .sup.11C, .sup.13N, 150,
respectively.
[0019] In certain embodiments, there are provided compounds of
formula (III):
##STR00005##
wherein R is selected from the group consisting of CH.sub.3,
CH.sub.2F, CHF.sub.2, and CF.sub.3, and wherein C is substituted by
.sup.11C or at least one of F is substituted by .sup.18F in R. For
instance, R is .sup.11CH.sub.3, CH.sub.2.sup.18F, CHF.sup.18F,
CH(.sup.18F).sub.2, C.sup.18FF.sub.2, C(.sup.18F).sub.2F, or
C(.sup.18F).sub.3.
[0020] Certain embodiments are directed to the compounds of formula
(IV):
##STR00006##
wherein R is selected from the group consisting of CF.sub.3,
CH.sub.2F, CH.sub.3CH.sub.2F, C(CH.sub.3).sub.3, and wherein at
least one of F or H in the R group is substituted by .sup.18F.
Non-limiting examples include CH.sub.2.sup.18F, CHF.sup.18F,
CH(.sup.18F).sub.2, C.sup.18FF.sub.2, C(.sup.18F).sub.2F,
C(.sup.18F).sub.3, CH.sub.3CH.sub.2.sup.18F, and
C(CH.sub.3).sub.2.sup.18F.
[0021] In some aspects, any of C, N, O in the compounds described
herein is optionally replaced by an isotope thereof. An isotope of
C, N, O may be any known C, N, O isotope. In particular aspects,
the isotope is a radioisotope. For example, any of C, N, O in the
compounds of formula (I)-(IV) may be optionally replaced by the
isotope .sup.11C, .sup.13N, .sup.15O, respectively.
[0022] In some embodiments there are provided pharmaceutical
compositions comprising one or more of the above compounds and a
pharmaceutically acceptable carrier. In some embodiments, the
pharmaceutical compositions further comprise one or more
pharmaceutically acceptable excipients. In some embodiments, the
composition is formulated for controlled release of any of the
compounds disclosed herein.
[0023] Certain embodiments are directed to a kit comprising one or
more of the above compounds. In further aspects, there are provided
a kit comprising one or more of the above compounds comprising a
radioisotope.
[0024] In some embodiments, there are provided imaging methods
comprising administering to a subject in need thereof the imaging
agent described herein and detecting the compound comprised in the
imaging agent in the subject. In some aspects, the amount of the
compound in the subject is quantified. In further aspects, a
demyelinated region in an axon in the subject is detected via a
detection of the compound in the subject. In still further aspects,
the compound administered to the subject may block potassium
channels located at the demyelinated region in an axon in the
subject.
[0025] In certain embodiments, the imaging is effected by a
radiodiagnostic method. The radiodiagnostic method may be performed
by any instrument capable of detecting radiation by the compounds.
Exemplary radiodiagnostic methods include, but not limited to,
Positron Emission Tomography (PET), PET-Time-Activity Curve (TAC)
or PET-Magnetic Resonance Imaging (MRI). In particular aspect, the
radiodiagnostic method is PET.
[0026] Certain embodiments are directed to an imaging agent
comprising a compound described herein wherein the compound
contains an isotope. In some embodiments, the isotopes are isotopes
of F, O, N and C. In particular aspects, the isotope is a fluorine
isotope. In further embodiments, the isotope is a radioisotope. In
still further embodiments, the radioisotope is .sup.18F, .sup.15O,
.sup.13N or .sup.11C. In particular embodiments, the isotope is
.sup.18F. For example, an imaging agent may comprise a derivative
of 4-AP, including, but not limited to,
[.sup.18F]-3-fluoro-4-aminopyridine,
[.sup.18F]-3-fluoro-methyl-4-aminopyridine, and
[.sup.18F]-3-fluoro-ethyl-4-aminopyridine.
[0027] In some embodiments, there are provided methods the use of
novel compounds as described herein, including for the treatment
and/or in vivo imaging of the central nervous system to diagnose
and/or assess the progression of MS or other diseases.
[0028] In some embodiments, there are provided methods for
diagnosing traumatic brain injury (TBI) or evaluating the
progression of TBI comprising administering to a subject in need
thereof an imaging agent described herein and detecting the
compound comprised in the imaging agent in the subject.
[0029] In some embodiments, there are provided methods of treating
a demyelinating disease or mitigating a symptom of a demyelinating
disease comprising administering to a subject in need thereof an
effective amount of a compound as defined above.
[0030] In further embodiments, there are provided methods of
treating TBI or mitigating a symptom of TBI comprising
administering to a subject in need thereof an effective amount of a
compound as defined above.
[0031] It is specifically contemplated that in certain embodiments,
methods related to therapy and/diagnostics involve a subject that
is a human patient.
[0032] "Treatment" or "treating" includes (1) inhibiting a disease
in a subject or patient experiencing or displaying the pathology or
symptomatology of the disease (e.g., arresting further development
of the pathology and/or symptomatology), (2) ameliorating a disease
in a subject or patient that is experiencing or displaying the
pathology or symptomatology of the disease (e.g., reversing the
pathology and/or symptomatology), and/or (3) effecting any
measurable decrease in a disease in a subject or patient that is
experiencing or displaying the pathology or symptomatology of the
disease.
[0033] "Effective amount" or "therapeutically effective amount" or
"pharmaceutically effective amount" means that amount which, when
administered to a subject or patient for treating a disease, is
sufficient to effect such treatment for the disease. In some
embodiments, the subject is administered at least about 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 60, 70, 80, 90, or 100 mg/kg (or any range derivable
therein).
[0034] The amount of the compound that is administered or taken by
the patient may be based on the patient's weight (in kilograms).
Therefore, in some embodiments, the patient is administered or
takes a dose or multiple doses amounting to about, at least about,
or at most about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,
0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,
2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8,
3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,
5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,
7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0,
9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0,
11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5,
17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135,
140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200,
205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,
270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330,
335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395,
400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490,
500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600,
610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710,
720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820,
825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925,
930, 940, 950, 960, 970, 975, 980, 990, 1000 micrograms/kilogram
(kg) or mg/kg, or any range derivable therein. In some aspects, the
pharmaceutically effective amount comprises a dose from about
0.0001 mg/kg/day to about 100 mg/kg/day. In further aspects, the
effective amount comprises a dose from about 0.01 mg/kg/day to
about 5 mg/kg/day. In still further aspects, the dose is about 0.25
mg/kg/day.
[0035] The composition may be administered to (or taken by) the
patient 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20 or more times, or any range derivable therein, and they
may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1, 2, 3,
4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12 months, or any range derivable therein. It is
specifically contemplated that the composition may be administered
once daily, twice daily, three times daily, four times daily, five
times daily, or six times daily (or any range derivable therein)
and/or as needed to the patient. Alternatively, the composition may
be administered every 2, 4, 6, 8, 12 or 24 hours (or any range
derivable therein) to or by the patient.
[0036] In some embodiments, the compounds described herein are
comprised in a pharmaceutical composition. In further embodiments,
the compounds described herein and optional one or more additional
active agents, can be optionally combined with one or more
pharmaceutically acceptable excipients and formulated for
administration via epidural, introperitoneal, intramuscular,
cutaneous, subcutaneous or intravenous injection. In some aspects,
the compounds or the composition is administered by aerosol,
infusion, or topical, nasal, oral, anal, ocular, or otic delivery.
In further embodiments, the pharmaceutical composition is
formulated for controlled release.
[0037] "Pharmaceutically acceptable" means that which is useful in
preparing a pharmaceutical composition that is generally safe,
non-toxic and neither biologically nor otherwise undesirable and
includes that which is acceptable for veterinary use as well as
human pharmaceutical use.
[0038] "Pharmaceutically acceptable salts" means salts of compounds
of the present invention which are pharmaceutically acceptable, as
defined above, and which possess the desired pharmacological
activity. Such salts include acid addition salts formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid, and the like; or with
organic acids such as 1,2-ethanedisulfonic acid,
2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid,
3-phenylpropionic acid,
4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),
4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,
aliphatic mono- and dicarboxylicacids, aliphatic sulfuric acids,
aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,
camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,
cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,
glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,
heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,
laurylsulfuric acid, maleic acid, malic acid, malonic acid,
mandelic acid, methanesulfonic acid, muconic acid,
o-(4-hydroxybenzoyl)benzoic acid, oxalic acid,
p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids,
propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic
acid, stearic acid, succinic acid, tartaric acid,
tertiarybutylacetic acid, trimethylacetic acid, and the like.
Pharmaceutically acceptable salts also include base addition salts
which may be formed when acidic protons present are capable of
reacting with inorganic or organic bases. Acceptable inorganic
bases include sodium hydroxide, sodium carbonate, potassium
hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable
organic bases include ethanolamine, diethanolamine,
triethanolamine, tromethamine, N-methylglucamine and the like. It
should be recognized that the particular anion or cation forming a
part of any salt of this invention is not critical, so long as the
salt, as a whole, is pharmacologically acceptable. Additional
examples of pharmaceutically acceptable salts and their methods of
preparation and use are presented in Handbook of Pharmaceutical
Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds.,
Verlag Helvetica Chimica Acta, 2002).
[0039] In certain embodiments, the demyelinating disease includes,
but is not limited to, multiple sclerosis, spinal cord compression,
ischemia, acute disseminated encephalomyelitis, optic
neuromyelitis, leukodystrophy, progressive multifocal
leukoencephalopathy, metabolic disorders, toxic exposure,
congenital demylinating disease, peripheral neuropathy,
encephalomyelitis, central pontine myelolysis, Anti-MAG Disease,
Guillain-Barre syndrome, chronic inflammatory demyelinating
polyneuropathy, or multifocal motor neuropathy (MMN). In particular
embodiments, the demyelinating disease is multiple sclerosis.
[0040] In additional embodiments, leukodystrophy includes, but is
not limited to, adrenoleukodystrophy, Alexander's Disease, Canavan
Disease, Krabbe Disease, Metachromatic Leukodystrophy,
Pelizaeus-Merzbacher Disease, vanishing white matter disease,
Refsum Disease, Cockayne Syndrome, Van der Knapp Syndrome, or
Zellweger Syndrome.
[0041] In some embodiments, there are provided methods for
diagnosing a demyelinating disease or evaluating the progression of
a demyelinating disease comprising administering to a subject in
need thereof the imaging agent described herein and detecting the
compound comprised in the imaging agent in the subject. In certain
aspects, the compound is detected by a radiodiagnostic method,
including, but not limited to PET, TAC, or PET-MRI. In particular
aspects, the compound is detected by PET.
[0042] In some aspects, the subject is a mammal. In particular
aspects, the subject is a human. In additional aspects, the subject
is a healthy individual. In further aspects, the subject is a
verified or putative animal model of myelin-associated neuropathy.
For example, in some embodiments, the animal model is DTA model,
cuprizone-induced demyelination model, a lysolecithin injection
model or experimental autoimmune encephalomyelitis (EAE) model. In
still further aspects, the animal model is a mouse mutant with
altered nodal environ, including, but not limited to, shiverer,
trembler, jimpy, P0 null, E-cadherin null, Mag null, Dystrophic
laminin .alpha.2, Cgt null, Contactin null, Caspr null, Cst null,
Caspr2 null, Tag1 null, Dystroglycan, quivering Spectrin .beta.IV,
Nrcam null, and Na+ channel .beta.2 null.
[0043] In some embodiments, the subject is at risk for traumatic
brain injury or a concussion. In some embodiments, the subject has
a concussion or has symptoms of a concussion. In some embodiments,
the subject is an athlete or participates in athletic activities
such as football, hockey, soccer, lacrosse, rugby, field hockey,
horseback riding, bull riding, cheerleading, gymnastics, motocross,
boxing, wrestling, base jumping, mountaineering, mixed martial
arts, parkour, sky diving, free climing, skateboarding, surfing,
luge, cliff diving, snowboarding, skiing, pole vault, martial arts,
cycling, racing, mountain biking, skating, cricket, basketball,
roller derby, softball, baseball, polo, water polo, or other
activities.
[0044] In some embodiments, there are provided methods for
synthesizing the compounds described herein. For example,
3-fluoromethyl-4-aminopyridine or 3-fluoroethyl-4-aminopyridine is
produced by a method comprising (a) protecting the amino group of
4-aminopyridine-3-methanol or 4-aminopyridine-3-ethanol with a
protection group to form a first intermediate compound, (b)
fluorinating the first intermediate compound by using a
fluoro-containing reagent to form a second intermediate compound,
and (c) removing the protection group from the second intermediate
compound to form 3-fluoromethyl-4-aminopyridine or
3-fluoroethyl-4-aminopyridine. In certain aspects, the protection
group is Boc (N-tert-butoxycarbonyl). In further aspects, the
fluoro-containing reagent is XtalFluor E
((Diethylamino)difluorosulfonium tetrafluoroborate).
[0045] In additional aspects, 3-fluoroethyl-4-aminopyridine may be
synthesized by a method comprising (a) converting
4-(Boc-amino)pyridine to 4-(Boc-amino)pyridine-3-ethanol, (b)
fluorinating 4-(Boc-amino)pyridine-3-ethanol by using Xtal-Fluor,
and c) removing Boc to form 3-fluoroethyl-4-aminopyridine.
[0046] Methods for producing the fluorine isotope containing
compounds describe herein are also contemplated. For example,
[.sup.18F]-3-fluoro-4-aminopyridine is produced by a method
comprising (a) converting a compound having the structure A
(4-(Boc-amino)pyridine) to an intermediate compound with structure
B, and (b) fluorinating the intermediate structure B to form
[.sup.18F]-3-fluoro-4-aminopyridine. A [.sup.18F]-containing
reagent is supplied in the fluorination step.
##STR00007##
[0047] A method for producing
[.sup.18F]-3-fluoromethyl-4-aminopyridine or
[.sup.18F]-3-fluoroethyl-4-aminopyridine is also provided. The
method comprises (a) protecting the amino group of
4-aminopyridine-3-methanol or 4-aminopyridine-3-ethanol with a
protection group to form a first intermediate compound, (b)
fluorinating the first intermediate compound by using a
[.sup.18F]-containing reagent to form a second intermediate
compound, and (c) removing the protection group from the second
intermediate compound to form
[.sup.18F]-3-fluoromethyl-4-aminopyridine or
[.sup.18F]-3-fluoroethyl-4 aminopyridine. In particular aspects,
the protection group is Boc (N-tert-butoxycarbonyl).
[0048] In additional aspects,
[.sup.18F]-3-fluoroethyl-4-aminopyridine may be synthesized by a
method comprising (a) converting 4-(Boc-amino)pyridine to
4-(Boc-amino)pyridine-3-ethanol, (b) fluorinating
4-(Boc-amino)pyridine-3-ethanol by using a [.sup.18F]-containing
reagent, and (c) removing Boc to form
[.sup.18F]-3-fluoroethyl-4-aminopyridine.
[0049] In certain embodiments, the [.sup.18F]-containing reagent
includes, but is not limited to, [.sup.18F]-Kryptofix,
[.sup.18F]-F2, [.sup.18F]-AcOF, [.sup.18F]F-TEDA,
[.sup.18F]-Benzo[h]quinolinyl (tetrapyrazolylborate) Pd(IV)
fluoride trifluoromethanesulfonate, [.sup.18F]-2-fluoroethyl
bromide, and [.sup.18F]-fluoromethyl-bromide. In particular
aspects, the [.sup.18F] containing reagent is [.sup.18F]
Kryptofix.
[0050] In some embodiments, an alternative method for producing
[.sup.18F]3-fluoro-4-aminopyridine is provided, comprising the
steps of (a) using Koser's reagent to iodonate
4-(Boc-amino)pyridin-3-ylboronic acid to form a first intermediate
compound, (b) fluorinating the intermediate compound by using a
[.sup.18F]fluor-containing reagent, and (c) using HCl to remove the
protecting group to yield [.sup.18F]3-fluoro-4-aminopyridine.
[0051] The methods for producing the compounds described herein are
not limited to the exemplary methods described herein. The
compounds may be synthesized by any suitable method known in the
art and it will be obvious to those skilled in the art that various
adaptations, changes, modifications, substitutions, deletions or
additions of procedures may be made without departing from the
spirit and scope of the invention.
[0052] In certain methods and compositions, embodiments concern the
use of a compound for research purposes involving a potassium
channel blocker. The compound may be used for its potassium channel
blocking activity. Therefore, in some embodiments, methods involve
exposing, contacting, or adding a compound discussed herein to a
channel or a polypeptide involved in channel activity and
determining calcium channel activity. In some embodiments, the
compound is a control. In other embodiments, the compound is used
to screen other compounds for an activity that affects channel
activity (such as by inhibiting or enhancing that activity).
[0053] Because of the biological activity of the compounds
disclosed herein, in additional embodiments, there are methods and
compositions for use of these compounds as an avicide. In some
embodiments, a compound discussed herein is formulated as grain
bait, a powder concentrate or a liquid for exposure to or ingestion
by birds. The LD50 for birds is generally in the range of about 100
parts per million (ppm) to 1000 parts per million, and dosages are
formulated to provide at least that much to birds. Embodiments also
include methods of using an avicide comprising providing to an
avian an effective amount of a composition comprising a compound
discussed herein, including but not limited to those having Formula
I or Formula II. In certain embodiments, providing the compound
comprises distributing the composition to places that birds can
access, including but not limited to distributing it in grass,
trees, bushes, on leaves, in bird feeders or in bird baths or other
food or water supplies for birds. In further embodiments,
distributing the composition may involve spraying a liquid or
powder composition, or depositing or placing a solid, liquid or
powder composition. In certain embodiments, a subject is a
bird.
[0054] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the measurement or quantitation method.
[0055] The use of the word "a" or "an" when used in conjunction
with the term "comprising" may mean "one," but it is also
consistent with the meaning of "one or more," "at least one," and
"one or more than one."
[0056] The words "comprising" (and any form of comprising, such as
"comprise" and "comprises"), "having" (and any form of having, such
as "have" and "has"), "including" (and any form of including, such
as "includes" and "include") or "containing" (and any form of
containing, such as "contains" and "contain") are inclusive or
open-ended and do not exclude additional, unrecited elements or
method steps.
[0057] The compositions and methods for their use can "comprise,"
"consist essentially of," or "consist of" any of the ingredients or
steps disclosed throughout the specification. Compositions and
methods "consisting essentially of" any of the ingredients or steps
disclosed limits the scope of the claim to the specified materials
or steps which do not materially affect the basic and novel
characteristic of the claimed invention.
[0058] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method or
composition of the invention, and vice versa. Furthermore,
compositions of the invention can be used to achieve methods of the
invention.
[0059] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific
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. Note that simply because a
particular compound is ascribed to one particular generic formula
doesn't mean that it cannot also belong to another generic
formula.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] FIGS. 1A-1C illustrate the mechanism of action of a
potassium channel blocker. (A) shows a scheme of a healthy neuron.
(B) shows a scheme of a demyelinated neuron. Aberrant leakage of
potassium ions from the axon results in poor conduction of
electrical impulses along the axon. (C) shows a demyelinated neuron
treated with a potassium channel blocker.
[0061] FIGS. 2A-2G show potassium channel blockers and fluorinated
4-AP derivatives. (A) 4-aminopyridine (B) 3,4-diaminopyridine (C)
3-methanol-4-aminopyridine (D) 3-fluoro-4-aminopyridine (E)
3-fluoromethyl-4-aminopyridine (F) 3-fluoroethyl-4-aminopyridine
(G) 2-fluoro-4-aminopyridine
[0062] FIGS. 3A-3F. (A) shows synthesis of
3-fluoromethyl-4-aminopyridine. (B) shows synthesis of
3-fluoroethyl-4-aminopyridine. (C) shows NMR of
3-fluoromethyl-4-aminopyridine. (D) shows high resolution Mass
Spectra of 3-fluoromethyl-4-aminopyridine. (E) shows NMR of
3-fluoroethyl-4-aminopyridine. (F) shows high resolution Mass
spectra of 3-fluoroethyl-4-aminopyridine.
[0063] FIGS. 4A-4C show enhancement of compound action potential
(CAP) by 4-AP derivatives. (A) CAP traces before (solid line) and
after (dashed line) addition of the drug. Scale bar: 5 mV/5 ms. (B)
Relative increase of maximum CAP amplitude vs. concentration for
each drug. Amplitude was normalized to the amplitude before the
drug. (C) Half-maximal effective concentration of each molecule and
95% confidence interval obtained from fitting the data to the Hill
equation. n=number of times each drug was tested.
[0064] FIGS. 5A-5C show inhibition of ionic current of Shaker
K.sup.+ channel by 4-AP derivatives. (A) K.sup.+ currents were
generated by a series of 50 ms pulses from -70 mV to +40 mV in
increments of 10 mV in the presence of cumulative concentrations of
4-AP derivatives. Each panel represents the K.sup.+ current
recorded from the same oocyte before and after addition of the
drug. Scale bar: 1 .mu.A/10 ms. (B) Relative K.sup.+ current vs.
concentration for each drug obtained at +20 mV. (C) Half-maximal
inhibitory concentration of each molecule and 95% confidence
interval obtained from fitting the data to the Hill equation.
n=number of times each drug was tested.
[0065] FIGS. 6A-6B show pharmacology of 4-AP derivatives. (A)
Pharmacological parameters for 4-AP derivatives and control
compounds. cLogP: calculated partition coefficient using VCCLAB (I.
V. Tetko et al., Virtual computational chemistry laboratory--design
and description. Journal of computer-aided molecular design 19, 453
(June, 2005)), Pe: permeability coefficient across artificial
membrane (n=3), t.sub.1/2: half-life in mouse microsomes (n=3). (B)
Pharmacokinetic profile of 4-AP and 3-F-4-AP in plasma and brain of
mice after intravenous injection of 0.75 mg of drug per mouse kg
(n=3 mice per time point).
[0066] FIGS. 7A-7E. Brain distribution of 4-AP in mice injected
with LPC. Top label represents mouse name+section number. (A)
Fluorescent microscopy of myelin basic protein (MBP)
immunostaining. Each small square represents one picture at
40.times.. Areas rich in MBP appear darker. Partial demyelination
is evident in certain areas of the corpus callosum. (B)
Autoradiography: areas where 4-AP localizes appear darker. 4-AP
mostly localizes in grey matter areas with almost no signal in
white mater areas. (C and D) 2.times. magnification of the corpus
callosum area from A and B. Areas of demyelination in the corpus
callosum appear darker than the rest of the corpus callosum. The
corpus callosum has been marked with a dashed white line and the
areas of demyelination within the corpus callosum have been circled
with a solid white line. Autoradiographic signal is more intense in
areas of demyelination compared to the rest of the corpus callosum.
Scale bar=2 um. All animals pictured here received LPC injections
however not all of them showed lesions at the level of sectioning
(ie. no lesions are observed in LPC1-12). (E) Quantification of the
mean pixel intensity in the whole corpus callosum and in the lesion
area (as determined by IHC).
[0067] FIG. 8. Possible radiosynthesis of [.sup.18F] 3-F-4-AP and
[.sup.18F] 3-MeF-4-AP
[0068] FIGS. 9A-9B--(A) Experimental scheme: TBI is induced in rats
using a controlled impact. (B) Autoradiography.
DETAILED DESCRIPTION OF THE INVENTION
[0069] Multiple sclerosis (MS) is the most common neurodegenerative
disease in young adults. The progressive demyelination of neurons
in the central nervous system (CNS) is the hallmark of MS
(Calabresi, 2007). When axons lose their myelin, K.sup.+ channels
in the axonal membrane become exposed and leak K.sup.+ ions (FIGS.
1A and B). The aberrant leakage of K.sup.+ ions from the axons
results in poor impulse conduction, which in turn causes the
appearance of neurological symptoms (Ritchie et al., 1981; Waxman
and Ritchie, 1993; Rasband et al., 1998; Arroyo et al., 2004).
[0070] Positron Emission Tomography (PET) allows imaging of
molecular changes before macroscopic changes have occurred and
therefore it provides an opportunity for early detection. It does
this by detecting a radiation coming from a radionuclide introduced
in the body in a biologically active molecule that selectively
localizes to the area of interest, also known as tracer. Images of
the tracer's distribution can be reconstructed using computer
analysis allowing precise mapping of its location. For example,
.sup.18F-fludeoxyglucose is widely used to image highly
metabolically active cells such as cancer cells inside an organism.
Similarly, it is conceivable that a PET-active molecule that
selectively localizes to injured areas in the brain could provide
accurate maps of TBI.
[0071] During TBI, compression or stretching of the brain often
causes axons to tear and oligodendrocytes (cells responsible for
producing and maintaining myelin) to break. Injury to
oligodendrocytes can leave axons devoid of myelin, which then
become more sensitive to degeneration. It is well known that loss
of myelin (as in conditions like multiple sclerosis, MS) causes
K.sup.+ channels, which are usually buried beneath the myelin
sheath to become exposed and leaky.
[0072] 4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP) are
well-known potassium channel blockers relatively selective for
voltage gated K.sup.+ channels of the K.sub.v1 family (Wulff et
al., 2009). 4-AP sensitive K+ channels, Kv1.1 and Kv1.2, are
localized in the juxtaparanodal region of myelinated axons. Upon
demyelination these channels redistribute throughout the intermodal
region of the axons as seen in tissue samples from MS patients and
in demyelinated animals. In demyelinated animals Kv1 channels have
been shown to be upregulated 2-4 fold. 4-AP and 3,4-DAP have been
used effectively in the treatment of Lambert-Eaton Syndrome and
Multiple Sclerosis (Murray and Newsom-Davis, 1981; Soni and Kam,
1982; Lundh et al., 1977). 4-AP and 3,4-DAP block K.sub.v1
potassium channels with affinities in the micromolar range. Binding
of 4-AP and 3,4-DAP to K.sub.v1 potassium channels restores impulse
conduction in demyelinated fibers (Yeh et al., 1976; Sherratt et
al., 1980; Kirsch and Narahashi, 1978). 4-aminopyridine-3-methanol
can also restore impulse conduction of demyelinated fibers (Sun et
al., 2010; Leung et al., 2011).
[0073] In 2010, the FDA approved a slow-release formulation of
4-aminopyridine (4-AP), to improve walking in MS patients (Ampyra,
Acorda Therapeutics, Inc., 2010). 4-AP is a relatively selective
blocker of K.sub.v1 family of K.sup.+ channels (Wulff et al.,
2009). The proposed mechanism of action of 4-AP in MS patients is
that 4-AP blocks K.sup.+ channels in demyelinated axons, which
leads to improved impulse conduction.
[0074] Fluorinated molecules generally display better
pharmacological properties such as increased membrane permeability
and metabolic stability than their non-fluorinated analogs.
Described herein are compounds of formula I or II, which contain
fluorine and efficiently block voltage gated potassium channels. In
particular, certain embodiments are directed to fluorinated 4-AP
derivatives, such as 3-fluoromethyl-4-aminopyridine, or
3-fluoroethyl-4-aminopyridine.
[0075] In addition, 4-AP can efficiently cross the blood brain
barrier. Application of a computational model for the estimation of
log BB (a parameter used to predict blood brain barrier
permeability by certain compounds) predicts that the compounds
described herein, in particular, fluorinated 4-AP derivatives, will
efficiently cross the blood brain barrier (Sun, 2004). It has also
been shown that 3-F-4-AP is more lipophylic than 4-AP
(Arzneimittelforschung, 1989)
[0076] 4-AP is safe within the concentrations used in therapy,
which indicates that the compounds described herein, in particular,
fluorinated 4-AP derivatives are likely to be safe tools when used
in humans.
[0077] To effectively treat a patient with a neurodegenerative
disease, such as MS, it is important to diagnose and evaluate the
progression of the disease in the patient. Currently, magnetic
resonance imaging (MRI) is the primary imaging techniques for the
diagnosis and the assessment of disorders that disrupt the myelin
sheath, including MS. Unfortunately, signal changes on an MRI are
non-specific and correlate only indirectly with the underlying
pathology. Moreover, current methods do not correlate well with the
underlying pathology of the disease and are not well-suited for use
in clinical trials.
[0078] PET is a non-invasive medical imaging technique that relies
on the detection of radiation emitted by a radionuclide
(radioactive tracer) introduced in the body of the subject on a
biologically active molecule. Images of the radioactive tracer's
localization can be reconstructed by computer analysis providing
quantitative maps of the radioactive tracer's distribution in the
body of the subject. Such images can provide valuable information
of the biochemistry and physiology of a subject. Because PET is a
molecular imaging technique, it can detect cellular abnormalities
before anatomical changes have occurred. For example,
18F-fluorodeoxyglucose (FDG) is widely used to distinguish highly
metabolically active cancer cells from other cells (Oriuchi et al.,
2006). Similarly, it is conceivable that a "PET-active" molecule
that selectively localizes to demyelinated axons could provide
accurate maps of the lesions early in the process.
[0079] The most common radioisotopes used in PET are .sup.18F,
.sup.15O, .sup.13N and .sup.11C, with half-lives of 110, 2, 10, and
20 min respectively. .sup.18F is usually preferred due to its
longer half-life and its lower positron energy which results in
better resolution. Despite the relatively short half-life of these
radioisotopes, they are widely used in medical diagnostics as many
hospitals have their own cyclotron to prepare the radioactive
tracers or have a nearby facility that can prepare the radioactive
tracers.
[0080] A recent review on PET markers for MS highlighted several
potential targets for PET imaging including 18 kDa Translocator
Protein, Cannabinoid Receptor Type 2, Myelin, Cerebral metabolic
rate of glucose utilization, Type A .gamma.-aminobutyric acid, and
Acetyl choline receptor (Owen et al., 2011). Nevertheless, all of
these markers have limitations: some of these tracers were
originally developed for other conditions and suffer from low
pathological specificity; others were developed to target myelin or
myelin related proteins and have limited signal-to-noise ratio and
the rest target inflammatory cells which do not necessarily
correlate with the underlying demyelination. More recently, a
report on [.sup.11C]PIB, a PET radioactive tracer that binds to
amyloid plaques originally developed for Alzheimer's Disease, has
been shown to be useful in quantifying myelin (Stankoff et al.,
2011). Nevertheless, since MS is a de-myelinating disorder, it
would be desirable to have access to a PET radioactive tracer
specific for de-myelinated axons. In particular, it would be
desirable to develop a PET radioactive tracer that targets
potassium channels for imaging demyelination or other
conditions.
[0081] Incorporation of a positron emitting radionuclide such as
.sup.18F into a potassium channel blocker, such as a 4-AP
derivative, allows visualization of the location and abundance of
exposed potassium channels and provides a better assessment of
demyelinated regions. The fact that 4-AP has proven therapeutically
beneficial indicates that it preferentially binds to potassium
channels of demyelinated neurons. Furthermore, the fact that there
are relatively few side effects of 4-AP indicates that there are
few off-target receptors at the concentrations currently used in
therapy. Such properties indicate that the compounds described
herein are suitable for imaging demyelinated neurons with adequate
signal-to-noise ratio.
[0082] Furthermore, the metabolic stability of
[.sup.18F]Fluoroalkylbiphenyls, which share a similar core
structure to the compounds described herein, have been examined and
were found to be stable for PET studies (Lee et al., 2004),
indicating that the compounds described herein are likely stable
for PET studies.
[0083] Substitution of .sup.18F for OH or H is common in the art.
Such substitutions generally preserve the biological properties of
the molecule and render the molecules suitable for imaging using
PET or SPECT cameras. For example substitution of the OH in
position 2 of glucose with .sup.18F does not alter the capability
to be uptaken by cells. Many examples of .sup.18F substitutions
that preserve the parent molecule's properties can be found on the
MICAD database (available on the world wide web at
ncbi.nlm.nih.gov/books/NBK5330/).
[0084] FIG. 1C shows a cartoon representation of the proposed
mechanism of action of the radioactive tracer. 4-AP as well as the
radioactive tracers described herein bind to potassium channels on
demyelinated axons decreasing efflux of K.sup.+. Visualization of
the localization of these molecules can inform of the localization
and extent of demyelinated axons.
[0085] Disclosed herein are new radioactive tracers for PET, which
serve as novel diagnostic markers to image demyelinated axons in a
subject. In particular embodiments, the new radioactive tracers for
PET are .sup.18F-labeled versions of 4-AP derivatives. Methods for
their manufacture and methods for their use in in vivo imaging of
the central nervous system to diagnose and/or assess the
progression of MS or other diseases are also provided. The present
disclosure also provides fluorine containing compounds that bind to
potassium channels, methods for their manufacture and methods for
their use in the treatment of neurodegenerative diseases.
I. DEFINITIONS
[0086] The term "radioactive isotope" refers to an isotope having
an unstable nucleus that decomposes spontaneously by emission of a
nuclear electron, positron, or helium nucleus and radiation, thus
achieving a more stable nuclear composition.
[0087] The term "deuterated version" as used herein means one or
more of hydrogen in a compound is replaced with .sup.2H, an isotope
of hydrogen.
[0088] As used herein, the term "radioactive tracer", or
"radioactive label", or "tracer", or "radiotracer" means a chemical
compound in which one or more atoms have been replaced by a
radioisotope. By virtue of its radioactivity, it can be used to
explore the mechanism of chemical reactions by tracing the path
that the radioisotope follows from reactants to products. A
radioactive tracer can also be used to track the distribution of a
substance within a natural system such as a cell or tissue.
Radioactive tracers form the basis of a variety of imaging systems,
such as PET scans and SPECT scans.
[0089] The use of the word "a" or "an," when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one."
[0090] The term "hydrate" when used as a modifier to a compound
means that the compound has less than one (e.g., hemihydrate), one
(e.g., monohydrate), or more than one (e.g., dihydrate) water
molecules associated with each compound molecule, such as in solid
forms of the compound.
[0091] An "isomer" of a first compound is a separate compound in
which each molecule contains the same constituent atoms as the
first compound, but where the configuration of those atoms in three
dimensions differs.
[0092] As used herein, the term "patient" or "subject" refers to a
living mammalian organism, such as a human, monkey, cow, sheep,
goat, dog, cat, mouse, rat, guinea pig, or transgenic species
thereof. In certain embodiments, the patient or subject is a
primate. Non-limiting examples of human subjects are adults,
juveniles, infants and fetuses.
[0093] As generally used herein "pharmaceutically acceptable"
refers to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues, organs, and/or bodily
fluids of human beings and animals without excessive toxicity,
irritation, allergic response, or other problems or complications
commensurate with a reasonable benefit/risk ratio.
[0094] "Prevention" or "preventing" includes: (1) inhibiting the
onset of a disease in a subject or patient which may be at risk
and/or predisposed to the disease but does not yet experience or
display any or all of the pathology or symptomatology of the
disease, and/or (2) slowing the onset of the pathology or
symptomatology of a disease in a subject or patient which may be at
risk and/or predisposed to the disease but does not yet experience
or display any or all of the pathology or symptomatology of the
disease.
[0095] "Prodrug" means a compound that is convertible in vivo
metabolically into an inhibitor according to the present invention.
The prodrug itself may or may not also have activity with respect
to a given target protein. For example, a compound comprising a
hydroxy group may be administered as an ester that is converted by
hydrolysis in vivo to the hydroxy compound. Suitable esters that
may be converted in vivo into hydroxy compounds include acetates,
citrates, lactates, phosphates, tartrates, malonates, oxalates,
salicylates, propionates, succinates, fumarates, maleates,
methylene-bis-.beta.-hydroxynaphthoate, gentisates, isethionates,
di-p-toluoyltartrates, methanesulfonates, ethanesulfonates,
benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates,
quinates, esters of amino acids, and the like. Similarly, a
compound comprising an amine group may be administered as an amide
that is converted by hydrolysis in vivo to the amine compound.
[0096] A "stereoisomer" or "optical isomer" is an isomer of a given
compound in which the same atoms are bonded to the same other
atoms, but where the configuration of those atoms in three
dimensions differs. "Enantiomers" are stereoisomers of a given
compound that are mirror images of each other, like left and right
hands. "Diastereomers" are stereoisomers of a given compound that
are not enantiomers. Chiral molecules contain a chiral center, also
referred to as a stereocenter or stereogenic center, which is any
point, though not necessarily an atom, in a molecule bearing groups
such that an interchanging of any two groups leads to a
stereoisomer. In organic compounds, the chiral center is typically
a carbon, phosphorus or sulfur atom, though it is also possible for
other atoms to be stereocenters in organic and inorganic compounds.
A molecule can have multiple stereocenters, giving it many
stereoisomers. In compounds whose stereoisomerism is due to
tetrahedral stereogenic centers (e.g., tetrahedral carbon), the
total number of hypothetically possible stereoisomers will not
exceed 2n, where n is the number of tetrahedral stereocenters.
Molecules with symmetry frequently have fewer than the maximum
possible number of stereoisomers. A 50:50 mixture of enantiomers is
referred to as a racemic mixture. Alternatively, a mixture of
enantiomers can be enantiomerically enriched so that one enantiomer
is present in an amount greater than 50%. Typically, enantiomers
and/or diasteromers can be resolved or separated using techniques
known in the art. It is contemplated that that for any stereocenter
or axis of chirality for which stereochemistry has not been
defined, that stereocenter or axis of chirality can be present in
its R form, S form, or as a mixture of the R and S forms, including
racemic and non-racemic mixtures. As used herein, the phrase
"substantially free from other stereoisomers" means that the
composition contains .ltoreq.15%, more preferably .ltoreq.10%, even
more preferably .ltoreq.5%, or most preferably .ltoreq.1% of
another stereoisomer(s).
[0097] "Effective amount," "Therapeutically effective amount" or
"pharmaceutically effective amount" means that amount which, when
administered to a subject or patient for treating a disease, is
sufficient to effect such treatment for the disease.
[0098] "Treatment" or "treating" includes (1) inhibiting a disease
in a subject or patient experiencing or displaying the pathology or
symptomatology of the disease (e.g., arresting further development
of the pathology and/or symptomatology), (2) ameliorating a disease
in a subject or patient that is experiencing or displaying the
pathology or symptomatology of the disease (e.g., reversing the
pathology and/or symptomatology), and/or (3) effecting any
measurable decrease in a disease in a subject or patient that is
experiencing or displaying the pathology or symptomatology of the
disease.
[0099] As used herein, the term "water soluble" means that the
compound dissolves in water at least to the extent of 0.010
mole/liter or is classified as soluble according to literature
precedence.
[0100] The above definitions supersede any conflicting definition
in any of the reference that is incorporated by reference herein.
The fact that certain terms are defined, however, should not be
considered as indicative that any term that is undefined is
indefinite. Rather, all terms used are believed to describe the
invention in terms such that one of ordinary skill can appreciate
the scope and practice the present invention.
II. COMPOUNDS THAT BLOCK POTASSIUM CHANNELS
[0101] Certain embodiments provide compounds that block potassium
channels having the following formula:
##STR00008##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently
selected from the group consisting of H, (CH.sub.2).sub.nX,
CH.sub.2OCH.sub.2CH.sub.2X, NH.sub.2, CH.sub.2OH, and CF.sub.3; and
R.sub.5 is selected from the group consisting of H,
(CH.sub.2).sub.mX, and OH, wherein n=0, 1, 2, 3, 4, or 5, and m=2,
3, 4, or 5; wherein X represents a fluorine atom or an isotope
thereof; wherein at least one of R.sub.1, R.sub.2, R.sub.3, R.sub.4
and R.sub.5 is not hydrogen; wherein at least one of R.sub.1,
R.sub.2, R.sub.3, R.sub.4 and R.sub.5 contains a fluorine atom or
an isotope thereof; wherein when R.sub.2 is NH.sub.2 or CH.sub.2OH
or a nonradioactive fluorine or CF.sub.3, at least one of R.sub.1,
R.sub.3, R.sub.4, and R.sub.5 is not hydrogen; wherein when R.sub.4
is NH.sub.2, CH.sub.2OH, a nonradioactive fluorine, or CF.sub.3, at
least one of R.sub.1, R.sub.2, R.sub.4, and R.sub.5 is not
hydrogen; and wherein any of C, N, O is optionally replaced by the
isotope .sup.11C, .sup.13N, .sup.15O, respectively, or a
pharmaceutical acceptable salt thereof, a tautomer thereof or a
deuterated version thereof.
[0102] In some embodiments, the compounds have the formulas found
in FIGS. 2A-G. In particular embodiments, the compounds have the
formulas of FIG. 2E and FIG. 2F, which are not commercially
available and have never been described before.
[0103] In further embodiments, 4-AP derivatives having the
following formula are provided:
##STR00009##
[0104] wherein M is (CH.sub.2).sub.nY, and wherein n=0, 1, or 2,
and Y is fluorine or an isotope thereof.
[0105] In certain embodiments, there are provided compounds of
formula (III):
##STR00010##
[0106] wherein R is selected from the group consisting of CH.sub.3,
CH.sub.2F, CHF.sub.2, and CF.sub.3, and wherein C is substituted by
.sup.11C or at least one of F is substituted by .sup.18F in R.
[0107] Further embodiments are directed to the compounds of formula
(IV):
##STR00011##
[0108] wherein R is selected from the group consisting of CF.sub.3,
CH.sub.2F, CH.sub.3CH.sub.2F, C(CH.sub.3).sub.3, and wherein at
least one of F or H in the R group is substituted by .sup.18F.
[0109] The compounds provided by the present disclosure are
described in the summary of the invention section and in the claims
below.
[0110] Compounds employed in methods described herein may contain
one or more asymmetrically-substituted carbon or nitrogen atoms,
and may be isolated in optically active or racemic form. Thus, all
chiral, diastereomeric, racemic form, epimeric form, and all
geometric isomeric forms of a structure are intended, unless the
specific stereochemistry or isomeric form is specifically
indicated. Compounds may occur as racemates and racemic mixtures,
single enantiomers, diastereomeric mixtures and individual
diastereomers. In some embodiments, a single diastereomer is
obtained. The compounds can be formulated as a mixture of one or
more diastereomers. Alternatively, the diastereomers can be
separated and one or more of the diastereomers can be formulated
individually. The chiral centers of the compounds disclosed herein
can have the S or the R configuration, as defined by the IUPAC 1974
Recommendations. For example, mixtures of stereoisomers may be
separated using techniques known to those of skill in the art.
[0111] Atoms making up the compounds of the present invention are
intended to include all isotopic forms of such atoms. Compounds of
the present invention include those with one or more atoms that
have been isotopically modified or enriched, in particular those
with pharmaceutically acceptable isotopes or those useful for
pharmaceutical research. Isotopes, as used herein, include those
atoms having the same atomic number but different mass numbers. By
way of general example and without limitation, isotopes of hydrogen
include deuterium and tritium, and isotopes of carbon include
.sup.11C, .sup.13C and .sup.14C. Similarly, it is contemplated that
one or more carbon atom(s) of a compound of the present invention
may be replaced by a silicon atom(s). Furthermore, it is
contemplated that one or more oxygen atom(s) of a compound of the
present invention may be replaced by a sulfur or selenium
atom(s).
[0112] Compounds disclosed herein may also exist in prodrug form.
Since prodrugs are known to enhance numerous desirable qualities of
pharmaceuticals (e.g., solubility, bioavailability, manufacturing,
etc.), the compounds employed in some methods of the invention may,
if desired, be delivered in prodrug form. Thus, certain embodiments
contemplate prodrugs of compounds described herein as well as
methods of delivering prodrugs. Prodrugs of the compounds may be
prepared by modifying functional groups present in the compound in
such a way that the modifications are cleaved, either in routine
manipulation or in vivo, to the parent compound. Accordingly,
prodrugs include, for example, compounds described herein in which
a hydroxy, amino, or carboxy group is bonded to any group that,
when the prodrug is administered to a subject, cleaves to form a
hydroxy, amino, or carboxylic acid, respectively.
[0113] It should be recognized that the particular anion or cation
forming a part of any salt of this invention is not critical, so
long as the salt, as a whole, is pharmacologically acceptable.
Additional examples of pharmaceutically acceptable salts and their
methods of preparation and use are presented in Handbook of
Pharmaceutical Salts: Properties, and Use (2002), which is
incorporated herein by reference.
[0114] It should be further recognized that the compounds of the
present invention include those that have been further modified to
comprise substituents that are convertible to hydrogen in vivo.
This includes those groups that may be convertible to a hydrogen
atom by enzymological or chemical means including, but not limited
to, hydrolysis and hydrogenolysis. Examples include hydrolyzable
groups, such as acyl groups, groups having an oxycarbonyl group,
amino acid residues, peptide residues, o-nitrophenylsulfenyl,
trimethylsilyl, tetrahydropyranyl, diphenylphosphinyl, and the
like. Examples of acyl groups include formyl, acetyl,
trifluoroacetyl, and the like. Examples of groups having an
oxycarbonyl group include ethoxycarbonyl, tert-butoxycarbonyl
(--C(O)OC(CH.sub.3).sub.3), benzyloxycarbonyl,
p-methoxy-benzyloxycarbonyl, vinyloxycarbonyl,
.beta.-(p-toluenesulfonyl)ethoxycarbonyl, and the like. Suitable
amino acid residues include, but are not limited to, residues of
Gly (glycine), Ala (alanine), Arg (arginine), Asn (asparagine), Asp
(aspartic acid), Cys (cysteine), Glu (glutamic acid), His
(histidine), Ile (isoleucine), Leu (leucine), Lys (lysine), Met
(methionine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr
(threonine), Trp (tryptophan), Tyr (tyrosine), Val (valine), Nva
(norvaline), Hse (homoserine), 4-Hyp (4-hydroxyproline), 5-Hyl
(5-hydroxylysine), Orn (ornithine) and .beta.-Ala. Examples of
suitable amino acid residues also include amino acid residues that
are protected with a protecting group. Examples of suitable
protecting groups include those typically employed in peptide
synthesis, including acyl groups (such as formyl and acetyl),
arylmethoxycarbonyl groups (such as benzyloxycarbonyl and
p-nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups
(--C(O)OC(CH.sub.3).sub.3), and the like. Suitable peptide residues
include peptide residues comprising two to five amino acid
residues. The residues of these amino acids or peptides can be
present in stereochemical configurations of the D-form, the L-form
or mixtures thereof. In addition, the amino acid or peptide residue
may have an asymmetric carbon atom. Examples of suitable amino acid
residues having an asymmetric carbon atom include residues of Ala,
Leu, Phe, Trp, Nva, Val, Met, Ser, Lys, Thr and Tyr. Peptide
residues having an asymmetric carbon atom include peptide residues
having one or more constituent amino acid residues having an
asymmetric carbon atom. Examples of suitable amino acid protecting
groups include those typically employed in peptide synthesis,
including acyl groups (such as formyl and acetyl),
arylmethoxycarbonyl groups (such as benzyloxycarbonyl and
p-nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups
(--C(O)OC(CH.sub.3).sub.3), and the like. Other examples of
substituents "convertible to hydrogen in vivo" include reductively
eliminable hydrogenolyzable groups. Examples of suitable
reductively eliminable hydrogenolyzable groups include, but are not
limited to, arylsulfonyl groups (such as o-toluenesulfonyl); methyl
groups substituted with phenyl or benzyloxy (such as benzyl, trityl
and benzyloxymethyl); arylmethoxycarbonyl groups (such as
benzyloxycarbonyl and o-methoxy-benzyloxycarbonyl); and
haloethoxycarbonyl groups (such as
.beta.,.beta.,.beta.-trichloroethoxycarbonyl and
.beta.-iodoethoxycarbonyl).
[0115] The compounds described herein may exist in unsolvated forms
as well as solvated forms, including hydrated forms. In general,
the solvated forms are equivalent to unsolvated forms and are
within the scope of the compounds described herein. The compounds
described herein may exist in multiple crystalline or amorphous
forms. In general, all physical forms are equivalent for the uses
described herein and are intended to be within the scope of the
compounds described herein.
[0116] Compounds provided herein may also have the advantage that
they may be more efficacious than, be less toxic than, be longer
acting than, be more potent than, produce fewer side effects than,
be more easily absorbed than, and/or have a better pharmacokinetic
profile (e.g., higher oral bioavailability and/or lower clearance)
than, and/or have other useful pharmacological, physical, or
chemical properties over, compounds known in the prior art, whether
for use in the indications stated herein or otherwise.
III. FORMULATIONS
[0117] The compounds described herein can be formulated for
enteral, parenteral, topical, or pulmonary administration. In other
embodiments, the formulation is for administration to a subject,
but it may not be directly to the subject. The compounds can be
combined with one or more pharmaceutically acceptable carriers
and/or excipients that are considered safe and effective and may be
administered to an individual without causing undesirable
biological side effects or unwanted interactions. The carrier is
all components present in the pharmaceutical formulation other than
the active ingredient or ingredients.
[0118] A. Parenteral Formulations
[0119] The compounds described herein can be formulated for
parenteral administration. "Parenteral administration", as used
herein, means administration by any method other than through the
digestive tract or non-invasive topical or regional routes. For
example, parenteral administration may include administration to a
patient intravenously, intradermally, intraarterially,
intraperitoneally, intralesionally, intracranially,
intraarticularly, intraprostatically, intrapleurally,
intratracheally, intravitreally, intratumorally, intramuscularly,
subcutaneously, subconjunctivally, intravesicularly,
intrapericardially, intraumbilically, by injection, and by
infusion.
[0120] Parenteral formulations can be prepared as aqueous
compositions using techniques is known in the art. Typically, such
compositions can be prepared as injectable formulations, for
example, solutions or suspensions; solid forms suitable for using
to prepare solutions or suspensions upon the addition of a
reconstitution medium prior to injection; emulsions, such as
water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and
microemulsions thereof, liposomes, or emulsomes.
[0121] The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, one or more polyols (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol), oils,
such as vegetable oils (e.g., peanut oil, corn oil, sesame oil,
etc.), and combinations thereof. The proper fluidity can be
maintained, for example, by the use of a coating, such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and/or by the use of surfactants. In many cases, it will
be preferable to include isotonic agents, for example, sugars or
sodium chloride.
[0122] Solutions and dispersions of the active compounds as the
free acid or base or pharmacologically acceptable salts thereof can
be prepared in water or another solvent or dispersing medium
suitably mixed with one or more pharmaceutically acceptable
excipients including, but not limited to, surfactants, dispersants,
emulsifiers, pH modifying agents, and combination thereof.
[0123] Suitable surfactants may be anionic, cationic, amphoteric or
nonionic surface active agents. Suitable anionic surfactants
include, but are not limited to, those containing carboxylate,
sulfonate and sulfate ions. Examples of anionic surfactants include
sodium, potassium, ammonium of long chain alkyl sulfonates and
alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate;
dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene
sulfonate; dialkyl sodium sulfosuccinates, such as sodium
bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as
sodium lauryl sulfate. Cationic surfactants include, but are not
limited to, quaternary ammonium compounds such as benzalkonium
chloride, benzethonium chloride, cetrimonium bromide, stearyl
dimethylbenzyl ammonium chloride, polyoxyethylene and coconut
amine. Examples of nonionic surfactants include ethylene glycol
monostearate, propylene glycol myristate, glyceryl monostearate,
glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose
acylate, PEG-150 laurate, PEG-400 monolaurate, polyoxyethylene
monolaurate, polysorbates, polyoxyethylene octylphenylether,
PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene
glycol butyl ether, Poloxamer.RTM. 401, stearoyl
monoisopropanolamide, and polyoxyethylene hydrogenated tallow
amide. Examples of amphoteric surfactants include sodium
N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.-iminodipropionate,
myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.
[0124] The formulation can contain a preservative to prevent the
growth of microorganisms. Suitable preservatives include, but are
not limited to, parabens, chlorobutanol, phenol, sorbic acid, and
thimerosal. The formulation may also contain an antioxidant to
prevent degradation of the active agent(s).
[0125] The formulation is typically buffered to a pH of 3-8 for
parenteral administration upon reconstitution. Suitable buffers
include, but are not limited to, phosphate buffers, acetate
buffers, and citrate buffers.
[0126] Water soluble polymers are often used in formulations for
parenteral administration. Suitable water-soluble polymers include,
but are not limited to, polyvinylpyrrolidone, dextran,
carboxymethylcellulose, and polyethylene glycol.
[0127] Sterile injectable solutions can be prepared by
incorporating the active compounds in the required amount in the
appropriate solvent or dispersion medium with one or more of the
excipients listed above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the various sterilized active ingredients into a sterile vehicle
which contains the basic dispersion medium and the required other
ingredients from those listed above. In the case of sterile powders
for the preparation of sterile injectable solutions, the preferred
methods of preparation are vacuum-drying and freeze-drying
techniques which yield a powder of the active ingredient plus any
additional desired ingredient from a previously sterile-filtered
solution thereof. The powders can be prepared in such a manner that
the particles are porous in nature, which can increase dissolution
of the particles. Methods for making porous particles are well
known in the art.
[0128] Formulations may be stable over a period of 6 months when
stored at room temperature or 4.degree. C.
[0129] B. Enteral Formulations
[0130] Suitable oral dosage forms include tablets, capsules,
solutions, suspensions, syrups, and lozenges. Tablets can be made
using compression or molding techniques well known in the art.
Gelatin or non-gelatin capsules can prepared as hard or soft
capsule shells, which can encapsulate liquid, solid, and semi-solid
fill materials, using techniques well known in the art.
[0131] Formulations may be prepared using a pharmaceutically
acceptable carrier. As generally used herein "carrier" includes,
but is not limited to, diluents, preservatives, binders,
lubricants, disintegrators, swelling agents, fillers, stabilizers,
and combinations thereof.
[0132] Carrier also includes all components of the coating
composition which may include plasticizers, pigments, colorants,
stabilizing agents, and glidants. Delayed release dosage
formulations may be prepared as described in standard references
such as "Pharmaceutical dosage form tablets" (1989),
"Remington--The science and practice of pharmacy" (2000), and
"Pharmaceutical dosage forms and drug delivery systems" (1995).
These references provide information on carriers, materials,
equipment and process for preparing tablets and capsules and
delayed release dosage forms of tablets, capsules, and
granules.
[0133] Examples of suitable coating materials include, but are not
limited to, cellulose polymers such as cellulose acetate phthalate,
hydroxypropyl cellulose, hydroxypropyl methylcellulose,
hydroxypropyl methylcellulose phthalate and hydroxypropyl
methylcellulose acetate succinate; polyvinyl acetate phthalate,
acrylic acid polymers and copolymers, and methacrylic resins that
are commercially available under the trade name EUDRAGIT.RTM. (Roth
Pharma, Westerstadt, Germany), zein, shellac, and
polysaccharides.
[0134] Additionally, the coating material may contain conventional
carriers such as plasticizers, pigments, colorants, glidants,
stabilization agents, pore formers and surfactants.
[0135] Optional pharmaceutically acceptable excipients include, but
are not limited to, diluents, binders, lubricants, disintegrants,
colorants, stabilizers, and surfactants. Diluents, also referred to
as "fillers," are typically necessary to increase the bulk of a
solid dosage form so that a practical size is provided for
compression of tablets or formation of beads and granules. Suitable
diluents include, but are not limited to, dicalcium phosphate
dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol,
cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry
starch, hydrolyzed starches, pregelatinized starch, silicone
dioxide, titanium oxide, magnesium aluminum silicate and powdered
sugar.
[0136] Binders are used to impart cohesive qualities to a solid
dosage formulation, and thus ensure that a tablet or bead or
granule remains intact after the formation of the dosage forms.
Suitable binder materials include, but are not limited to, starch,
pregelatinized starch, gelatin, sugars (including sucrose, glucose,
dextrose, lactose and sorbitol), polyethylene glycol, waxes,
natural and synthetic gums such as acacia, tragacanth, sodium
alginate, cellulose, including hydroxypropylmethylcellulose,
hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic
polymers such as acrylic acid and methacrylic acid copolymers,
methacrylic acid copolymers, methyl methacrylate copolymers,
aminoalkyl methacrylate copolymers, polyacrylic
acid/polymethacrylic acid and polyvinylpyrrolidone.
[0137] Lubricants are used to facilitate tablet manufacture.
Examples of suitable lubricants include, but are not limited to,
magnesium stearate, calcium stearate, stearic acid, glycerol
behenate, polyethylene glycol, talc, and mineral oil.
[0138] Disintegrants are used to facilitate dosage form
disintegration or "breakup" after administration, and generally
include, but are not limited to, starch, sodium starch glycolate,
sodium carboxymethyl starch, sodium carboxymethylcellulose,
hydroxypropyl cellulose, pregelatinized starch, clays, cellulose,
alginine, gums or cross linked polymers, such as cross-linked PVP
(Polyplasdone.RTM. XL from GAF Chemical Corp).
[0139] Stabilizers are used to inhibit or retard drug decomposition
reactions which include, by way of example, oxidative reactions.
Suitable stabilizers include, but are not limited to, antioxidants,
butylated hydroxytoluene (BHT); ascorbic acid, its salts and
esters; Vitamin E, tocopherol and its salts; sulfites such as
sodium metabisulphite; cysteine and its derivatives; citric acid;
propyl gallate, and butylated hydroxyanisole (BHA).
[0140] Oral dosage forms, such as capsules, tablets, solutions, and
suspensions, can for formulated for controlled release. For
example, the one or more 4-AP derivatives and optional one or more
additional active agents can be formulated into nanoparticles,
microparticles, and combinations thereof, and encapsulated in a
soft or hard gelatin or non-gelatin capsule or dispersed in a
dispersing medium to form an oral suspension or syrup. The
particles can be formed of the drug and a controlled release
polymer or matrix. Alternatively, the drug particles can be coated
with one or more controlled release coatings prior to incorporation
in to the finished dosage form.
[0141] In another embodiment, the one or more 4-AP derivatives and
optional one or more additional active agents are dispersed in a
matrix material, which gels or emulsifies upon contact with an
aqueous medium, such as physiological fluids. In the case of gels,
the matrix swells entrapping the active agents, which are released
slowly over time by diffusion and/or degradation of the matrix
material. Such matrices can be formulated as tablets or as fill
materials for hard and soft capsules.
[0142] In still another embodiment, the one or more 4-AP
derivatives, and optional one or more additional active agents are
formulated into a sold oral dosage form, such as a tablet or
capsule, and the solid dosage form is coated with one or more
controlled release coatings, such as a delayed release coatings or
extended release coatings. The coating or coatings may also contain
the 4-AP derivatives and/or additional active agents.
[0143] C. Topical Formulations
[0144] Suitable dosage forms for topical administration include
creams, ointments, salves, sprays, gels, lotions, emulsions, and
transdermal patches. The formulation may be formulated for
transmucosal, transepithelial, transendothelial, or transdermal
administration. The compounds can also be formulated for intranasal
delivery, pulmonary delivery, or inhalation. The compositions may
further contain one or more chemical penetration enhancers,
membrane permeability agents, membrane transport agents,
emollients, surfactants, stabilizers, and combination thereof.
[0145] D. Other Formulations
[0146] Any of the formulations discussed above may be used for a
formulation that is not a pharmaceutical formulation. In some
embodiments, a formulation may be prepared for administration to a
subject by a method that is direct or by a method that is indirect.
In certain embodiments, a compound is provided in a liquid, solid,
or powder formulation. The compound may be in a composition that is
sprayed or otherwise applied to a surface or location. In some
embodiments, the composition is placed in a location that is
accessible to the subject so that the subject ingests or comes into
contact with the composition. In certain embodiments, the compound
is absorbed or adsorbed by the subject.
IV. METHODS OF MAKING COMPOUNDS THAT BLOCK POTASSIUM CHANNELS
[0147] The compounds provided by the present disclosure are
described in the summary of the invention section and in the claims
below. They may be made using the methods outlined in the summary
of the invention section and in the Examples section. These methods
can be further modified and optimized using the principles and
techniques of organic chemistry as applied by a person skilled in
the art. Such principles and techniques are taught, for example, in
March's Advanced Organic Chemistry: Reactions, Mechanisms, and
Structure (2007), which is incorporated by reference herein.
[0148] The compounds or compositions described herein can also be
prepared by any of the applicable techniques of organic synthesis
and polymer chemistry. Many such techniques are well known in the
art. Many of the known techniques are elaborated in Compendium of
Organic Synthetic Methods, Vol 1, 1971; Vol. 2, 1974; Vol. 3, 1977;
Vol. 4, 1980; Vol. 5, 1984; and Vol. 6, 1985; Comprehensive Organic
Synthesis Selectivity, Strategy & Efficiency in Modern Organic
Chemistry, 1993; Advanced Organic Chemistry, Part B: Reactions and
Synthesis, 4.sup.th Ed., 2001; Advanced Organic Chemistry,
Reactions, Mechanisms, and Structure, 2.sup.nd Ed., 1977;
Protecting Groups in Organic Synthesis, 2.sup.nd Ed., 991;
Comprehensive Organic Transformations, 2.sup.nd Ed., 1999, Textbook
of Polymer Chemistry, 3.sup.rd Ed., 1984, Organic Polymer
Chemistry, 2nd Ed., 1973, and Polymer Science, 1986. These are
incorporated herein by reference.
V. METHODS OF USING COMPOUNDS THAT BLOCK POTASSIUM CHANNELS
[0149] A. Treatment
[0150] The compounds described herein can be administered to
provide an effective amount to prevent, treat or mitigate a symptom
of a variety of diseases and disorders, in particular, a disease
associated with demyelination, such as multiple sclerosis. The
compounds described herein can be administered to a subject in need
thereof to treat the subject either prophylactically (e.g., to
prevent a demyelination disease) or therapeutically (e.g., to treat
a demyelination disease after it has been detected), including, but
not limited to, ameliorating the symptoms of a disease, reducing
the pain of the patient, delaying the progression of the disease,
preventing new attacks or recurring of the disease, preventing
disability and/or increasing survival time of the patient.
[0151] The compounds described herein can bind to potassium
channels, such as Kv1 potassium channels located in the axonal
membrane to partially or completely restore the impulse conduction
along the axon. In some embodiments, the compounds described herein
may also be used to treat non-neurological diseases when blocking
of potassium channels in the heart or other tissues expressing
potassium channels is desired.
[0152] By administering the compounds described herein to a patient
suffering from a demyelinating disease, one or more symptoms
associated with demyelination may be mitigated or eliminated. The
symptoms include changes in sensation such as loss of sensitivity
or tingling, pricking or numbness (hypoesthesia and paresthesia),
muscle weakness, clonus, muscle spasms, or difficulty in moving;
difficulties with coordination and balance (ataxia); problems in
speech (dysarthria) or swallowing (dysphagia), visual problems
(nystagmus, optic neuritis including phosphenes, or diplopia),
fatigue, acute or chronic pain, and bladder and bowel difficulties.
The symptoms may further include cognitive impairment of varying
degrees and emotional symptoms of depression or unstable mood are
also common, Uhthoffs phenomenon, an exacerbation of extant
symptoms due to an exposure to higher than usual ambient
temperatures, and Lhermitte's sign, an electrical sensation that
runs down the back when bending the neck.
[0153] Exemplary demyelination diseases which can be treated by the
compounds described herein include, but are not limited to,
multiple sclerosis, spinal cord compression, ischemia, acute
disseminated encephalomyelitis, optic neuromyelitis,
leukodystrophy, progressive multifocal leukoencephalopathy,
metabolic disorders, toxic exposure, congenital demylinating
disease, peripheral neuropathy, encephalomyelitis, central pontine
myelolysis, Anti-MAG Disease, Guillain-Barre syndrome, chronic
inflammatory demyelinating polyneuropathy, or multifocal motor
neuropathy (MMN). Exemplary leukodystrophy includes, but is not
limited to adrenoleukodystrophy, Alexander's Disease, Canavan
Disease, Krabbe Disease, Metachromatic Leukodystrophy,
Pelizaeus-Merzbacher Disease, vanishing white matter disease,
Refsum Disease, Cockayne Syndrome, Van der Knapp Syndrome, or
Zellweger Syndrome.
[0154] Patients can be treated using a variety of routes of
administration including systemic administration, such as
intravenous administration or subcutaneous administration, oral
administration or by intratumoral injection.
[0155] In certain embodiments, it may be desirable to provide
continuous delivery of one or more compounds described herein to a
patient in need thereof. For intravenous or intraarterial routes,
this can be accomplished using drip systems, such as by intravenous
administration. For topical applications, repeated application can
be done or a patch can be used to provide continuous administration
of the compounds described herein, including 4-AP derivatives over
an extended period of time. Extended release formulations can also
be used to provide limited but stable amounts of the drug over an
extended period of time.
[0156] For internal applications, continuous perfusion of the
region of interest may be desirable. This could be accomplished by
catheterization, post-operatively in some cases, followed by
continuous administration of the one or more 4-AP derivatives. The
time period for perfusion can be readily determined by the
attending physician clinician for a particular subject. Perfusion
times typically range from about 1-2 hours, to 2-6 hours, to about
6-10 hours, to about 10-24 hours, to about 1-2 days, to about 1-2
weeks or longer. Generally, the dose of the therapeutic composition
via continuous perfusion will be equivalent to that given by single
or multiple injections, adjusted for the period of time over which
the injections are administered.
[0157] The compositions described herein contain an effective
amount of the one or more compounds described herein. The amount to
be administered can be readily determined by the attending
physician based on a variety of factors including, but not limited
to, age of the patient, weight of the patient, disease or disorder
to be treated, presence of a pre-existing condition, and dosage
form to be administered (e.g., immediate release versus modified
release dosage form). Typically, the effective amount is from about
0.01 mg/kg/day to about 100 mg/kg/day, from 0.1 mg/kg/day to 50
mg/kg/day, from 0.1 mg/kg/day to 25 mg/kg/day, 0.1 mg/kg/day to 10
mg/kg/day, from 0.1 mg/kg/day to 1 mg/kg/day or any range derivable
therein. Dosages greater or less than this may be administered
depending on the diseases or disorder to be treated.
[0158] The therapeutically effective doses could also be determined
by using an animal model. For example, a mouse bearing experimental
autoimmune encephalomyelitis (EAE) could be used to optimize
appropriate therapeutic doses prior to translating to a clinical
environment.
[0159] The therapeutically effective doses could also be determined
by using an animal model. For example, a rodent bearing Traumatic
Brain Injury could be used to optimize appropriate therapeutic
doses prior to translating to a clinical environment.
[0160] In some embodiments, the compounds and compositions
disclosed herein may be useful in a variety of manners. In some
embodiments, the compounds and compositions disclosed herein may be
useful for improving gait in stroke patients. In some embodiments,
the compounds and compositions disclosed herein may be useful in
research to induce seizures. In some embodiments, the compounds and
compositions disclosed herein may be useful as pest control agents.
In some embodiments, the compounds and compositions disclosed
herein may be useful for Parkinson's Disease, pediatric and adult
Cerebral Palsy, Spinal Cord Injury, Lambert Eaton syndrome, and
MS.
[0161] In some embodiments, derivatives of 4-AP will have improved
pharmacological properties over 4-AP. 3-F-4-AP has better
permeability into the CNS than 4-AP and, therefore, it may be
better for CNS diseases. Fluorinated 4-APs may have longer half
life than 4-AP, may be less toxic, and may be more stable to
metabolic degradation.
[0162] B. Imaging
[0163] The compounds described herein can also be used as imaging
agents in medical imaging applications. Medical imaging is the
technique and process used to create images of the human body (or
parts and function thereof) for clinical purposes (medical
procedures seeking to reveal, diagnose or examine disease) or
medical science (including the study of normal anatomy and
physiology). Medical imaging may also be applied to an animal body.
Commonly used medical imaging techniques include, but are not
limited to, radiography, magnetic resonance imaging (MRI),
fiduciary markers, nuclear medicine, photo acoustic imaging, breast
thermography, tomography, and ultrasound.
[0164] 1. Radiography
[0165] Projection radiograph, also known as x-rays, and fluoroscopy
are two forms of radiographic images used in medical imaging; with
the latter being useful for catheter guidance. This imaging
modality utilizes a wide beam of x rays for image acquisition and
is the first imaging technique available in modern medicine.
[0166] 2. Magnetic Resonance Imaging (MRI)
[0167] Magnetic resonance imaging instrument (MRI scanner), or
"nuclear magnetic resonance (NMR) imaging" scanner as it was
originally known, uses powerful magnets to polarise and excite
hydrogen nuclei (single proton) in water molecules in human tissue,
producing a detectable signal which is spatially encoded, resulting
in images of the body.
[0168] 3. Fiduciary Markers
[0169] Fiduciary markers are used in a wide range of medical
imaging applications. Images of the same subject produced with two
different imaging systems may be correlated (called image
registration) by placing a fiduciary marker in the area imaged by
both systems. In this case, a marker which is visible in the images
produced by both imaging modalities must be used.
[0170] 4. Nuclear Medicine
[0171] Nuclear medicine encompasses both diagnostic imaging and
treatment of disease. Nuclear medicine uses certain properties of
isotopes and the energetic particles emitted from radioactive
material to diagnose or treat various pathology. This approach is
often used in e.g., scintigraphy, SPECT and PET to detect regions
of biologic activity that may be associated with disease. Isotopes
are often preferentially absorbed by biologically active tissue in
the body, and can be used to identify tumors or fracture points in
bone. Images are acquired after collimated photons are detected by
a crystal that gives off a light signal, which is in turn amplified
and converted into count data.
[0172] Scintigraphy ("scint") is a form of diagnostic test wherein
radioisotopes are taken internally, for example intravenously or
orally. Then, gamma cameras capture and form two-dimensional images
from the radiation emitted by the radiopharmaceuticals.
[0173] SPECT is a 3D tomographic technique that uses gamma camera
data from many projections and can be reconstructed in different
planes. In SPECT imaging, the patient is injected with a
radioisotope, most commonly Thallium 201TI, Technetium 99mTC,
Iodine 1231, and Gallium 67Ga.
[0174] Positron emission tomography (PET) uses coincidence
detection to image functional processes. Short-lived positron
emitting isotopes, such as .sup.18F, are incorporated with an
organic substance such as glucose, creating F18-fluorodeoxyglucose,
which can be used as a marker of metabolic utilization. Images of
activity distribution throughout the body can show rapidly growing
tissue, like tumor, metastasis, or infection. PET images can be
viewed in comparison to computed tomography scans to determine an
anatomic correlate. Modern scanners combine PET with a CT, or even
MRI, to optimize the image reconstruction involved with positron
imaging. This is performed on the same equipment without physically
moving the patient off of the gantry. The resultant hybrid of
functional and anatomic imaging information is a useful tool in
non-invasive diagnosis and patient management.
[0175] 5. Tomography
[0176] Tomography is the method of imaging a single plane, or
slice, of an object resulting in a tomogram. There are several
forms of tomography, including linear tomography, poly tomography,
zonography, orthopantomograph (OPT or OPG), and computed tomography
(CT).
[0177] 6. Ultrasound
[0178] Medical ultrasonography uses high frequency broadband sound
waves in the megahertz range that are reflected by tissue to
varying degrees to produce (up to 3D) images. This is commonly
associated with imaging the fetus in pregnant women. Uses of
ultrasound are much broader, however. Other important uses include
imaging the abdominal organs, heart, breast, muscles, tendons,
arteries and veins.
[0179] In certain embodiments, the compounds described herein are
used for in vivo imaging of the central nervous system. More
specifically, the compounds described herein bind to potassium
channels, including Kv1 channels. The compounds disclosed herein
contain one or more radioisotopes. Exemplary radioisotopes include,
but are not limited to, .sup.18F, .sup.11C, .sup.13N, and .sup.15O.
It would be within an artisan's ordinary skill to choose
appropriate radioisotope suitable for the imaging technique
intended to use. In some embodiments, one or more imaging
techniques may be combined for imaging purposes. For example, PET
may be combined with MRI. In some aspects, PET is used to image
demyelination and MRI is used to image inflammation. PET and MRI
are complement to each other and can provide valuable information
on progression of the MS disease in a patient.
[0180] One particular embodiment is directed to radiolabelled 4-AP
derivatives that target potassium channels of demyelinated neurons.
These radiotracers may be used as in vivo imaging agents for
demyelination. In particular embodiments, these radiotracers are
suitable for PET imaging technique. In one embodiment, the
radiotracers described herein contain .sup.18F.
[0181] Since the compounds described herein are capable of blocking
potassium channels, such as Kv1 potassium channels located in the
axonal membrane, the use of .sup.18F-labeled 4-AP derivatives such
as [.sup.18F]-3-fluoromethyl-4-aminopyridine, or
[.sup.18F]-3-fluoro-4-aminopyridine, or other radiotracers
described herein allows visualization of demyelinated axons in live
animals by proper medical imaging techniques, such as PET.
Therefore, the compounds described herein may be used to diagnose a
demyelinating disease or assessing the progression of a
demyelinating disease by administering the compounds to a subject
in need thereof and detecting the compounds in the subject by
proper medical imaging technique, such as PET, PET-Time-Activity
Curve (TAC), PET-MRI, in particular, PET. In some embodiments, one
or more medical imaging techniques disclosed herein may be used to
diagnose or evaluate the progression of a disease.
VI. KITS
[0182] In various aspects, a kit is envisioned containing one or
more compounds described herein. The kit may contain one or more
sealed containers, such as a vial, containing any of the compounds
described herein and/or reagents for preparing any of the compounds
described herein. In some embodiments, the kit may also contain a
suitable container means, which is a container that will not react
with components of the kit, such as an Eppendorf tube, an assay
plate, a syringe, a bottle, or a tube. The container may be made
from sterilizable materials such as plastic or glass.
[0183] The kit may further include instructions that outline the
procedural steps for methods of treatment or prevention of disease,
and will follow substantially the same procedures as described
herein or are known to those of ordinary skill. The instruction
information may be in a computer readable media containing
machine-readable instructions that, when executed using a computer,
cause the display of a real or virtual procedure of delivering a
pharmaceutically effective amount of one or more compounds
described herein.
VII. EXAMPLES
[0184] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
[0185] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
Publications cited herein and the materials for which they are
cited are specifically incorporated by reference.
[0186] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
claims.
Example 1. Synthesis of Fluorinated Derivative of 4-AP
[0187] 4-AP (FIG. 2A) is a relatively specific blocker of
voltage-gated K.sup.+ channels (K.sub.v1 family). 4-AP is a
membrane permeable molecule that binds to the intracellular mouth
of the K.sup.+ channel blocking ionic currents (Sherratt et al.,
1980). In demyelinated axons these channels are exposed and easily
accessible to the drug. For this reason, the inventors believe that
labeling 4-AP with a positron emitting radionuclide will enable
imaging of demyelinated regions.
[0188] Furthermore, since Kv1 channels are upregulated 2-4 fold in
demyelinated animals such as shiverer mice and upregulation of Kv1
channels in demyelinated axons suggests greater signal, the
inventors expect that the signal proceeding from demyelinated axons
will be greater.
[0189] A common approach in the design of PET radioactive tracers
is to replace a hydrogen (H), hydroxyl (OH) or methyl (CH.sub.3)
group with fluorine-18 (Ametamey et al., 2008). Fluorine-18 is the
preferred radionuclide due to its low positron energy and its
longer half-life. The low positron energy gives it greater spatial
resolution and its longer half-life facilitates off-site production
and distribution.
[0190] In an effort to design 4-AP derivatives that maintain
activity and are suitable for PET imaging, the inventors began by
examining 4-AP derivatives that are known to block K.sup.+
channels. 4-AP (FIG. 2A) and 3,4-diaminopyridine (FIG. 2B), are
well-known K.sup.+ channel blockers discovered in the 1970's
(Sherratt et al., 1980; Kirsch and Narahashi, 1978). More recently
Shi et al described 3-methanol-4-aminopyridine (FIG. 2C) as a novel
K.sup.+ channel blocker (Sun et al., 2010; Leung et al., 2011).
From these structures, it appears that some variation is permitted
on the 3 position of the pyridine ring. It has also been suggested
that certain variations on the 4 position of the pyridine 4 appear
to be acceptable as well (Smith et al., 2005). Thus, the inventors
hypothesized that 3-fluoro-4-aminopyridine (FIG. 2D),
3-fluoromethyl-4-aminopyridine (FIG. 2E) and
3-fluoroethyl-4-aminopyridine (FIG. 2F) could be suitable K.sup.+
channel blockers. These structures (FIGS. 2D-2F) contain different
substitutions on position 3 of the pyridine ring which does not
alter its function. The inventors also proposed that compound
2-fluoro-4-aminopyridine (FIG. 2G) could also bind to K.sup.+
channels.
[0191] Before radioactive labeling, it is important to test whether
these fluorinated derivatives are still able to bind to K.sup.+
channels. Non-radioactive 3-fluoro-4-aminopyridine (FIG. 2D) is
commercially available from Sigma. Synthesis of fluorinated
pyridines which share a core structure similar to the compounds
described herein has been reported previously (Lee and Chi, 1999;
Mobinikhaledi and Foroughifar, 2006). However,
3-fluoromethyl-4-aminopyridine (FIG. 2E) and
3-fluoroethyl-4-aminopyridine (FIG. 2F) have never been made
before. These compounds were synthesized according to syntheses
outlined in FIGS. 3A-3B. The production of final products
3-fluoromethyl-4-aminopyridine and 3-fluoroethyl-4-aminopyridine
are verified by NMR (FIGS. 3C, 3E) and high resolution Mass Spectra
(FIGS. 3D, 3F), respectively.
Example 2. Confirm Binding to Potassium Channels
[0192] In order to confirm that the fluorinated 4-AP derivatives
maintain the ability to block K.sup.+ channels and select the most
suitable compound for imaging studies, the inventors measured
inhibition of K.sup.+ currents in Shaker channels (Kv1.2) using
cut-open oocyte voltage clamp (Stefani and Bezanilla, 1998) (FIGS.
5A-5B). Using the cut-open oocyte voltage clamp studies to screen
compounds to determine their potency towards Kv1 cannels was
previously described in Starace and Bezanilla et al. (2004).
[0193] Remarkably, 3-fluoro-4-aminopyridine (3F-4AP) has very
similar affinity to 4-AP for K.sup.+ channels.
3-fluoromethyl-4-aminopyridine (3MeF-4AP) is similar to
3-methanol-4-aminopyridine (3MeOH-4AP) and around 10-fold less
potent than 4-AP. 3-fluoroethyl-4-aminopyridine (3EtF-4AP) and
2-fluoro-4-aminopyridine (2F-4AP) are at least a hundred fold-less
potent than 4-AP. Based on these results, 3F-4AP and 3-MeF-4AP are
the preferred molecules for imaging and therapy.
[0194] One advantage of 4-AP, and presumably of its analogs, is
that they bind to all channels from the K.sub.v1 family. It is
known that neurons express several of these channels (K.sub.v1.1,
K.sub.v1.2, K.sub.v1.4, K.sub.v1.5, K.sub.v1.6, K.sub.v1.7 and
K.sub.v1.8, among which the most important neuronal voltage gated
K+ channels are Kv1.1 and Kv1.2) and that these channels can form
hererotetramers. However, it is unclear which one or several are
responsible for the aberrant efflux of K.sup.+ ions from
demyelinated axons and thus, a broad-spectrum channel may be
beneficial.
[0195] A desired property for radioactive tracers is high affinity.
It is striking that 4-AP and 3,4-diaminopyridine possess a
relatively modest affinity to K.sup.+ channels (M to mM) and yet
that they are useful in therapeutics (Murray and Newsom-Davis,
1981; Maddison and Newsom-Davis, 2003; Goodman et al., 2009). It is
possible that these molecules have a higher effective affinity in
vivo as they bind quasi-irreversibly to the channel. Once bound to
the channel these molecules become trapped inside the channel and
do not dissociate. Thus, it is expected that despite their modest
affinity, the PET markers described herein will display a high
signal-to-noise ratio.
[0196] It is important to note that even though the in vitro
affinity of 4-AP is low (.about.250 .mu.M), 4-AP is active at much
lower concentrations in vivo (.about.0.5 .mu.M). This difference
might be because blockage of a small fraction of channels already
leads to an effect and because 4-AP binds to the channels when they
are open and once the channels close, 4-AP becomes trapped inside,
functioning as a non-reversible ligand (Armstrong and Loboda,
2001).
Example 3. In Vivo Effects of Fluorinated 4-AP Derivative
[0197] A consequence of excessive K.sup.+ channel blockage is the
advent of seizures. 4-AP is known to cause seizures in mice at high
doses. The inventors tested some 4-AP derivatives described herein
and compared them to 4-AP in their ability to cause seizures (Table
1).
[0198] The fact that only the molecules that are active by cut-open
voltage clamp are able to cause seizures strongly suggests that
these molecules are targeting K+ channels receptors in vivo.
[0199] In addition, the inventors noticed that Shiverer mice, which
harbor a mutation on myelin basic protein and suffer from
demyelination of the CNS, appear to be less sensitive to 4-AP
induced seizures. Previous studies have shown that Shiverers and
other demyelinated mice display an abnormal localization pattern of
K.sub.v1 channels and a 2-4 fold increase in expression of
K.sub.v1.1 and K.sub.v1.2 channels in axons (Wang et al., 1995).
The inventors believe that the higher expression of K.sub.v1
channels in Shiverer mice is the reason for why these animals are
less sensitive to 4-AP. It is not known whether K.sub.v1 channels
are also upregulated in MS patients, but it is known that K.sub.v1
channels in MS lesions present a similar localization patterns as
in demyelinated animals (Coman et al., 2006). Therefore, similar
upregulation is anticipated. Accordingly, upregulation of K.sub.v1
channels in MS patient lesions combined with the presumed lower
accessibility of 4-AP to K.sub.v1 channels in myelinated axons make
K.sub.v1 channels an attractive target for PET imaging.
[0200] At high doses, 4-AP causes tremors, muscle spasms and
seizures. A summary of the observations is shown in Table 1.
TABLE-US-00001 TABLE 1 Effects after intraperitoneal injection of
4-AP derivatives (100 .mu.L per 10 g of mouse) MW Dose Drug g/mol
.mu.mol/kg mg/kg Effect 4-AP 94.11 30 2.82 Mild tremor, mouse quiet
60 5.65 Severe tremor, mild jerks, salivation 90 8.47 Severe
tremors and seizures that start 10 min post-injection and last ca.
2 h 3-F-4-AP 112.11 30 3.36 Mouse quiet 60 6.73 Mild tremor, mouse
quiet 90 10.1 Severe tremors and seizures that start 10 s
post-injection and last ca. 30 min. 1 of 5 mice died of seizure
3-MeF-4-AP 126.13 30 3.78 No effect 60 7.57 Very mild tremor.
Normal after 30 min 90 11.4 Tremor and occasional jerks that last
ca. 45 min 120 15.1 Severe tremor and seizures that start 5 min
post-injection and last ca. 45 min. 1 of 5 mice died of seizure
3-MeOH-4-AP 124.14 960 119.1 No effect 2-F-4-AP 112.11 1920 215.2
No effect N= 5 per group.
[0201] From this experiment it can be seen that 3-F-4-AP and
3-MeF-4-AP have very similar effects as 4-AP in mice. Both of these
drugs cause salivation, tremors, jerks, extension of the hind limbs
and seizures. 3-F-4-AP has very similar potency to that of 4-AP and
acts much faster (onset of seizures at highest dose 10 s vs. 10
min) which is consistent with a faster absorption and a higher
permeation of the blood-brain barrier. 3-MeF-4-AP is slightly less
potent than 4-AP but remarkably potent considering that in the
voltage-clamp and optic nerve experiments it was found to be 6-20
times less potent than 4-AP. In contrast, 2-F-4-AP and 3-MeOH-4-AP
did not cause any effects at doses up to 20 times higher. The
inventors also tested the effect of the drugs given by oral gavage
on a small number of animals and found the same effects (data not
shown).
Example 4 (Prophetic Example). Synthesis of [.sup.18F]
4-Amino-3-(Fluoromethyl)Pyridine
##STR00012##
[0203] Based on the report by Lee et al on the synthesis of
fluoroalkyl pyridines with fluorine-18 (Lee et al., 1999), this
example depicts a possible synthetic route to generate
[.sup.18F]-3-fluoromethyl-4-aminopyridine by protecting the amine
of 4-aminopyridine-3-methanol with Boc, followed by nucleophilic
substitution of the benzyl alcohol with .sup.18F.sup.-, and boc
deprotection.
[0204] A solution of Boc.sub.2O (0.20 mol) in CH.sub.2Cl.sub.2 (100
mL, not anhydrous) is added over 20 min to a stirred suspension of
4-aminopyridine-3-methanol (0.20 mol) in CH.sub.2Cl.sub.2 (200 mL).
The resulting solution is stirred at room temperature for 25 min
(TLC) and acidified with 1 M HCl (230 mL, 0.23 mol). The phases are
separated, and the aqueous layer is washed with CH.sub.2Cl.sub.2.
The combined organic extracts are dried (MgSO.sub.4) and evaporated
in vacuum to give compound 2 which is used in the next step without
further purification.
[0205] .sup.18F water is added to the reaction vessel followed by
[.sup.18F.sup.-] K222 (2 mg) in acetonitrile (500 ml), and
K.sub.2CO.sub.3 (0.1 mol dm.sup.-3, 50 ml) and dried at 100.degree.
C. for 20-30 min. Compound 3 (1 mg) in acetonitrile (1000 mL) is
added. The reaction vessel is sealed and heated at 100.degree. C.
for 10 min. The reaction mixture is cooled, washed from the
reaction vessel with water (1.5 mL) and collected in a vial. 3 mL
of CF.sub.3COOH are added to the vial and the reaction is heated in
a microwave (75 W, 140.degree. C.) for 3 min. Subsequently, the
reaction mixture is purified by reverse phase HPLC. Finally, the
fractions containing the product are diluted to 5 mL in PBS.
Example 5 (Prophetic Example). Synthesis of [.sup.18F]
4-Amino-3-(Fluoroethyl)Pyridine
##STR00013##
[0207] The synthesis of compound 8 starting from compound 5 may be
performed using the same procedure to Example 4.
Example 6 (Prophetic Example). Synthesis of [.sup.18F]
4-Amino-3-Fluoropyridine
##STR00014##
[0209] This example depicts a possible synthetic route to generate
[.sup.18F]-3-fluoro-4-aminopyridine by double deprotonation of
N-boc protected 4-aminopyridine followed by reaction with
[.sup.18F]-F.sub.2 and Boc deprotection. Other synthesis including
using the recently reported Pd-mediated electrophilic synthesis
(Lee et al., 2011) may also be applicable for synthesizing
[.sup.18F]-3-fluoro-4-aminopyridine.
[0210] To a solution of 9 (200 mmol) in THF (500 mL) at -78.degree.
C. is added t-BuLi (282 mL, 1.7 M, 480 mmol) in pentane over 70
min. The resulting bright yellow suspension is stirred at
-78.degree. C. for 20 min and at -15.degree. C. for 2 h.
Subsequently, [.sup.18F]-F.sub.2 is bubbled through 5 ml of
solution containing lithiated species 10. After 20 min, 3 mL of
CF.sub.3COOH are added to the solution and the reaction is heated
in a microwave (75 W, 140.degree. C.) for 3 min to afford compound
11.
[0211] Alternatively, the .sup.18F-labeled versions of 3-F-4-AP and
3-MeF-4-AP are synthesized as depicted below based on the synthesis
of similar PET markers (FIG. 8) (Zhou, et al., 2009; Lee, et al.,
1999; Dolle, et al., 2005; Cai, et al., 2008; Chun, et al.,
2012).
[0212] The proposed synthesis of [.sup.18F] 3-F-4-AP uses iodonium
salts for high efficiency synthesis of aryl fluorides (Chun, et
al., 2012). This tracer may be useful to evaluate lesion size and
lesion load in multiple sclerosis patients. This tracer may also be
useful in patients with Parkinson's disease, stroke, Cerebral
palsy, Alzheimer disease, ALS, Lambert Eaton, brain tumors and
other diseases.
##STR00015##
[0213] Synthesis of [.sup.18F] 3-F-4-AP (13): Koser reagent is
added to a solution of
4-[(tert-butoxycarbonyl)amino]pyridin-3-ylboronic acid (10) in
CH.sub.2Cl.sub.2 and stirred until formation of
{4-[(tert-butoxycarbonyl)amino]pyridin-3-yl}(phenyl)iodonium (11).
After 16 h the solvent is removed under vacuo and the product (11)
purified using standard techniques. Next
{4-[(tert-butoxycarbonyl)amino]pyridin-3-yl}(phenyl)iodonium (11)
is dissolved in DMF and [(crypt-222)K].sup.+18F.sup.- is added to
generate [.sup.18F] N-(3-fluoropyridin-4-yl)carbamate (12).
Finally, [.sup.18F] N-(3-fluoropyridin-4-yl)carbamate (12) is
treated with aqueous HCl to deprotect the amino group and yield the
final product [.sup.18F] 3-fluoropyridin-4-amine also known as
[.sup.18F] 3-fluoro-4-aminopyridinine or [.sup.18F] 3-F-4-AP
Example 7 (Prophetic Example). Use of the Compounds for Imaging in
Animal Models of Demyelination
[0214] Once the radiolabeled markers are obtained, the inventors
will perform imaging studies in several mouse models of
demyelination. The compounds are tested for imaging demyelination
as previously described (Stankoff et al., 2011). The use of
different mouse models will enable the inventors to assess whether
4-AP mainly targets potassium channels in neurons or also channels
in other cells such as lymphocytes. Suitable animal models, in
particular, mouse models, are contemplated as follows.
[0215] DTA Model.
[0216] The inventors have generated a new mouse model (DTA) of
widespread CNS demyelination wherein the ablation of
oligodendrocytes is accomplished via cell-specific activation of
diphtheria toxin (DT-A) expression in young adult animals (Traka et
al., 2010). This approach results in widespread DT-A-mediated death
of mature oligodendrocytes and extensive demyelination throughout
the CNS (Traka et al., 2010). At the peak of disease the DTA mice
developed severe tremor and ataxia. These mice demonstrate a
gradual recovery that culminates in full attenuation of the disease
symptoms by approximately 70 dpi, which correlates with the
repopulation of oligodendrocytes and remyelination. This model
provides widespread and extensive demyelination of the CNS.
[0217] Cuprizone-Induced Demyelination.
[0218] Feeding of cuprizone (bis-cyclohexanone oxaldihydrazone) to
young adult mice induces a synchronous and consistent demyelination
of the corpus callosum (Matsushima and Morel, 2001; Stidworthy et
al., 2004). Demyelination and oligodendrocyte apoptosis do not
involve T cells or breakdown of the blood-brain barrier in this
model and the mice do not display any clinical symptoms. Following
the loss of oligodendrocytes and demyelination, there is a
repopulation of the oligodendrocytes in the corpus callosum and
robust remyelination. The inventors have considerable experience
using the cuprizone protocol to examine the demyelination and
remyelination processes (Gao et al., 1999; Lin et al., 2004). The
cuprizone model provides a nice system in which to image highly
reproducible, focal demyelinated lesions that do not involve
peripheral immune system infiltration.
[0219] Experimental Autoimmune Encephalomyelitis (EAE).
[0220] EAE which is considered the best animal model of MS, can be
induced in a variety of species of laboratory animals by
immunization with either myelin or one of its components (Prog.
Clin. Biol. Res., 1984; Zamvil and Steinman, 1990; Martin and
McFarland, 1995). EAE is an immune-mediated demyelinating disease
that displays many of the clinical, pathologic, and immunological
features of MS (Behi et al., 2005). Clinical symptoms correlate
with focal inflammatory demyelinated lesions in the spinal cord of
the affected animals. The EAE model is capable of providing the
most MS-like lesions, which include loss of oligodendrocytes,
demyelination and T cell infiltration.
[0221] Other animal models of demyelination, such as a lysolecithin
injection model, may also be used in the study of demyelination
associated diseases. Animal models other than mouse models may also
be used. It will be obvious to those skilled in the art to choose
an appropriate animal model to adapt to intended research
purposes.
[0222] Traumatic brain injury models. In these models a traumatic
brain injury is caused in mice or rats by a controlled impact
(using a pendulum or a weight) or by an explosion.
Example 8. (Prophetic Example) Use of the Compounds Described
Herein for Diagnosis and Evaluation of Ms Progression
[0223] A detectable amount of the compound described herein, such
as [.sup.18F]-3-fluoromethyl-4-aminopyridine, or
[.sup.18F]-3-fluoro-4-aminopyridine, is introduced in the patient
body via a pharmaceutically acceptable route known in the art. The
patient is positioned inside a PET scanner or an instrument capable
of detecting radiation emitted by the compound as typically done in
the art. The localization of the radioactive tracer is done using a
computer, which can provide images of the localization and extent
of demyelinated axons.
Example 9. Methods
[0224] Non-Radioactive Synthesis.
[0225] Non-radioactive synthesis has been performed using standard
techniques in organic chemistry. Reactions were monitored by TLC
and products characterized by .sup.1H, .sup.13C and .sup.19F NMR,
and high-resolution mass spectroscopy.
[0226] All chemicals were ordered from Sigma unless otherwise
specified. Animal protocols were approved by IACUC.
##STR00016##
Synthesis of tert-butyl N-[3-(hydroxymethyl)pyridin-4-yl]carbamate
(8)
[0227] To a solution of 4-aminopyridine-3-methanol (3) (Alfa Aesar)
(806 mg, 6.5 mmol) in CH.sub.2Cl.sub.2 (10 mL) a solution of
di-tert-butyl-dicarbonate (1.43 g, 6.56 mmol) in CH.sub.2Cl.sub.2
(5 mL) was added and stirred at room temperature for 1 h (TLC).
After 1 h, the solution was acidified with 1 N HCl (7.4 mL, 7.4
mmol). The phases were separated and the aqueous phase was washed
with CH.sub.2Cl.sub.2. The mg, 5.1 mmol). The phases were separated
and treated with additional amounts of CH.sub.2Cl.sub.2. The
combined organic extracts were dried (MgSO.sub.4) and evaporated in
vacuo to give a 60:40 mixture containing desired product 8 and the
O-linked carbonate. Attempts to purify the N-carbamate by flash
chromatography from the O-carbonate were unsuccessful due
interconversion between these two species in solution at room
temperature. .sup.1H-NMR (CDCl.sub.3, 500 MHz) .delta.: 1.53 (9H,
s), 4.67 (2H, s), 4.83 (2H, br s), 7.95 (1H, s), 8.07 (1H, d, J=5.5
Hz), 8.28 (1H, d, J=5.5 Hz), 8.48 (1H, s). This product has been
previously synthesized through a different route (Mochizuki, et
al., 2011).
Synthesis of tert-butyl N-[3-(fluoromethyl)pyridin-4-yl]carbamate
(9)
[0228] To a solution of triethylamine (450 .mu.L, 2.76 mmol) in
CH.sub.2Cl.sub.2 (5 mL) at -78.degree. C. was added
XtalFluor-E.RTM. (473 mg, 207 mmol) and the product from the
previous reaction (310 mg, 1.38 mmol in 5 mL CH.sub.2Cl.sub.2). The
reaction was stirred at 0.degree. C. for 15 min (TLC).
Subsequently, the reaction mixture was washed with NaHCO.sub.3 (10
mL) and brine (10 mL). The organic phase was dried (MgSO.sub.4) and
concentrated in vacuo. The crude product was purified by flash
chromatography to afford 9 (113 mg, 36% yield). R.sub.f=0.5 (1:1,
hexanes: EtOAc). .sup.1H-NMR (CDCl.sub.3, 500 MHz) .delta. ppm:
1.56 (9H, s), 5.48 (2H, d, J=48 Hz), 7.12 (1H, br s), 8.12 (1H, d,
J=5.5 Hz), 8.38 (1H, d, J=3 Hz), 8.32 (1H, s), 8.53 (1H, dd,
J.sub.2=5.5 Hz, J.sub.1=1.0 Hz). .sup.13C-NMR (CDCl.sub.3, 125 MHz)
.delta.: 28.2, 80.6, 82.0 (d, J=15.5 Hz), 113.2, 118.0 (d, J=15.5
Hz), 145.6, 150.1, 149.7, 151.7, 152.4. .sup.19F-NMR (CDCl.sub.3,
470 MHz) .delta.: -209.3 (t, J=48 Hz). HR-MS m/z: 227.1190
(M+H).sup.+.
Synthesis of 3-fluoromethyl-4-aminopyridine (5)
[0229] To a solution of 9 (56 mg, 0.25 mmol) in CH.sub.2Cl.sub.2 (3
mL) was added TFA (194 .mu.L, 2.5 mmol) at 0.degree. C. and stirred
at room temperature for 5 h (TLC). After 5 h the reaction was
quenched with excess NaOH (1 M). The solvent was evaporated to
afford 8 quantitatively. R.sub.f=0.2 (MeOH). .sup.1H-NMR (500 MHz,
D.sub.2O) .delta.: 5.40 (2H, d, J=48 Hz), 6.87 (1H, d, J=7 Hz),
7.95 (1H, d, J=7 Hz), 8.09 (1H, s). .sup.19F-NMR (CDCl.sub.3, 470
MHz) .delta.: -215.9 (t, J=48 Hz). HR-MS m/z: 127.0666
(M+H).sup.+.
##STR00017##
Synthesis of tert-butyl N-[3-(2-hydroxyethyl)pyridin-4-yl]carbamate
(11)
[0230] Adapted from Spivey et al: To a solution of
4-(Boc-amino)pyridine (1) (2.0 g, 10.3 mmol, 1 eq.) in 25 mL of dry
THF at -78.degree. C. was added t-BuLi (14.5 mL, 1.7 M, 24.7 mmol,
2.4 eq.) in pentane over 30 min. The resulting bright yellow
suspension was stirred at -78.degree. C. for 15 min, at -15.degree.
C. for 2 h and then recooled to -78.degree. C. In a separate flask,
n-BuLi (7.38 mL, 2.5M, 18.54 mmol, 1.8 eq) in hexanes was added to
a solution of 2-bromoethanol (1.294 mL, 15.45 mmol, 1.5 eq) in 20
mL of dry THF at -78.degree. C. and stirred for 10 min. After 10
min, the bromoethanol solution was transferred via cannula to the
flask containing lithiated N-boc-4-aminopyridine over 10 min. The
reaction was allowed to warm to room temperature and the mixture
was stirred for 2 h. The reaction was recooled to -78.degree. C.
and quenched with 5 mL of water. The solution was partitioned
between water (20 mL) and CH.sub.2Cl.sub.2 (30 mL). The phases were
separated and the extraction was completed with additional portions
of CH.sub.2Cl.sub.2. The combined organic extracts were washed with
brine, dried over MgSO.sub.4 and evaporated under vacuum. The crude
product was dissolved in a small amount of CH.sub.2Cl.sub.2
purified by silica gel chromatography (EtOAc) to afford the product
11 (0.678 g, 36% yield). R.sub.f=0.15 (EtOAc). .sup.1H-NMR (500
MHz, CDCl.sub.3) .delta.: 1.51 (9H, s), 2.81 (2H, t, J=5.0 Hz),
3.80 (1H, br s), 3.94 (2H, t, J=5.0 Hz), 7.92 (1H, d, J=5.5 Hz),
8.15 (1H, s), 8.27 (1H, d, J=5.5 Hz), 8.63 (1H, s).
Synthesis of tert-butyl N-[3-(2-fluoroethyl)pyridin-4-yl]carbamate
(12)
[0231] To a solution of Et.sub.3N-3HF (715 .mu.L, 4.39 mmol, 2 eq.)
in 5 mL of dry CH.sub.2Cl.sub.2 at 0.degree. C., XtalFluor E.RTM.
(753 mg, 3.29 mmol, 1.5 eq) was added and stirred for 5 min. After
5 min, 2 (525 mg, 2.195 mmol, 1 eq) was added and the reaction was
monitored by TLC (1:1, hexanes:EtOAc). 15 min later the reaction
was washed with NaHCO.sub.3 (5 mL) and brine (5 mL), dried with
MgSO.sub.4 and the solvent evaporated under vacuum. The crude
product was dissolved in a small amount of CH.sub.2Cl.sub.2 and
purified by silica gel chromatography to afford 3 (457 mg, 71%
yield). R.sub.f=0.4 (1:1, hexanes:EtOAc). Mp=103.degree. C.
.sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.: 1.53 (9H, s), 2.98 (2H,
dt, J.sub.2=29 Hz, J=5.8 Hz), 4.72 (2H, dt, J.sub.2=47 Hz, J=5.8
Hz), 7.04 (1H, d, J=7.5 Hz), 7.99 (1H, d, J=5.0 Hz), 8.32 (1H, s),
8.40 (1H, d, J=5.0 Hz). .sup.13C-NMR (CDCl.sub.3, 125 MHz) .delta.:
28.2, 30.4 (d, J=20.1 Hz), 81.6, 84.9 (d, J=165 Hz), 113.7, 121.0,
144.7, 149.7, 151.2. .sup.19F-NMR (CDCl.sub.3, 470 MHz) .delta.:
-213.3 (tt, J.sub.2=47 Hz, J=29 Hz). HR-MS m/z: 241.1347
(M+H).sup.+.
Synthesis of 3-fluoroethyl-4-aminopyridine (6)
[0232] To a solution of 12 (120 mg, 0.5 mmol, 1 eq.) in 5 mL of
CH.sub.2Cl.sub.2 was added TFA (191 .mu.L, 2.5 mmol, 5 eq) at
0.degree. C. The reaction was allowed to warm up to room
temperature and stirred for 5 h (TLC). After 5 h, the reaction was
quenched with excess NaOH (1 M) and extracted multiple times with
CH.sub.2Cl.sub.2. The solvent was evaporated to afford 4
quantitatively. R.sub.f=0.5 (MeOH). .sup.1H-NMR (500 MHz,
CDCl.sub.3) .delta.: 2.91 (2H, dt, J.sub.2=26.5 Hz, J.sub.1=6 Hz),
4.26 (2H, br s), 4.69 (2H, dt, J.sub.2=47 Hz, J=6 Hz), 6.54 (1H, d,
J=5.5 Hz), 8.13 (1H, s), 8.15 (1H, d, J=5.5 Hz). .sup.13C-NMR
(CDCl.sub.3, 125 MHz) .delta.: 30.2 (d, J=20.6 Hz), 84.2 (d, J=166
Hz), 110.1, 149.1, 151.0, 151.7. .sup.19F-NMR (CDCl.sub.3, 470 MHz)
-213.3 (tt, J.sub.2=47 Hz, J.sub.1=26.5 Hz). HR-MS m/z: 141.0823
(M+H).sup.+.
[0233] [.sup.18F] Labeling (Prophetic)
[0234] .sup.18F-labeling will be performed using
cyclotron-generated reagents for nucleophilic and electrophilic
fluorination.
[0235] Measure Compound Action Potential of Demyelinated
Nerves:
[0236] 4-AP can enhance the Compound Action Potential in
demyelinated nerves. The effects of the 4-AP derivatives in the
compound action potential of optic nerves and/or spinal cords from
demyelinated animals will be measured according the protocol by
Stys et al. Briefly, optic nerves will be removed postmortem and
placed in an oxygenated aCSF solution. Suction electrodes will be
used to measure CAP in the presence and absence of the test
compounds (Stys et al., 1991).
[0237] Imaging (Prophetic):
[0238] Six mice of each group (DT-A, Cuprizone, EAE and healthy
controls) will be used for the Imaging study. 100 .mu.Ci/100 .mu.L
of [.sup.18F]-labeled 4-AP derivative will be injected into the
tail vein of anesthesized mice. The imaging sessions will be
carried out as 1 h dynamic scan using the MicroPET scanner. The
MicroPET data will be processed using filter back projection
algorithm with attenuation and scatter corrections. In vitro
stability studies of the radioactive tracers will be performed
according to the protocol by Zhou et al. (2009). Briefly, 2 mL of
heparinized mouse blood (C57BL/6N mice) are incubated with the
radioactive tracer (.about.400 .mu.Ci) for 5 min, 30 min, 1 h and 2
h at 37.degree. C. At each time point the blood will be lysed with
3 volumes of ethanol and centrifuged. The radioactive species in
the supernatant will be analyzed by radio-TLC and compared to the
radioactive tracer's control. In vivo stability: .about.400 Ci of
[.sup.18F] 4-AP in 200 .mu.L of saline are injected into a mouse by
iv injection in the tail vein of an immobilized mouse. Blood
samples (0.5 mL) are obtained via cardiac puncture under anesthesia
at 5 and 30 min post-injection. Afterwards, the plasma is treated
with 3 equivalents of acetonitrile and the pellet separated by
centrifugation. The radioactivity species of the supernatant will
be analyzed by radio-TLC and compared to control. In vivo
biodistribution time-course study: .about.400 Ci of [.sup.18F] 4-AP
in 200 .mu.L of saline will be injected into a mouse by iv
injection. At 5 min, 30 min, 1 h and 2 h post injection the mice
will be sacrificed and blood tissues and organs removed, weighed
and counted using a Beckman counter with standard diluted aliquots
of the sample. The percent injected dose per gram of tissue will be
calculated.
[0239] Neurological Evaluation (Prophetic):
[0240] the effects of the 4-AP derivatives in demyelinated animals
will be evaluated using a rotorod to test for balance and
coordination, the inventors will also measure changes in tremor and
other functions.
[0241] Pharmacology of 4-AP Derivatives (Prophetic):
[0242] metabolic stability, membrane permeability, toxicity,
pharmacokinetic and drug distribution studies will be conducted
with the assistance of a third party research contract
organization.
[0243] Measurement of 4-AP and Derivatives Distribution in Mice
(Prophetic):
[0244] The distribution of 4-AP and derivatives will be measured
using MALDI-IMS, whole body autoradiography, or organ
autoradiography.
Example 10. Blockage of K.sup.+ Channels by 4-AP Derivatives Using
Voltage Clamp
[0245] The inventors tested the ability of compounds 1-7 to block
voltage-gated K.sup.+ channels expressed in Xenopus oocytes using
the cut-open voltage clamp technique described by Stefani and
Bezanilla. For this experiment, Shaker K.sup.+ channel from D.
megalonaster was chosen as the archetypical voltage gated K.sup.+
channel that gives name to the family. Shaker shares an identity
ranging from 69%-79% with neuronal K.sub.v1.1, K.sub.v1.2,
K.sub.v1.3, K.sub.v1.4, K.sub.v1.6 that are among the presumed
targets of 4-AP and its sensitivity to 4-AP is comparable to other
K.sub.v1 channels (Gutman, et al., 2005; McCormack, et al., 1994).
In order to compare the relative potency of the different 4-AP
derivatives, each drug was applied at increasing concentrations and
the ratio between the K+ current with and without drug was computed
(FIG. 5).
[0246] Electrophysiology:
[0247] Electrophysiology studies are conducted according to the
protocol by Stefani and Bezanilla (1998). Briefly, K.sup.+ channel
Shaker cRNA is injected into Xenopus oocytes 24 h after their
surgical extraction from adult frogs. 1-5 days after injection
channel currents are recorded using the cut-open voltage-clamp.
Each molecule is added to the external solution at a range of
concentrations and K+ currents recorded and compared to those with
4-AP.
[0248] Expression of Shaker K+ Channel in Xenopus laevis
Oocytes:
[0249] K.sup.+ channel expression in Xenopus oocytes membranes was
achieved by injecting approximately 50 ng of WT Shaker cRNA (kit
Ambion) into the oocytes 24 h after surgical extraction from adult
frogs and collagenase treatment. Injected oocytes were maintained
in a standard oocytes solution (100 mM NaCl, 5 mM KCl, 2 mM
CaCl.sub.2, and 10 mM Hepes at pH 7.5) at 16.5.degree. C. and
recordings were performed 1-3 days after injection.
[0250] Recording of K.sup.+ Currents in Xenopus Oocytes:
[0251] K.sup.+ currents were recorded from oocytes expressing
Shaker K.sup.+ channels using the cut-open voltage clamp technique
as described by Stefani and Bezanilla. The internal solution was
120 mM KOH, 20 mM HEPES-methyl sulfonate (MES) pH 7.4, 2 mM EGTA.
The external solution was 12 mM KOH, 105 mM
N-methyl-D-glucamine-MES pH 7.4, 20 mM HEPES, 2 mM CaOH. To assess
the effects of the 4-AP derivatives, the drug under study was added
in incremental concentrations by exchanging the external solution
(top and guard chambers) several times. After application, cells
were voltage-clamped at least 5 min at 0 mV, then voltage-clamped
at -80 mV for 1-2 min. K.sup.+ currents were generated by applying
series of 50 ms pulses from -70 mV to +40 mV in increments of 10
mV. The effect of the drug was assessed by measuring the relative
intensity of the K.sup.+ current before and after applying varying
drug concentration at a constant voltage (typically +20 mV) and at
the end of the test-pulse. Analysis of the traces was done using an
in-house software. The half-maximal inhibitory concentration
(IC.sub.50) for each drug was calculated by plotting the relative
K.sup.+ current vs. concentration and fitted to the Hill equation
using the software Origin.
[0252] In this experiment, it was demonstrated that 4-AP and
3-F-4AP are the most potent compounds with half-maximal inhibitory
concentrations (IC.sub.50) around 0.27 mM (4-AP: IC.sub.50=0.29 mM,
95% C.I.=0.21-0.41 mM; 3-F-4-AP: IC.sub.50=0.25 mM, 95%
C.I.=0.13-0.44 mM), which is similar to what has been reported for
other Shaker-like channels (Gutman, et al., 2005). Although Berger
et al described 3-F-4-AP as being less potent than 4-AP in
eliciting muscle twitches in dissected mouse hemidiaphragms, it was
found to have comparable potency in blocking Shaker K.sup.+
channel. In this assay, 3-MeOH-4-AP and 3-MeF-4-AP were found to be
between 15 and 25 times less potent than 4-AP (3-MeOH-4-AP:
IC.sub.50=4.38 mM, 95% C.I.=3.4-5.6 mM; 3-MeF-4-AP: IC.sub.50=7.45
mM, 95% C.I.=6.2-9.0 mM). In contrast, 3-EtF-4-AP and 2-F-4-AP have
IC.sub.50 values greater than 10 mM (95% C.I. not determined).
These results demonstrate that only small modifications in the 2
position of 4-AP are permitted (e.g. 3-F-4-AP, 3-MeF-4-AP,
3-MeOH-4-AP) whereas larger modifications such as 3-EtF-4-AP or
substitution in the 2 position such as 2-F-4-AP significantly
diminish activity. In this experiment, it was also observed that
these drugs are difficult to wash, out which is similar to what has
been reported for 4-AP (McCormack, et al., 1994), suggesting a
similar mode of binding.
Example 11. Effects of 4-AP Derivatives on the Compound Action
Potential of Dissected Optic Nerve
[0253] It is known that 4-AP can significantly enhance action
potential of demyelinated fibers (Sherratt, et al., 1980; Devaux,
et al., 2002). In order to determine if the drugs could also be
effective in enhancing compound action potentials (CAP), the
effects of these compounds on the CAP of hypomyelinated optic
nerves from Shiverer mice (shi.sup.-/-) and control mice
(shi.sup.+/-, shi.sup.+/+) were tested. Shiverer mice lack compact
myelin in the CNS due to a null mutation of the myelin basic
protein gene (MBP). The results of this experiment are shown in
FIG. 5.
[0254] Dissection of Optic Nerves from Shiverer Mice:
[0255] optic nerves were dissected from 12-16 week old Shiverer
(shi.sup.-/-) and control mice (shi.sup.+/- and shi.sup.+/+). Mice
were euthanized by CO.sub.2 overdose and the optic nerves were
quickly dissected between the eyeball and the optic chiasm. The
nerves were incubated for 30 min at 37.degree. C. in oxygenated
(95% 02, 5% CO.sub.2) aCSF solution (126 mM NaCl, 3 mM KCl, 2 mM
MgSO.sub.4, 26 mM NaHCO.sub.3, 2 mMCaCl.sub.2, 10 mM dextrose, pH
7.5) before the experiment.
[0256] Optic Nerve Electrophysiology:
[0257] compound action potentials (CAP) from hypomyelinated nerves
(Shiverer mice, shi.sup.-/-) and myelinated nerves (litermate
controls, sh.sup.+/- and shi.sup.+/+) were recorded using suction
electrodes as described by Stys et al. Briefly, the dissected optic
nerve was placed inside a chamber containing oxygenated (5%
CO.sub.2, 95% O.sub.2) aCSF (300 .mu.L) between two suction
electrodes (stimulus and recording electrodes) forming a tight seal
on each end. Two additional electrodes were placed in the bath for
reference. A supramaximal pulse (250 mV, 20 s) was applied at the
stimulating end of the nerve. The resulting CAP was amplified from
the recording electrode using a high impedance low-noise amplifier
(EG&G Princeton Applied Research Corporation) and filtered and
sampled at 10-100 kHz. To assess the effects of the 4-AP
derivatives on the CAP, the drug under study was added in
incremental concentrations to the recording chamber after the CAP
was allowed to stabilize for 5 min while pulsing repeatedly. After
each measurement the chamber was washed for 5 min (flow 1 mL/min)
with oxygenated aCSF. The study was conducted at
22.2.+-.1.3.degree. C. to allow for slower conduction and the
temperature was monitored throughout the experiment. CAP recordings
were acquired with a SBC6711 board (Innovative Integration)
controlled by in-house written software. Analysis of the traces was
done using an in-house software. The half-maximal effective
concentration (EC.sub.50) for each drug was calculated by plotting
the final over initial amplitude vs. concentration and fitted to
the Hill equation using Origin.
[0258] This experiment shows the typical differences between
normally myelinated nerves and hypomyelinated nerves. The
Shiverer's hypomyelinated nerves conducted much slower (average
conduction velocity 0.59.+-.0.10 m/s vs. 1.4.+-.0.3 m/s at
22.degree. C.), had a smaller CAP amplitude (20-30% compared to
myelinated nerves) and showed a larger undershoot than control
nerves. Addition of 4-AP to normally myelinated nerves caused small
increases in CAP amplitude of around 5% and broadening of the
signal, whereas addition of 4-AP to hypomyelinated nerves caused
large increases in CAP amplitude of 2-4 fold generating a CAP that,
although delayed, almost looked like a normally myelinated nerve
(FIG. 4A). As for the effect of the different derivatives, 4-AP and
3-F-4-AP were found to be the most potent in enhancing the CAP with
half-maximal effective concentrations (EC.sub.50) of 59.2 .mu.M
(95% C.I. 43-81 .mu.M) and 96 .mu.M (95% C.I. 29-323 .mu.M)
respectively. The derivatives 3-MeOH-4-AP and 3-MeF-4AP were around
4-6 times less potent with EC.sub.50's around 390 .mu.M
(3-MeOH-4-AP: IC.sub.50=386 .mu.M, 95% C.I.=295-505 .mu.M;
3-MeF-4-AP: IC.sub.50=286 .mu.M, 95% C.I.=234-648 .mu.M). As
expected, 2-F-4AP had no effect demonstrating that the observed
effects with the other derivatives are specific. 3-EtF-4-AP was not
included in this experiment since it was already found to be
inactive by voltage clamp. The trend observed in this experiment
was consistent with what was observed in the voltage-clamp
experiment indicating that the increase in CAP is due to blockage
of voltage-gated K.sup.+ channels. Interestingly, the EC.sub.50
values calculated from this experiment were significantly lower
than what was measured using voltage-clamp.
Example 12. Pharmacology of 3-F-4-AP and 4-AP
[0259] The permeability of 3-F-4-AP and 4-AP to an artificial
membrane made of porcine brain polar lipids was tested. In this
experiment, the inventors included highly permeable verapamil and
lowly permeable theophylline as controls. The inventors found
3-F-4-AP to be 6.6-times more permeable than 4-AP (Pe: 15.6.+-.0.6
nm/s vs. 2.36.+-.0.03 nm/s, FIG. 6A). This value correlates well
with the predicted partition coefficients in octanol/water for
these drugs (cLogP: 0.26 vs. 0.03, Pearson r=0.997, P
value=0.0033).
[0260] The stability of these drugs in mouse plasma and mouse liver
microsomes was also tested. Liver microsomes contain large amounts
of cytochrome P450 and can be used to estimate the metabolic
stability of drugs. In this experiment, highly stable verapamil and
lowly stable propanolol were included as controls. Both drugs were
found to be stable in plasma (>93% remaining after 1 h) and
3-F-4-AP was found to be 2.7-times more stable than 4-AP in
microsomes (4-AP: t.sub.1/2=53.+-.10 min; 3-F-4-AP:
t.sub.1/2=144.+-.11 min) (FIG. 6A).
[0261] Pharmacokinetic profiling of 4-AP and 3-F-4-AP in mice after
a single intravenous dose of the drugs at 0.75 mg/kg (FIG. 6B) was
also performed. In this experiment, it was found that 4-AP and
3-F-4-AP have a short half-life in plasma (0.33 h and 0.34 h,
respectively) and a moderate half-life in brain (1.9 h and 1.43 h,
respectively). Interestingly, it was observed that 3-F-4-AP reaches
a significantly higher concentration in the brain indicated by the
ratio between the maximum dose in the brain over the maximum dose
in plasma (C.sub.brain/C.sub.plasma: 0.214+0.17 vs. 0.10+0.05).
This result is consistent with the previous experiment which showed
that 3-F-4-AP can diffuse faster across hydrophobic membranes.
Taken together these experiments demonstrate that 3-F-4-AP has
better stability and better brain permeability than 4-AP.
[0262] In Vivo Effects:
[0263] 10-week-old female C57Bl/6J mice were given an
intraperitoneal injection of the drug under investigation and
monitored continuously for 4 h. After 4 h no signs of drug effects
could be observed. At least 72 h passed between injections to the
same mice.
[0264] Parallel Artificial Membrane Permeability Assay (PAMPA):
[0265] Permeability studies were performed as previously described
by Sugano, et al., 2011. A 96-well microplate (acceptor
compartment) was filled with PBS containing 5% DMSO. A hydrophobic
filter plate (donor compartment) was placed atop the buffer-filled
plate and the filter surface was impregnated with 5 .mu.L solution
of porcine polar brain lipids (Avanti Lipids) in dodecane (1% w/v).
150 .mu.L of the test compounds dissolved in PBS containing 5% DMSO
(compound concentration 0.5 mM) was added to the donor compartment
and covered. The only barrier between the two compartments was the
artificial BBB membrane containing the porcine polar brain lipids.
The whole system was incubated for several hours. Time of
incubation was chosen considering cLogP of tested compounds (4 h
for Verapamil and 16 h for 4-aminopyridine,
3-fluoro-4-aminopyridine and theophylline). Samples from the
acceptor compartment were analyzed by UV-VIS spectrophotometry
(4-aminopyridine: 260 nm, 3-fluoro-4-aminopyridine: 265 nm) and
compared to reference solutions.
[0266] Stability in Plasma:
[0267] Plasma stability was conducted by incubating each compound
at initial concentration of 1 .mu.M in mouse plasma for 60 minutes.
Samples were collected at 0, 20, 40 and 60 minutes and the reaction
was stopped by addition of 1 vol. of acetonitrile. The loss of
compound was determined using LC-MS comparing the peak area at
several time points. Half-life time was calculated from linear
regression of time course data.
[0268] Stability in Microsomes:
[0269] study compounds were incubated at initial concentration of 1
.mu.M with liver microsomes from CD1 mouse (0.04 mg/mL) in PBS in
the presence or absence of enzyme cofactors (1.3 mM NADP.sup.+, 3.3
mM MgCl.sub.2, 3.3 mM G6P and 1 U/ml G6PDH, 1 mM UDPGA and 4.7
.mu.g/ml Alamethicin). After t=0, 20, 40 and 60 min, a sample was
removed and the reaction was stopped by adding 1 volume of
acetonitrile. The loss of compound was determined by LC-MS analysis
comparing the amount of compound in the sample to the respective
reference samples (without cofactors). To ensure that the assay is
reliable, Propanolol and Verapamil were included as control
compounds. The results were normalized for reaction volume and
protein concentration.
[0270] LC-MS: The following conditions were used for LC-MS
analysis. Solvent A: Water (0.1% Formic acid). Solvent B:
Acetonitrile (0.1% Formic acid). Flow rate: 0.5 ml/min. Gradient
conditions: 0.0-0.5 min 95% B, 0.5-6.0 min 5% B, 6.0-6.5 min 5% B,
6.5-6.6 min 95% B, 6.6-7.5 min 95% B. Running time: 7.5 min.
Injection volume: 40 .mu.l. Column: Luna HILIC, 150.times.4.6 mm, 3
.mu.m. Ionization mode: ESI positive. MS mode: Multiple Reaction
Monitoring (MRM). Capillary voltage: 4500 V. Nebuliser gas: 40 psi.
Dry gas: 9 L/min. Dry Temperature: 300.degree. C. HPLC and MS/MS
parameters: 4-aminopyridine retention time 3.5 min, ion product
94.9. 3-fluoro-4-aminopyridine retention time 4.0 min, ion
product=112.9.
[0271] Pharmacokinetic Study:
[0272] 39 mice (CD-1, 6-weeks old, female) were used in the study.
18 mice were administered 4-aminopyridine, 18 mice were
administered 3-fluoro-4-aminopyridine and 3 were left untreated.
The drugs were dissolved in PBS and administered via tail-vein
injection to achieve a dose of 0.75 mg/kg of body weight. At
specific times post injection (10 min, 30 min, 1 h, 2 h, 4 h and 24
h) blood and brain samples were collected. Blood samples were
transferred into tubes containing 5% EDTA, stored on ice, and
centrifuged (4.degree. C., 1000 rpm, 15 min). Plasma (upper phase)
was transferred to a new tube and stored at -80.degree. C. for
further analysis. Brain tissue samples were collected after
intracardial perfusion of the mouse. Brain tissue samples were
stored at -80.degree. C. for further analysis.
[0273] Drug Quantification in Plasma:
[0274] Briefly, to a 1.5 mL Eppendorf tube containing 50 .mu.l of
plasma, 200 .mu.l of ice-cold acetonitrile containing 1,000 ng/ml
Progesterone (used as internal standard) was added in order to
precipitate the proteins. The sample was vortexed, mixed and
centrifuged at 4000.times.g for 10 min at 4.degree. C. to remove
precipitates. 140 .mu.l supernatant was collected and transferred
to a 500 .mu.l 96-wel polypropylene plate and covered using
silicone plate mat. 40 .mu.l of sample was injected in into LC-MS.
Using the same sample preparation procedure 11 standard solutions
ranging from 50 ng/mL to 100 .mu.g/mL were prepared, analyzed by
LC-MS and used as a calibration curve to correlate peak area of the
samples to concentration.
[0275] Drug Quantification in Brain:
[0276] the mouse brain was weighted and 1 mL of water per 400 mg of
tissue was added. The sample was homogenized using an electric
tissue homogenizer. To a 1.5 mL Eppendorf tube containing 50 .mu.l
of homogenized sample, 200 .mu.l of ice-cold acetonitrile
containing 1000 ng/ml Demeclocycline (used as internal standard)
was added in order to precipitate the proteins. The sample was
vortexed, mixed and centrifuged at 4000.times.g for 10 min at
4.degree. C. to remove precipitates. 140 .mu.l supernatant was
collected and transferred to a 500 .mu.l 96-well polypropylene
plate and covered using silicone plate mat. 40 .mu.l of each sample
was injected into LC-MS. Using the same sample preparation
procedure 11 standard solutions ranging from 50 ng/mL to 100 g/mL
were prepared, analyzed by LC-MS and used as a calibration curve to
correlate peak area of the samples to concentration.
[0277] Data Analysis:
[0278] The Hill equation used to fit the data from the
voltage-clamp and the optic nerve experiments was as follows:
y=y.sub.0+(y.sub.f-y.sub.0)*x.sup.n/(k.sup.n+x.sup.n); where n
refers to the Hill coefficient (typically 1.+-.0.1) and k refers to
EC.sub.50 or IC.sub.50. y.sub.0 and y.sub.f refers to the origin
and final ordinate values and were fixed at 1.+-.0.1 or 0.+-.0.1
depending on the experiment. EC.sub.50=10.sup.<log EC50>;
where <log EC.sub.50>=average of log EC.sub.50 values from
all experiments with the same drug. 95% C.I.=[Upper Limit . . .
Lower Limit]; where U.L.=10.sup.(log EC50+s.d.) and
L.L.=10.sup.(log EC50-s.d.). The half-life (t.sub.1/2) in the
stability and pharmacokinetic experiments was calculated by fitting
the data to the equation C.sub.t=C.sub.0*exp(-k*t) where
C.sub.0=initial concentration, C.sub.t=concentration at time t, and
k=ln 2/t.sub.1/2. Student t-test was used to compare results and
P<0.05 was considered significant.
Example 13. Evaluation 4-AP Distribution in Partially Demyelinated
Brains Using Autoradiography
[0279] The inventors conducted an experiment to evaluate if 4-AP
selectively localizes in demyelinated (injured) areas. In this
experiment, tritium labeled 4-aminopyridine ([.sup.3H] 4-AP) was
injected into mice containing demyelinating lesions in the brain.
The lesions were caused by prior injection of
lysophosphatidylcholine (LPC), also called lysolecithin, into their
brains, which causes focal demyelination at the site of injection.
Two days after LPC injection, at the peak of demyelination, the
animals were injected with [.sup.3H] 4-AP (0.4 .mu.Ci/g) via tail
vein injection. Twenty to sixty minutes after injection of
[.sup.3H] 4-AP the mice were euthanized and their brains were
dissected and frozen. The frozen brains were then cut into 20 .mu.m
sections using a cryostat and the sections mounted in slides. The
slides were then exposed to radiation sensitive X-ray film at
-80.degree. C. in the dark for forty days to capture the
distribution of radioactivity coming from [.sup.3H] 4-AP throughout
the brain.
[0280] After autoradiographic exposure, the slides were stained for
myelin basic protein (MBP) using immunohistochemistry and imaged
using fluorescent microscopy to determine the areas of
demyelination. The results of this experiment are shown in FIGS.
7A-E.
[0281] In this experiment, partial demyelination in distinct areas
on the right side of the corpus callosum where lysolecithin was
injected (FIG. 7C, circled in red) were seen. In those areas, the
autoradiographic signal appears darker than the rest of the corpus
callosum (FIG. 7D). The inventors quantified the signal in those
areas and observed a statistically significant increase in signal
in demyelinated areas (FIG. 7E).
[0282] This experiment demonstrates that 4-AP selectively localizes
to grey matter areas and there's virtually no 4-AP in white matter
areas. It was also observed that demyelination of white matter
areas causes a local increase in the autoradiographic signal
indicating that 4-AP localizes to demyelinated areas but not
myelinated areas. The conclusion is that 4-AP does not bind to
white matter areas unless there is demyelination.
[0283] It was shown that fluorinated 4-APs can block Shaker K.sup.+
channel similar to 4-AP, that fluorinated 4-APs can enhance
compound action potential of dysmyelinated optic nerves but have
very little effect on normally myelinated optic nerves, and that
fluorinated 4-APs have very similar in vivo effects as 4-AP. It was
also shown that fluorinated 4-APs have enhanced permeability to the
CNS relative to 4-AP. As 4-AP localizes to demyelinated areas and
fluorinated 4-APs have very similar biological activity to 4-AP, it
can be inferred that fluorinated 4-APs also localizes to
demyelinated lesions. As fluorinated molecules can be used as PET
tracers simply by exchanging the natural isotope of fluorine
(.sup.19F) for the positron emitting isotope .sup.18F and this
exchange does not alter the biological properties of the molecule,
the evidence supports an inference that .sup.18F-labeled 4-APs can
serve as PET tracers for demyelination.
Example 14 (Prophetic). Distribution of [.sup.14C] 3-F-4-AP in
Partially Demyelinated Rat Brain and Spinal Cord Containing
Demyelinated Lesions Using Autoradiography
[0284] Rats will be injected with LPC in the brain and spinal cord
to create focal demyelinated areas at the sites of injection. 1-6
days after LPC injection, the rats will be injected intravenously
with [.sup.14C] 3-F-4-AP (0.5 mg/kg, 0.5 uCi/g). 20-90 min after
[.sup.14C] 3-F-4-AP injection, the rats will be euthanized and
their brains removed. Thin sections of the brain will be prepared
using a cryostat and mounted into glass slides. The slides will be
then exposed to a radiation sensitive film for up to 6 weeks. After
exposure, the film will be developed and the slides will be
processed for IHC. The distribution of the drug on the brain
revealed by autoradiographic signal and compared with the
distribution of myelin revealed by IHC. In some experiments, other
.sup.14C labeled fluorinated derivatives of 4-AP are used. In some
experiments, .sup.3H labeled fluorinated derivatives of 4-AP are
used. In some embodiments, different rodent models of demyelination
are used. In some embodiments, different species may be used.
Example 15 (Prophetic). Distribution of [.sup.18F] 3-F-4-AP in
Partially Demyelinated Rat Brain and Spinal Cord Using Pet
Scanner
[0285] The inventors will inject 0.005-50 mCi of [.sup.18F]
3-F-4-AP or other .sup.18F-labeled 4-AP derivative into LPC treated
rats. Immediately after injection of the tracer, dynamic emission
scan will be performed in 3D acquisition mode on the animal using a
GMI microPET/SPECT/CT system. In order to quantify the images, the
signal will be integrated in the lesion and compare it to the
signal in the same area in a control animal. The results will be
analyzed using statistical tests. In some experiments, other
.sup.11C labeled fluorinated derivatives of 4-AP are used. In some
embodiments, different rodent models of demyelination are used. In
some embodiments, different species may be used. In some
embodiments, the species will be humans.
Example 16. (Prophetic) 4-AP Preferentially Localizes to TBI
Lesions Using Autoradiography
[0286] 4-AP has a higher uptake in demyelinated (injured) white
matter areas than in the rest of the white matter. In order to
determine if 4-AP localizes to areas of the brain affected by a
traumatic brain injury, the inventors will conduct a similar
experiment in a rat model of TBI (FIG. 9A). The inventors will
induce traumatic brain injuries in rats using the controlled impact
model. Afterwards, the animals will be injected with
.sup.14C-labeled 4-AP (0.1-0.5 .mu.Ci/g). The animals will be
euthanized 30 to 90 min later and their brains removed. Thin
sections of their brains will be prepared and the localization of
the drug determined using autoradiography (FIG. 9B). Subsequently
the lesioned areas will be examined by immunohistochemistry to
identify the traumatic injured areas. The autoradiographic signal
corresponding to those regions will be quantified and the results
evaluated using analysis of variance (ANOVA).
[0287] We also plan to perform a similar experiment using
[.sup.14C] 2-deoxy-d-glucose (2-DG). 2-DG has a similar
distribution pattern in the brain (higher uptake in grey matter
areas than white matter areas). However, 2-DG it is not expected to
accumulate in injured areas of the brain to a significant degree.
This experiment will serve as a control and provide a preview of
the comparison of [.sup.18F]-labeled 4-AP with [.sup.18F] FDG using
PET.
Example 17 (Prophetic)--Compare the Distribution of 3-F-4-AP and
3-MeF-4-AP in the Brain of Animals with Demyelinating Lesions
[0288] Using the same autoradiographic technique we will evaluate
the brain distribution of [.sup.14C] 3-F-4-AP and [.sup.14C]
3-MeF-4-AP. These will be the molecules used for PET imaging. Both
of these molecules have similar affinity to K+ channels as 4-AP and
better brain permeability. This experiment will allow us to
identify the best candidate for imaging. In addition, we will
include [.sup.14C] 2-F-4-AP which has the same molecular weight as
[.sup.14C] 3-F-4-AP and very similar brain permeability but does
not bind to K.sup.+ channels to control for non-specific
localization of this type of molecules.
[0289] Similar as with 4-AP, we will perform a dose-response and
preblocking experiments in control rats to determine the best
conditions for imaging. Once we optimize the conditions we will
perform the experiments in lysolecithin-injected rats. Based on our
statistical calculation, we estimate that in this experiment we
will need around 6 rats per group. Similar as before, the results
will be analyzed using ANOVA.
Example 18 (Prophetic)--Synthetic Methodology for [.sup.18F]
3-F-4-AP and [.sup.18F] 3-MeF-4-AP
[0290] Fluorine-18 is the most appropriate radionuclide for PET as
its low positron energy allows for sharper resolution, and its
longer half-life (109.8 min vs. 20 min for .sup.11C) allows for
off-site production and commercialization. In order to be able to
use fluorinated 4-APs as PET tracers, a quick radiolabeling
strategy will be necessary (.sup.18F half-life: 109 min). See FIG.
8.
[0291] The proposed synthesis of [.sup.18F] 3-F-4-AP makes use of
the recently developed method of using iodonium salts for high
efficiency synthesis of aryl fluorides (Chun, et al., 2012). In
comparison, [.sup.18F] 3-MeF-4-AP possesses an aliphatic fluoride
that it is expected to be facile to synthesize.
Example 19 (Prophetic)--Pet Imaging in Small Animal Models of
TBI
[0292] Immediately after synthesizing the .sup.18F-labeled
compounds, a dynamic emission scan will be performed in 3D
acquisition mode in the TBI induced animals using a GMI
microPET/SPECT/CT system. The resolution of the microPET scanner is
limited to .about.1 mm. In addition, a post-mortem autoradiography
will be conducted after the scan to further verify the localization
of the tracer.
[0293] All of the methods and apparatuses disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill i