Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR THE TREATMENT OF
ANXIETY DISORDERS
Reference to Related Applications
This application claims priority to: U.S. Patent Application No. 11/251,724
filed
October 17, 2005; U.S. Patent Application No. 11/130,945, filed May 17, 2005;
and U.S.
Patent Application No. 11/101,000, filed April 7, 2005.
Technical Field of the Invention .
The present invention relates to methods and compositions for treating
selected
conditions of the central and peripheral nervous systems employing non-
synaptic
mechanisms. More specifically, the present invention relates to methods and
compositions for treating neuropsychiatric disorders by administering agents
that
modulate expression and/or activity of sodium-potassium-chloride co-
transporters.
Background of the Invention
Neuropathic pain and nociceptive pain differ in their etiology,
pathophysiology,
diagnosis and treatment. Nociceptive pain occurs in response to the activation
of a
specific subset of peripheral sensory neurons, the nociceptors. It is
generally acute (with
the exception of arthritic pain), self-limiting and serves a protective
biological function by
acting as a warning of on-going tissue damage. It is typically well localized
and often has
an aching or throbbing quality. Examples of nociceptive pain include post-
operative pain,
sprains, bone fractures, burns, bumps, bruises, inflammation (from an
infection or
arthritic disorder), obstructions and myofascial pain. Nociceptive pain can
usually be
treated with opioids and non-steroidal anti-inflammatory drugs (NSAIDS).
Neuropathic pain is a common type of chronic, non-malignant, pain, which is
the
result of an injury or malfunction in the peripheral or central nervous system
and serves
no protective biological function. It is estimated to affect more than 1.6
million people in
the U.S. population. Neuropathic pain has many different etiologies, and may
occur, for
example, due to trauma, diabetes, infection with herpes zoster (shingles),
HIV/AIDS
peripheral neuropathies, late-stage cancer, amputation (including mastectomy),
carpal
tunnel syndrome, chronic alcohol use, exposure to radiation, and as an
unintended side-
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effect of neurotoxic treatment agents, such as certain anti-HIV and
chemotherapeutic
drugs.
In contrast to nociceptive pain, neuropathic pain is frequently described as
"burning", "electric", "tingling" or "shooting" in nature. It is often
characterized by
chronic allodynia (defined as pain resulting from a stimulus that does not
ordinarily elicit
a painful response, such as light touch) and hyperalgesia (defined as an
increased
sensitivity to a normally painful stimulus), and may persist for months or
years beyond
the apparent healing of any damaged tissues.
Neuropathic pain is difficult to treat. Analgesic drugs that are effective
against
normal pain (e.g., opioid narcotics and non-steroidal anti-inflammatory drugs)
are rarely
effective against neuropathic pain. Similarly, drugs that have activity in
neuropathic pain
are not usually effective against nociceptive pain. The standard drugs that
have been used
to treat neuropathic pain appear to often act selectively to relieve certain
symptoms but
not others in a given patient (for example, relief of allodynia, but not
hyperalgesia). For
this reason, it has been suggested that successful therapy may require the use
of multiple
different combinations of drugs and individualized therapy (see, for example,
Bennett,
Hosp. Pract. (Off Ed). 33:95-98, 1998). Treatment agents typically employed in
the
management of neuropathic pain include tricylic antidepressants (for example,
amitriptyline, imipramine, desimipramine and clomipramine), systemic local
anesthetics,
and anti-convulsants (such as phenytoin, carbamazepine, valproic acid,
clonazepam and
gabapentin).
Many anti-convulsants originally developed for the treatment of epilepsy and
other seizure disorders have found application in the treatment of non-
epileptic
conditions, including neuropathic pain, mood disorders (such as bipolar
affective
disorder), and schizophrenia (for a review of the use of anti-epileptic drugs
in the
treatment of non-epileptic conditions, see Rogawski and Loscher, Nat.
Medicine, 10:685-
692, 2004). It has thus been suggested that epilepsy, neuropathic pain and
affective
disorders have a common pathophysiological mechanism (Rogawski & Loscher,
ibid;
Ruscheweyh & Sandkuhler, Pain 105:327-338, 2003), namely a pathological
increase in
neuronal excitability, with a corresponding inappropriately high frequency of
spontaneous
firing of neurons. However, only some, and not all, antiepileptic drugs are
effective in
treating neuropathic pain, and furthermore such antiepileptic drugs are only
effective in
certain subsets of patients with neuropathic pain (McCleane, Expert. Opin.
Pharmacother. 5:1299-1312, 2004).
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Epilepsy is characterized by abnormal discharges of cerebral neurons and is
typically manifested as various types of seizures. Epileptiform activity is
identified with
spontaneously occurring synchronized discharges of neuronal populations that
can be
measured using electrophysiological techniques. This synchronized activity,
which
distinguishes epileptiform from non-epileptiform activity, is referred to as
"hypersynchronization" because it describes the state in which individual
neurons become
increasingly likely to discharge in a time-locked manner with one another.
Hypersynchronized activity is typically induced in experimental models of
epilepsy by
either increasing excitatory or decreasing inhibitory synaptic currents, and
it was
therefore assumed that hyperexcitability per se was the defining feature
involved in the
generation and maintenance of epileptiform activity. Similarly, neuropathic
pain was
believed to involve conversion of neurons involved in pain transmission from a
state of
normal sensitivity to one of hypersensitivity (Costigan & Woolf, Jnl. Pain
1:35-44,
2000). The focus on developing treatments for both epilepsy and neuropathic
pain has
thus been on suppressing neuronal hyperexcitability by either: (a) suppressing
action
potential generation; (b) increasing inhibitory synaptic transmission; or (c)
decreasing
excitatory synaptic transmission. However, it has been shown that
hypersychronous
epileptiform activity can be dissociated from hyperexcitability and that the
cation chloride
cotransport inhibitor furosemide reversibly blocked synchronized discharges
without
reducing hyperexcited synaptic responses (Hochman et al. Science 270:99-102,
1995).
Both abnormal expression of sodium channel genes (Waxman, Pain 6:S133-140,
1999; Waxman et al. Proc. Natl. Acad. Sci USA 96:7635-7639, 1999) and
pacemaker
channels (Chaplan et al. J. Neurosci. 23:1169-1178, 2003) are believed to play
a role in
the molecular basis of neuropathic pain.
Neuropsychiatric disorders, including anxiety disorders, are generally treated
by
counseling and/or with drugs. Many of the drugs currently employed in the
treatment of
such disorders have significant negative side effects, such as tendencies to
induce
dependence.
The cation-chloride co-transporters (CCCs) are important regulators of
neuronal
chloride concentration that are believed to influence cell-to-cell
communication, and
various aspects of neuronal development, plasticity and trauma. The CCC gene
family
consists of three broad groups: Na+-C1- co-transporters (NCCs), K+-Cl- co-
transporters
(KCCs) and Na+-K+-2C1" co-transporters (NKCCs). Two NKCC isoforms have been
identified: NKCC 1 is found in a wide variety of secretory epithelia and non-
epithelial
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cells, whereas NKCC2 is principally expressed in the kidney. For a review of
NKCC I
structure, function and regulation see, Haas and Forbush, Annu. Rev. Physiol.
62:515-534,
2000. Randall et al. have identified two splice variants of the Slc12a2 gene
that encodes
NKCC 1, referred to as NKCC 1 a and NKCC 1 b(Am. J Physiol. 273 (Cell Physiol.
42):C1267-1277, 1997). The NKCC1 a gene has 27 exons, while the splice variant
NKCCIb lacks exon 21. The NKCCIb splice variant is expressed primarily in the
brain.
NKCC 1 b is believed to be more than 10% more active than NKCC 1 a, although
it is
proportionally present in a much smaller amount in the brain than is NKCCIa.
It has
been suggested that differential splicing of the NKCC 1 transcript may play a
regulatory
role in human tissues (Vibat et al. Anal. Biochem. 298:218-230, 2001). Na-K-Cl
co-
transport in all cell and tissues is inhibited by loop diuretics, including
furosemide,
bumetanide and benzmetanide.
Na-K-2C1 co-transporter knock-out mice have been shown to have impaired
nociception phenotypes as well as abnormal gait and locomotion (Sung et al.
Jnl.
Neurosci. 20:7531-7538, 2000). Delpire and Mount have suggested that NKCCI may
be
involved in pain perception (Ann. Rev. Physiol. 64:803-843, 2002). Laird et
al. recently
described studies demonstrating reduced stroking hyperalgesia in NKCC 1 knock-
out
mice compared to wild-type and heterozygous mice (Neurosci. Letts. 361:200-
203, 2004).
However, in this acute pain model no difference in punctuate hyperalgesia was
observed
between the three groups of mice. Morales-Aza et al. have suggested that, in
arthritis,
altered expression of NKCCI and the K-Cl co-transporter KCC2 may contribute to
the
control of spinal cord excitability and may thus represent therapeutic targets
for the
treatment of inflammatory pain (Neurobiol. Dis. 17:62-69, 2004). Granados-Soto
et al.
have described studies in rats in which formalin-induced nociception was
reduced by
administration of the NKCC inhibitors bumetanide, furosemide or piretanide
(Pain
114:231-238, 2005). While the formalin-induced acute pain model is extensively
used, it
is believed to have little relevance to chronic pain conditions (Walker et al.
Mol. Med.
Today 5:319-321, 1999). Co-treatment of brain damage induced by episodic
alcohol
exposure with an NMDA receptor antagonist, non-NMDA receptor and CaZ+ channel
antagonists together with furosemide has been shown to reduce alcohol-
dependent
cerebrocortical damage by 75-85%, while preventing brain hydration and
electrolyte
elevations (Collins et al, FASEB J., 12:221-230, 1998). The authors stated
that the results
suggest that furosemide and related agents might be useful as neuroprotective
agents in
alcohol abuse. Willis et al. have published studies indicating that nedocromil
sodium,
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furosemide and bumetanide inhibit sensory nerve activation to reduce the itch
and flare
responses induced by histamine in human skin in vivo. Espinosa et al. and
Ahmad et al.
have previously suggested that furosemide might be useful in the treatment of
certain
types of epilepsy (Medicina Espanola 61:280-281, 1969; and Brit. J. Clin.
Pharmacol.
3:621-625, 1976).
As with epilepsy, the focus of pharmacological intervention in neuropathic
pain
has been on reducing neuronal hyperexcitability. Most agents currently used to
treat
neuropathic pain target synaptic activity in excitatory pathways by, for
example,
modulating the release or activity of excitatory neurotransmitters,
potentiating inhibitory
pathways, blocking ion channels involved in impulse generation, and/or acting
as
membrane stabilizers. Conventional agents and therapeutic approaches for the
treatment
of neuropathic pain and neuropsychiatric disorders thus reduce neuronal
excitability and
inhibit synaptic firing. One serious drawback of these therapies is that they
are
nonselective and exert their actions on both normal and abnormal neuronal
populations.
This leads to negative and unintended side effects, which may affect normal
CNS
functions, such as cognition, learning and memory, and produce adverse
physiological
and psychological effects in the treated patient. Common side effects include
over-
sedation, dizziness, loss of memory and liver damage. There is therefore a
continuing
need for methods and compositions for treating neuronal disorders that disrupt
hypersynchronized neuronal activity without diminishing the neuronal
excitability and
spontaneous synchronization required for normal functioning of the peripheral
and central
nervous systems.
Summary of the Invention
The treatment compositions and methods of the present invention are useful for
treating conditions including neuropathic pain and neuropsychiatric disorders,
such as
bipolar disorders, anxiety disorders (including panic disorder, social anxiety
disorder,
obsessive compulsive disorder, posttraumatic stress disorder, generalized
anxiety disorder
and specific phobia (American Psychiatric Association, Diagnostic and
Statistical Manual
of Mental Disorders, 4'f' edition - Text Revision, 2000)), depression and
schizophrenia, ,
that are characterized by neuronal hypersynchrony. The inventive compositions
and
methods may be employed to reduce neuronal hypersynchrony associated with
neuropathic pain and/or neuropsychiatric disorders without suppressing
neuronal
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excitability, thereby avoiding the unwanted side effects often associated with
agents
currently employed for the treatment of neuropathic pain and neuropsychiatric
disorders.
The methods and compositions disclosed herein generally involve via non-
synaptic mechanisms and modulate, generally reduce, the synchronization of
neuronal
population activity. The synchronization of neuronal population activity is
modulated by
manipulating anionic concentrations and gradients in the central and/or
peripheral
nervous systems. More specifically, the inventive compositions are capable of
reducing
the effective amount, inactivating, and/or inhibiting the activity of a Na+-K+-
2Cl" (NKCC)
co-transporter. Especially preferred treatment agents of the present
invention, exhibit a
high degree of NKCC co-transporter antagonist activity in cells of the central
and/or
peripheral nervous system, e.g., glial cells, Schwann cells and/or neuronal
cell
populations, and exhibit a lesser degree of activity in renal cell
populations. In one
embodiment, the inventive compositions are, capable of reducing the effective
amount,
inactivating, and/or inhibiting the activity of the co-transporter NKCC1.
NKCC1
antagonists are especially preferred treatment agents for use in the inventive
methods.
NKCC co-transporter antagonists that may be usefully employed in the inventive
treatment compositions include, but are not limited to, CNS-targeted NKCC co-
transporter antagonists such as furosemide, bumetanide, ethacrynic acid,
torsemide,
azosemide, muzolimine, piretanide, tripamide and the like, as well as thiazide
and
thiazide-like diuretics, such as bendroflumethiazide, benzthiazide,
chlorothiazide,.
hydrochlorothiazide, hydroflumethiazide, methylclothiazide, polythiazide,
trichlormethiazide, chlorthalidone, indapamide, metolazone and quinethazone,
together
with analogs and functional derivatives of such components.
Analogs of CNS-targeted NKCC co-transporter antagonists such as furosemide,
bumetanide, piretanide, azosemide and torsemide that may be usefully employed
in the
inventive compositions and methods include those provided below as Formulas I-
V. The
inventors believe that such analogs have increased lipophilicity and reduced
diuretic
effects compared to the CNS-targeted NKCC co-transporter antagonists from
which they
are derived and thus result in fewer undesirable side effects when employed in
the
inventive treatment methods.
In one embodiment, the level of diuresis that occurs following administration
of
an effective amount of an analog provided below as Formula I-V, is less than
99%, 90%,
80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of that which occurs following
administration of an effective amount of the parent molecule from which the
analog is
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derived. For example, the analog may be less diuretic than the parent molecule
when
administered at the same mg/kg dose. Alternatively, the analog may be more
potent than
the parent molecule from which it is derived, so that a smaller dose of the
analog is
required for effective relief of symptoms, thereby eliciting less of a
diuretic effect.
Similarly, the analog may have a longer duration of effect in treating
disorders than the
parent molecule, so that the analog may be administered less frequently than
the parent
molecule, thus leading to a lower total diuretic effect within any given
period of time.
Other treatment agents that may be usefully employed in the inventive
compositions and methods include, but are not limited to: antibodies, or
antigen-binding
fragments thereof, that specifically bind to NKCC 1; soluble NKCCI ligands;
small
molecule inhibitors ofNKCC1; anti-sense oligonucleotides to NKCC1; NKCC1-
specific
small interfering RNA molecules (siRNA or RNAi); and engineered soluble NKCCI
molecules. Preferably, such antibodies, or antigen-binding fragments thereof,
and small
molecule inhibitors of NKCC1 specifically bind to the domains of NKCC1
involved in
bumetanide binding, as described, for example, in Haas and Forbush II, Annu.
Rev.
Physiol. 62:515-534, 2000. The polypeptide sequence for human NKCC 1 is
provided in
SEQ ID NO: 1, with the corresponding cDNA sequence being provided in SEQ ID
NO:
2.
As the methods and treatment agents of the present invention employ "non-
synaptic" mechanisms, little or no suppression of neuronal excitability
occurs. More
specifically, the inventive treatment agents cause little (less than a 1%
change compared
to pre-administration levels) or no suppression of action potential generation
or excitatory
synaptic transmission. In fact, a slight increase in neuronal excitability may
occur upon
administration of certain of the inventive treatment agents. This is in marked
contrast to
conventional anti-epileptic drugs currently used in the treatment of
neuropathic pain,
which do suppress neuronal excitability. The methods and treatment agents of
the present
invention affect the synchronization, or relative synchrony, of neuronal
population
activity. Preferred methods and treatment agents modulate the extracellular
anionic
chloride concentration and/or the gradients in the central or peripheral
nervous system to
reduce neuronal synchronization, or relative synchrony, without substantially
affecting
neuronal excitability.
In one aspect, the present invention relates to methods and agents for
relieving
neuropathic pain, or the abnormal perception of pain, by affecting or
modulating
spontaneous hypersynchronized bursts of neuronal activity and the propagation
of action
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potentials or conduction of impulses in certain cells and nerve fibers of the
peripheral
nervous system, for example, primary sensory afferent fibers, pain fibers,
dorsal horn
neurons, and supraspinal sensory and pain pathways.
The inventive treatment agents may be employed in combination with other,
known, treatment agents, such as those presently used in the treatment of
neuropsychiatric
l0 disorders. One of skill in the art will appreciate that the combination of
a treatment agent
of the present invention with another, known, treatment agent may involve both
synaptic
and non-synaptic mechanisms.
Treatment compositions and methods of the present invention may be used
therapeutically and episodically following the onset of symptoms or
prophylactically,
prior to the onset of specific symptoms. For example, treatment agents of the
present
invention can be used to treat existing neuropathic pain or to protect nerves
from
neurotoxic injury and neuropathic pain secondary to chemotherapy,
radiotherapy,
exposure to infectious agents, and the like.
In certain embodiments, the treatment agents employed in the inventive methods
are capable of crossing the blood brain barrier, and/or are administered using
delivery
systems that facilitate delivery of the agents to the central nervous system.
For example,
various blood brain barrier (BBB) permeability enhancers can be used, if
desired, to
transiently and reversibly increase the permeability of the blood brain
barrier to a
treatment agent. Such BBB permeability enhancers may include leukotrienes,
bradykinin
agonists, histamine, tight junction disruptors (e.g., zonulin), hyperosmotic
solutions (e.g.,
mannitol), cytoskeletal contracting agents, short chain alkylglycerols (e.g.,
1-0-
pentylglycerol), and others which are currently known in the art.
The above-mentioned and additional features of the present invention, together
with the manner of obtaining them, will be best understood by reference to the
following
more detailed description. All references disclosed herein are hereby
incorporated by
reference in their entirety as if each was incorporated individually.
Brief Description of the Drawings
Figs. IA, 1 A 1, 1 B, 1 B 1, IC, 1 C 1 and 1 D show the effect of furosemide
on
stimulation evoked after discharge activity in rat hippocampal slices.
Figs. 2A - 2R show furosemide blockade of spontaneous epileptiform burst
discharges across a spectrum of in vitro models.
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Figs. 3A - 3H show furosemide blockade of kainic acid-evoked electrical
"status
epilepticus" in urethane-anesthetized rats, with EKG recordings shown in the
upper traces
and cortical EEG recordings shown in the bottom traces.
Figs. 4A and 4B show a schematic diagram of ion co-transport under conditions
of
reduced chloride concentration.
Fig. 5 shows that significantly less freezing was observed in animals treated
with
either bumetanide or furosemide than in animals receiving vehicle alone in a
test of
contextual fear conditioning in rats.
Fig. 6 shows baseline startle amplitudes in a fear potentiated startle test in
rats
Fig. 7 shows the amplitude of response in rats on startle alone trials
determined
immediately following administration of either DMSO alone, bumetanide or
furosemide.
Fig. 8 shows the difference score (startle alone - fear potentiated startle)
on the
test day in rats treated with either DMSO, bumetanide or furosemide
Fig. 9 shows the startle alone amplitude in rats one week after administration
of
either DMSO, bumetanide or furosemide.
Fig. 10 shows the difference score in rats one week after administration of
either
DMSO, bumetanide or furosemide.
Fig. 11 shows the difference score (startle alone - fear potentiated startle)
on the
test day in rats treated with one of the following bumetanide analogs:
bumetanide N,N-
diethylglycolamide ester (referred to as 2MIK053); bumetanide methyl ester
(referred to
as 3MIK054); bumetanide N,N-dimethylglycolamide ester (referred to as 3MIK069-
11);
bumetanide morpholinodethyl ester (referred to as 3MIK066-4); bumetanide
pivaxetil
ester (referred to as 3MIK069-12); bumetanide cyanomethyl ester (referred to
as
3MIK047); bumetanide dibenzylamide (referred to as 3MIK065); and bumetanide 3-
(dimethyl-aminoproply) ester (referred to as 3MIK066-5). The vehicle was DMSO.
Fig. 12 shows the difference score in rats treated with different doses of
bumetanide.
Fig. 13 shows the output of urine in rats following administration of either
bumetanide from two different sources (columns 1 and 5), bumetanide N,N-
diethylglycolamide ester (column 2), bumetanide pivaxetil ester (column 3),
bumetanide
cyanomethyl ester (column 4) of saline (column 6).
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Detailed Description of the Invention
As discussed above, preferred treatment agents and methods of the present
invention, for use in treating neuropathic pain and/or neuropsychiatric
disorders,
modulate or disrupt the synchrony of neuronal population activity in areas of
heightened
synchronization by reducing the activity of NKCC co-transporters. As described
in detail
below and illustrated in the examples, movement of ions and modulation of
ionic
gradients by means of ion-dependent co-transporters, preferably cation-
chloride
dependent co-transporters, is critical to regulation of neuronal
synchronization. Chloride
co-transport function has long been thought to be directed primarily to
movement of
chloride out of cells. The sodium independent transporter, which has been
shown to be
neuronally localized, moves chloride ions out of neurons. Blockade of this
transporter,
such as by administration of the loop diuretic furosemide, leads to
hyperexcitability,
which is the short-term response to cation-chloride co-transporters such as
furosemide.
However, the long-term response to furosemide demonstrates that the inward,
sodium-
dependent movement of chloride ions, mediated by the glial associated Na+-K+-
2C1- co-
transporter NKCCI, plays an active role in blocking neuronal synchronization,
without
affecting excitability and stimulus-evoked cellular activity. Haglund and
Hochman have
demonstrated that furosemide is able to block epileptic activity in humans
while not
affecting normal brain activity (J. Neurophysiol. (Feb. 23, 2005) doi:10.1152/
jn.00944.2004). These results provide support for the belief that the
inventive methods
and compositions may be effectively employed in the treatment of neuropathic
pain
without giving rise to undesirable side effects often seen with conventional
treatments.
As discussed above, the NKCC1 splice variant referred to as NKCCIb is more
active than the NKCCIa variant. A central or peripheral nervous system which
expresses
a few more percentage NKCCIb may thus be more prone to disorders such as
neuropathic
pain and epilepsy. Similarly, a treatment agent that is more specific for
NKCCIb
compared to NKCCIa may be more effective in the treatment of such disorders.
The inventive methods may be used for the treatment and/or prophylaxis of
neuropathic pain having, for example, the following etiologies: alcohol abuse;
diabetes;
eosinophilia-myalgia syndrome; Guillain-Barre syndrome; exposure to heavy
metals such
as arsenic, lead, mercury, and thallium; HIV/AIDS; exposure to anti-HIV/AIDS
drugs;
malignant tumors; medications including amiodarone, aurothioglucose,
cisplatinum,
dapsone, stavudine, zalcitabine, didanosine, disulfiram, FK506, hydralazine,
isoniazid,
metronidazole, nitrofurantoin, paclitaxel, phenytoin and vincristine;
monoclonal
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gammopathies; multiple sclerosis; post-stroke central pain, postherpetic
neuralgia; trauma
including carpal tunnel syndrome, cervical or lumbar radiculopathy, complex
regional
pain syndrome, spinal cord injury and stump pain; trigeminal neuralgia;
vasculitis;
vitamin B6 megadosing; and certain vitamin deficiencies (B12, BI, B6, E).
Neuropsychiatric disorders that may be effectively treated using the inventive
methods
include, but are not limited to, bipolar disorders, anxiety disorders, panic
disorders,
depression, schizophrenia, obsessive-compulsive disorders and post-traumatic
stress
syndrome.
Compositions that may be effectively employed in the inventive methods are
capable of reducing the effective amount, inactivating, and/or inhibiting the
activity of a
Na+-K+-2C1" (NKCC) co-transporter. Preferably such compositions are capable of
reducing the effective amount, inactivating, and/or inhibiting the activity of
the co-
transporter NKCCI. In certain embodiments, the inventive compositions comprise
at
least one treatment agent selected from the group consisting of: antagonists
of NKCC1
(including but not limited to, small molecule inhibitors of NKCC 1,
antibodies, or antigen-
binding fragments thereof, that specifically bind to NKCC1 and soluble NKCCI
ligands);
anti-sense oligonucleotides to NKCCI; NKCCI-specific small interfering RNA
molecules (siRNA or RNAi); and engineered soluble NKCC1 molecules. In
preferred
embodiments, the treatment agent is selected from the group consisting of: CNS-
targeted
NKCC co-transporter antagonists such as furosemide, bumetanide, ethacrynic
acid,
torsemide, azosemide, muzolimine, piretanide, tripamide and the like; thiazide
and
thiazide-like diuretics, such as bendroflumethiazide, benzthiazide,
chlorothiazide,
hydrochlorothiazide, hydro-flumethiazide, methylclothiazide, polythiazide,
trichlormethiazide, chlorthalidone, indapamide, metolazone and quinethazone;
and
analogs and functional derivatives of such components.
Analogs of CNS-targeted NKCC co-transporter antagonists that may be employed
in the inventive methods include compounds according to formula I, II and/or
III:
O R,R2
R5R4NO2S N H3
H
R3
I
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O RjR2
\
I
N O
R5R4NO2S
R3
II
0 RjR2
R5R4NO2S N
R3
III
or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof,
wherein
Ri is not present, H or 0;
R2 is H or when R, is 0, is selected from the group consisting of:
alkylaminodialkyl, alkylaminocarbonyldialkyl, alkyloxycarbonylalkyl,
alkylaldehyde,
alkylketoalkyl, alkylamide, an alkylammonium group, alkylcarboxylic acid,
alkylheteroaryls, alkylhydroxy, a biocompatible polymer such as
alkyloxy(polyalkyloxy)alkylhydroxyl, a polyethylene glycol (PEG), a
polyethylene glycol
ester (PEG ester), a polyethylene glycol ether (PEG ether), methyloxyalkyl,
methyloxyalkaryl, methylthioalkylalkyl and methylthioalkaryl, unsubstituted or
substituted,
and when Ri is not present, R2 is selected from the group consisting of:
hydrogen,
dialkylamino, diarylamino, dialkylaminodialkyl, dialkylcarbonylaminodialkyl,
dialkylesteralkyl, dialkylaldehyde, dialkylketoalkyl, dialkylamido,
dialkylcarboxylic acid,
and dialkylheteroaryls, unsubstituted or substituted;
R3 is selected from the group consisting of: aryl, halo, hydroxy, alkoxy, and
aryloxy, unsubstituted or substituted; and
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R.4 and R5 are each independently selected from the group consisting of:
hydrogen, alkylaminodialkyl, alkylhydroxyaminodiakyl, unsubstituted or
substituted:
In some embodiments of the present invention, the analog can be bumetanide
aldehyde, bumetanide dibenzylamide, bumetanide diethylamide, bumetanide
morpholinoethyl ester, bumetanide 3-(dimethylaminopropyl) ester, bumetanide
N,N-
diethylglycolamide ester, bumetanide dimethylglycolamide ester, bumetanide
pivaxetil
ester, bumetanide methoxy(polyethyleneoxy)õ_i-ethyl ester, bumetanide
benzyltrimethyl-
ammonium salt, and bumetanide cetyltrimethylammonium salt.
According to further embodiments of the present invention, the analog can be
furosemide aldehyde, furosemide ethyl ester, furosemide cyanomethyl ester,
furosemide
benzyl ester, furosemide morpholinoethyl ester, furosemide 3-
(dimethylaminopropyl)
ester, furosemide N,N-diethylglycolamide ester, furosemide dibenzylamide,
furosemide
benzyltrimethylammonium salt, furosemide cetyltrimethylammonium salt,
furosemide
N,N-dimethylglycolamide ester, furosemide methoxy(polyethyleneoxy)õ_~-ethyl
ester,
furosemide pivaxetil ester and furosemide propaxetil ester.
In still further embodiments of the present invention, the analog can be
piretanide
aldehyde, piretanide methyl ester, piretanide cyanomethyl ester, piretanide
benzyl ester,
piretanide morpholinoethyl ester, piretanide 3-(dimethylaminopropyl) ester,
piretanide
N,N-diethylglycolamide ester, piretanide diethylamide, piretanide
dibenzylamide,
piretanide benzylltrimethylammonium salt, piretanide cetylltrimethylammonium
salt,
piretanide N,N-dimethylglycolamide ester, piretanide
methoxy(polyethyleneoxy),,_i-ethyl
ester, piretanide pivaxetil ester and/or piretanide propaxetil ester.
Analogs of CNS-targeted NKCC co-transporter anatagonists that may be usefully
employed in the methods of the present invention further include compounds
according to
formula IV:
N=N
R6
H
N S
R5R4NO2S
R3
IV
13
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WO 2006/110187 PCT/US2005/043177
or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof,
wherein
R3, R4 and R5 are defined above; and
R6 is selected from the group consisting of: alkyloxycarbonylalkyl,
alkylaminocarbonyldialkyl, alkylaminodialkyl, alkylhydroxy, a biocompatible
polymer
such as alkyloxy(polyalkyloxy)alkylhydroxyl, a polyethylene glycol (PEG), a
polyethylene glycol ester (PEG ester), a polyethylene glycol ether (PEG
ether),
methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl,
unsubstituted
or substituted.
In some embodiments of the present invention, the analog may be selected from
the group consisting of: tetrazolyl-substituted azosemides (such as
methoxymethyl
tetrazolyl-substituted azosemides, methylthiomethyl tetrazolyl-substituted
azosemides
and N-mPEG350-tetrazolyl-substituted azosemides), azosemide
benzyltrimethylammonium salt and/or azosemide cetyltrimethylammonium salt.
Analogs that may usefully be employed in the inventive methods further include
compounds according to formula V:
R7
I
N+ X'
SOZ ",
HN HN ~ CH3
I
HN O /
H3CCH3
v
or a pharmaceutically acceptable salt, solvate, tautomer or hydrate thereof,
wherein
R7 is selected from the group consisting of: alkyloxycarbonylalkyl,
alkylaminocarbonyldialkyl, alkylaminodialkyl, alkylhydroxy, a biocompatible
polymer
such as alkyloxy(polyalkyloxy)alkylhydroxyl, a polyethylene glycol (PEG), a
polyethylene glycol ester (PEG ester), a polyethylene glycol ether (PEG
ether),
methyloxyalkyl, methyloxyalkaryl, methylthioalkyl and methylthioalkaryl,
unsubstituted
or substituted; and
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X- is a halide such as bromide, chloride, fluoride, iodide or an anionic
moiety such
as mesylate or tosylate; alternatively, X" is not present and the compound
forms an
"inner.".or zwitterionic salt by loss of a proton from the sulfonylurea moiety
(-S02-NH-
CO-).
In some embodiments of the present invention, the analog may be selected from
the group consisting of: pyridine-substituted torsemide quaternary ammonium
salts or the
corresponding inner salts (zwitterions). Examples include, but are not limited
to,
methoxymethyl pyridinium torsemide salts, methylthiomethyl pyridinium
torsemide salts
and N-mPEG350-pyridinium torsemide salts.
Any of the R groups as defined herein can be excluded from the analogs
disclosed
herein.
Intermediate compounds formed through the synthetic methods described below
to provide the compounds of formula I, II, III, IV and/or V may also possess
utility as a
therapeutic agent for neuropsychiatric disorders described herein.
Modification of the CNS-targeted NKCC co-transport antagonists employed in the
inventive methods can include reacting the antagonist with a functional group
and/or
compound selected from the group consisting of: an aluminum hydride, alkyl
halide,
alcohol, aldehyde, aryl halide, alkyl amide, aryl amine and quaternary
ammonium salt,
unsubstituted or substituted, or combinations thereof. Non-limiting examples
of
compounds that may be used as a starting material are exemplified below.
O OH
0 OH
H
HZNO2S N~\CH3 N
H
llizz~:
H2NO2S
/ CI
bumetanide furosemide
Merck Index, 13th Merck Index, 13th
Edition, 2001, 1471. Edition, 2001, 4330.
CA 02604446 2007-10-09
WO 2006/110187 PCT/US2005/043177
0 OH
N=N
HN ~N
HZNOZS N H
O N
\
H2NOZS
~
CI
piretanide azosemide
Merck Index, 13th Merck Index, 13th
Edition, 2001, 7575. Edition, 2001, 924.
N
I \
SOz ~
HN HN :rCH3
"~O
HN
'1~ toresemide
H3C CH3 Merck Index, 13th
Edition, 2001, 9629.
The term "alkyl" as used herein refers to a straight or branched chain
saturated or
partially unsaturated hydrocarbon radical. Examples of alkyl groups include,
but are not
limited to, methyl, ethyl, isopropyl, tert-butyl, n-pentyl and the like. By
"unsaturated" is
meant the presence of 1, 2 or 3 double or triple bonds, or a combination
thereof. Such
alkyl groups may be optionally substituted as described below.
The term "alkylene" as used herein refers to a straight or branched chain
having
two terminal monovalent radical centers derived by the removal of one hydrogen
atom
from each of the two terminal carbon atoms of straight-chain parent alkane.
The term "aryl" as used herein refers to an aromatic group or to an optionally
substituted aromatic group fused to one or more optionally substituted
aromatic groups,
optionally substituted with suitable substituents including, but not limited
to, lower alkyl,
lower alkoxy, lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl,
oxo,
hydroxy, mercapto, amino optionally substituted by alkyl, carboxy, tetrazolyl,
carbamoyl
optionally substituted by alkyl, aminosulfonyl optionally substituted by
alkyl, acyl, aroyl,
heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano,
halogen, or
lower perfluoroalkyl, multiple degrees of substitution being allowed. Examples
of aryl
include, but are not limited to, phenyl, 2-naphthyl, 1-naphthyl, and the like.
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The term "halo" as used herein refers to bromo, chloro, fluoro or iodo.
Alternatively, the term "halide" as used herein refers to bromide, chloride,
fluoride or
iodide.
The term "hydroxy" as used herein refers to the group -OH.
The term "alkoxy" as used herein alone or as part of another group, refers to
an
alkyl group, as defined herein, appended to the parent molecular moiety
through an oxy
group. Representative examples of alkoxy include, but are not limited to,
methoxy,
ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the
like.
The term "aryloxy" as used herein refers to the group -ArO wherein Ar is aryl
or
heteroaryl. Examples include, but are not limited to, phenoxy, benzyloxy and 2-
naphthyloxy.
The term "amino" as used herein refers to -NH2 in which one or both of the
hydrogen atoms may optionally be replaced by alkyl or aryl or one of each,
optionally
substituted.
The term "alkylthio" as used herein alone or as part of another group, refers
to an
alkyl group, as defined herein, appended to the parent molecular moiety
through a sulfur
moiety. Representative examples of alkylthio include, but are not limited to,
methylthio,
ethylthio, n-propylthio, isopropylthio, n-butylthio, and the like.
The term "carboxy" as used herein refers to the group -COZH.
The term "quaternary ammonium" as used herein refers to a chemical structure
having four bonds to the nitrogen with a positive charge on the nitrogen in
the "onium"
state, i.e., "R4N+" or "quaternary nitrogen," wherein R is an organic
substituent such as
alkyl or aryl. The term "quaternary ammonium salt" as used herein refers to
the
association of the quaternary ammonium with a cation.
The term "substituted" as used herein refers to replacement of one or more of
the
hydrogen atoms of the group replaced by substituents known to those skilled in
the art
and resulting in a stable compound as described below. Examples of suitable
replacement
groups include, but are not limited to, alkyl, acyl, alkenyl, alkynyl
cycloalkyl, aryl,
hydroxy, alkoxy, aryloxy, acyl, amino, amido, carboxy, carboxyalkyl,
carboxyaryl, halo,
oxo, mercapto, sulfinyl, sulfonyl, sulfonamido, amidino, carbamoyl,
dialkoxymethyl,
cycloalkyl, heterocycloalkyl, dialkylaminoalkyl, carboxylic acid, carboxamido,
haloalkyl,
alkylthio, aralkyl, alkylsulfonyl, arylthio, amino, alkylamino, dialkylamino,
guanidino,
ureido and the like. Substitutions are permissible when such combinations
result in
compounds stable for the intended purpose. For example, substitutions are
permissible
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WO 2006/110187 PCT/US2005/043177
when the resultant compound is sufficiently robust to survive isolation to a
useful degree
of purity from a reaction mixture, and formulation into a therapeutic or
diagnostic agent.
The term "solvate" as used herein is intended to refer to a pharmaceutically
acceptable solvate form of a specified compound that retains the biological
effectiveness
of such compound, for example, resulting from a physical association of the
compound
with one or more solvent molecules. Examples of solvates, without limitation,
include
compounds of the invention in combination with water, isopropanol, ethanol,
methanol,
DMSO, ethyl acetate, acetic acid, or ethanolamine.
The term "hydrate" as used herein refers to the compound when the solvent is
water.
The term "biocompatible polymer" as used herein refers to a polymer moiety
that
is substantially non-toxic and does not tend to produce substantial immune
responses,
clotting or other undesirable effects. Polyalkylene glycol is a biocompatible
polymer
where, as used herein, polyalkylene glycol refers to straight or branched
polyalkylene
glycol polymers such as polyethylene glycol, polypropylene glycol, and
polybutylene
glycol, and further includes the monoalkylether of the polyalkylene glycol. In
some
embodiments of the present invention, the polyalkylene glycol polymer is a
lower alkyl
polyalkylene glycol moiety such as a polyethylene glycol moiety (PEG), a
polypropylene
glycol moiety, or a polybutylene glycol moiety. PEG has the formula -
HO(CH2CH2O),H, where n can range from about I to about 4000 or more. In some
embodiments, n is 1 to 100, and in other embodiments, n is 5 to 30. The PEG
moiety can
be linear or branched. In further embodiments, PEG can be attached to groups
such as
hydroxyl, alkyl, aryl, acyl or ester. In some embodiments, PEG can be an
alkoxy PEG,
such as methoxy-PEG (or mPEG), where one terminus is a relatively inert alkoxy
group,
while the other terminus is a hydroxyl group.
The compounds of formula I, II, III, IV and/or V can be synthesized using
traditional synthesis techniques well known to those skilled in the art. More
specific
synthesis routes are described below.
The bumetanide analogs are synthesized by reacting the carboxylic acid moiety
of
bumetanide with various reagents. For example, bumetanide may undergo
esterification
via reaction with alcohols, including linear, branched, substituted, or
unsubstituted
alcohols. Bumetanide may also be alkylated via reaction with suitable
substituted and
unsubstituted alkyl halides and aryl halides, including chloroacetonitrile,
benzyl chloride,
1-(dimethylamino)propyl chloride, 2-chloro-N,N-diethylacetamide, and the like.
PEG-
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type esters may be formed by alkylation using alkyloxy(polyalkyloxy)alkyl
halides such
as MeO-PEG350-Cl and the like, or alkyloxy(polyalkyloxy)alkyl tosylates such
as MeO-
PEG1000-OTs-and the like. "Axetil"-type esters may also be formed by
alkylation using
alkyl halides such as chloromethyl pivalate or chloromethyl propionate.
Bumetanide may
also undergo amidation by reaction with suitable substituted or unsubstituted
alkyl amines
1 o or aryl amines, either after conversion to the acid chloride or by using
an activator, such
as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Bumetanide may also be
reacted with a quaternary ammonium hydroxide, such as benzyltrimethylammonium
hydroxide or cetyltrimethylammonium hydroxide, to form bumetanide quaternary
ammonium salts. Schemes 1 and 7 below presents a synthesis scheme of some
exemplary
compounds according to formula I.
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Scheme 1. Synthesis of Exemplary Compounds According to Formula I.
0 OH 0 OR
esterification
ROH, H'
H NO S I/ N~\CHs or alkylation route H2NOZS N~~CH3
Z 2 H XCH2R, base H
I
O I \ 0~
/ /
1 bumetanide 2 bumetanide alkyl esters
_ R = methyl, ethyl, propyl,
i-propyl, butyl, i-butyl, ..... ,
alkylation route -CH2CH2(OCH2CH2)õ-1-Y
to esters
0 O(CH2)m Ar
X(CH2) Ar,
Et3N base
alkylation route to ~
pro-drug esters H2N02S H CH3
amidation
XCH(R)OCOR', 1) SOCIz 0 I \
Et3N base 2) HNRR'
/
\R,N- temary
salt formation 3 bumetanide aryl and heteroaryl esters
OH- R = phenyl, benzyl, phenethyl,
2-pyridyl, 3-pyridinyl, .....
(m=0, 1,2,.......)
RI_rOyR' O 0-'NRR'R"RO NRR'
0 0 0 I\ I\
HZNO2S H~\CH3 HZNOZS H-~\CH3
O 0 \
H2NO2S H~\CHs I/ ~/
O"a
4 bumetanide simple amides
5 bumetanide quaternary ammonium salts R = H, methyl, ethyl, benzyl, .....
R benzyl, cetyl, methyl, ethyl ..... R' = H, methyl, ethyl, benzyl, .....
=
6 bumetanide "axetil"-type esters R' methyl, ethyl, propyl , ......
R" = methyl, ethyl, propyl, .....
R= H, methyl; R"' = methyl, ethyl, propyl, .....
R' = H, methyl, ethyl, t-butyl, .....
The furosemide analogs are synthesized by methods analogous to those used in
the synthesis of the bumetanide analogs. Specifically, furosemide may undergo
esterification via reaction with alcohols, including linear, branched,
substituted, or
unsubstituted alcohols. Furosemide may also be alkylated via reaction with
suitable
substituted and unsubstituted alkyl halides and aryl halides, including for
example,
chloroacetonitrile, benzyl chloride, 1-(dimethylamino)propyl chloride, 2-
chloro-N,N-
diethylacetamide, and the like. PEG-type esters may be formed by alkylation
using
alkyloxy(polyalkyloxy)alkyl halides such as MeO-PEG350-Cl and the like, or
CA 02604446 2007-10-09
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alkyloxy(polyalkyloxy)alkyl tosylates such as MeO-PEG1000-OTs and the like.
"Axetil"-type esters may also be formed by alkylation using alkyl halides such
as
chloromethyl pivalate or chloromethyl propionate. Furosemide may also undergo
amidation by reaction with suitable substituted or unsubstituted alkyl amines
or aryl
amines, either after conversion to the acid chloride or by using an activator,
such as 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Furosemide may also be
reacted
with a quaternary ammonium hydroxide, such as benzyltrimethylammonium
hydroxide or
cetyltrimethylanunonium hydroxide, to form furosemide quaternary ammonium
salts.
Scheme 2 below presents a synthesis scheme of some exemplary compounds
according to
formula II.
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Scheme 2. Synthesis of Exemplary Compounds According to Formula II.
H O OR H
COZH
N esterification
\ O ROH, H'
I / or alkylation route
H2NO2S XCH2R, base H2NO2S
CI CI
1 furosemide 2 furosemide alkyl esters
- R = methyl, ethyl, propyl,
i-propyl, butyl, i-butyl, ..... ,
alkylation route -CH2CH2(OCH2CH2)õ_1-Y
to esters
X(CH2)mAr, Q O(CHZ)m-Ar
Et3N base
H
N
alkylation route to amidation
pro-drug esters 1) SOCIZ
2) HNRR' H2NO2S
XCH(R)OCOR', ci
Et3N base salt uatema
formation 3 furosemide aryl and heteroaryl esters
R4N' OH- R = phenyl, benzyl, phenethyl,
2-pyridyl, 3-pyrdinyl, ...
(m = 0, 1, 2, .......)
R,,"LI_ /OyR' O O-*NRR'R"RI \ O NRR'
~ H H
0 0 0 N N
p 1
2NO2S
N HZNOZS H
~ / CI CI
HzNOZS p
5 furosemide quaternary ammonium salts 4 furosemide simple amides
CI R benzyl, cetyl, methyl, ethyl ..... R = H, methyl, ethyl, benzyl, .....
R' = methyl, ethyl, propyl, ...... R' = H, methyl, ethyl, benzyl, .....
R" = methyl, ethyl, propyl, .....
6 furosemide "axetil"-type esters R"' = methyl, ethyl, propyl, .....
R = H, methyl;
R' = H, methyl, ethyl, t-butyl, .....
The piretanide analogs are synthesized by methods analogous to those used in
the
synthesis of the bumetanide analogs. Specifically, piretanide may undergo
esterification
via reaction with alcohols, including linear, branched, substituted, or
unsubstituted
alcohols. Piretanide may also be alkylated via reaction with suitable
substituted and
unsubstituted alkyl halides and aryl halides, including chloroacetonitrile,
benzyl chloride,
1-(dimethylamino)propyl chloride, 2-chloro-N,N-diethylacetamide, and the like.
PEG-
type esters may be formed by alkylation using alkyloxy(polyalkyloxy)alkyl
halides such
as MeO-PEG350-Cl and the like, or alkyloxy(polyalkyloxy)alkyl tosylates such
as MeO-
PEG1000-OTs and the like. "Axetil"-type esters may also be formed by
alkylation by
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WO 2006/110187 PCT/US2005/043177
using alkyl halides such as chloromethyl pivalate or chloromethyl propionate.
Piretanide
may also undergo amidation by reaction with suitable substituted or
unsubstituted alkyl
amines or aryl amines, either after conversion to the acid chloride or by
using an
activator, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
Piretanide
may also be reacted with a quaternary ammonium hydroxide, such as
benzyltrimethylammonium hydroxide or cetyltrimethylammonium hydroxide, to form
piretanide quaternary ammonium salts. Scheme 3 below presents a synthesis
scheme of
some exemplary compounds according to formula III.
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Scheme 3. Synthesis of Exemplary Compounds According to Formula III.
COzH CO2R
esterification
ROH, H' I \
Jor alkylation route H NO S / N~
H2NOZS N1 ~ XCH2R, base Z 2
IV/ O
~ I \
1 2 piretanide alkyl esters
piretanide R = methyl, ethyl, propyl,
i-propyl, butyl, i-butyl, ..... ,
alkylation route -CH2CH2(OCH2CH2)1,.,-Y
to esters
O(CH2)m Ar
X(CHp)mAr,
Et3N base
alkylation route to
pro-drug esters H2NO2S N
XCH(R)OCOR', ~
Et3N base III J'
q/
foN3 piretanide aryl and heteroaryl esters
, benzyl, phenethyl,
R = phenyl
2-pyridyl, 3-pyridinyl, .....
(m=0.1,2,.......
)
R,,L,rOyR' O O''NRR'R"RO NRR'
O O I \ I \
H2NO2S N H2NO2S N
I \ ~ \
HZNO2S / N
O
4 piretanide simple amides
5 piretanide quaternary ammonium salts R = H, methyl, ethyl, benzyl, .....
R = benzyl, cetyl, methyl, ethyl ..... R' = H, methyl, ethyl, benzyl, .....
R' = methyl, ethyl, propyl, ......
6 piretanide "axetil"-type esters R" = methyl, ethyl, propyl, .....
R = H, methyl; R"' = methyl, ethyl, propyl, .....
R' = H, methyl, ethyl, t-butyl, .....
The azosemide analogs are synthesized by the reaction of various reagents with
the tetrazolyl moiety of azosemide. Azosemide may undergo hydroxyalkylation
with the
addition of an aldehyde, whereby a hydroxylalkyl functionality is formed. An
alcohol
may optionally be reacted along with the aldehyde to obtain an ether. An alkyl
thiol may
optionally be added with the aldehyde to form a thioether. Azosemide may also
be
alkylated by the addition of suitable alkyl halides or aryl halides, including
alkyl or aryl
halides comprising an ether or thioether linkage, such as methyl chloromethyl
ether and
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WO 2006/110187 PCT/US2005/043177
benzyl chloromethyl thioether. PEG-type ethers may be formed by alkylation
using
alkyloxy(polyalkyloxy)alkyl halides such as MeO-PEG350-Cl and the like, or
alkyloxy(polyalkyloxy)alkyl tosylates such as MeO-PEG1000-OTs and the like.
"Axetil"-type analogs may also be formed via addition of alkyl or aryl
halides, such as
chloromethyl pivalate or chloromethyl propionate. Azosemide may be reacted
with a
quaternary ammonium salt, such as benzyltrimethylammoniumbromide and base such
as
sodium hydroxide or cetyltrimethylammonium bromide and base such as sodium
hydroxide, in order to form an azosemide quaternary ammonium salt. Scheme 4
below
presents a synthesis scheme of some exemplary compounds according to formula
IV.
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Scheme 4. Synthesis of Exemplary Compounds According to Formula IV.
N=N N=N
HN N HO N N
N I ~ hydroxyalkylation Y N
S R S
I RCHO,H' I
H2NO2S ~ H2NO2S CI CI
2 azosemide N-substituted alkanols
1 azosemide alkylation route to R H, methyl, ethyl, .....
N-substituted ethers
X(CHZ)mOR,
Et3N base
N=N
N / N
alkylation route to m N CS)
N-substituted thioethers X(CH2)R,SR,
Et3N base
N-alkylation route H2NO2S
XCH(R)OCOR', quaternary CI
Et3N base salt formation
R,N' er' 3 azosemide N-substituted ethers
NaOH R = methyl, ethyl, benzyl, ...... ,
CH2CH2(OCH2CH2)n_1-Y
m=1-5;n1-100
N=N
N=N
S N N
R1~
R4N' N' N m H c
H I ~ N S
N N S
R' O N/ N H2NO2S Y ~ N HZNO2SI CI
O R S CI
4 azosemide N-substituted thioethers
H2NO2S R = methyl, ethyl, benzyl, ...... ,
CI -CH2CH2(OCH2CH2),; Y
m = 1-5; n = 1-100
5 azosemide quatemary ammonium salts
R = benzyl, cetyl, methyl, ethyl, .....
6 azosemide N-substituted "axetil"-type
R = H, methyl
R' = H, methyl, ethyl, t-butyl, .....
The torsemide (also known as torasemide) analogs are synthesized by the
reaction of various reagents with the pyridine moiety of torsemide. Torsemide
may
undergo alkylation by the addition of suitable alkyl or aryl halides,
including benzyl
chloride, to form N-substituted quaternary ammonium salts. Alkyl halides and
aryl
halides comprising an ether linkage, including methyl chloromethyl ether and
benzyl
chloromethyl ether, may be used to form N-substituted ether quaternary
ammonium salts.
Alkyl halides and aryl halides comprising a thioether linkage, including
methyl
chloromethyl thioether and benzyl chloromethyl thioether, may be used to form
N-
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substituted thioether quaternary ammonium salts. PEG-type ether-containing
quaternary
ammonium salts may be formed by alkylation using alkyloxy(polyalkyloxy)alkyl
halides
such as_MeO-PEG350-Cl and the like, or alkyloxy(polyalkyloxy)alkyl tosylates
such as
MeO-PEG 1000-OTs and the like. "Axetil"-type quaternary ammonium salts may
also be
formed via the addition of alkyl halides such as chloromethyl pivalate or
chloromethyl
propionate. Scheme 5 below presents a synthesis scheme of some exemplary
compounds
according to formula V.
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Scheme 5. Synthesis of Exemplary Compounds According to Formula V.
CH2R
N N+ X
~ alkylation route to
~ N-substituted ~
02S / quaternary salts OZS
HN HN CH3 XCH2R HN' HN CH3
HN O HN O ~
H3CCH3 H3CCH3
alkylation route to 2 torsemide N-substituted
N-substituted ether - quatemary salts
1 torsemide quaternary salts R H, methyl, ethyl, benzyl, ......
X(CH2)mOR
\1k ute to R T~I )m
d thioether X_
salts N+
SR ' I
alkylation route to
"axetil" quatemary salts 02S
XCH(R)OCOR' HN' HNCH3
HN O
R )m H3C"~CH3
R'yO R ~* 3 torsemide N-sub stituted ether
~ quaternary salts
X- ~/ R methyl, ethyl, benzyl, ..... ,
+
N~ OZS -CHZCH2(OCH2CHZ)n-rY
I HN' HN CH3 m 1-5; n 1-100
OZS / ~ I \
HN HN CH3 HN O /
HN O H3C"~CH3
H3CCH3 4 torsemide N-substituted thioether
quaternary salts
R = methyl, ethyl, benzyl, ..... ,
5 torsemide N-substituted "axetil"-type -CHZCH2(OCH2CHZ)õ_1-Y
quaternary salts m = 1-5; n = 1-100
R = H, methyl
R' = H, methyl, ethyl, t-butyl, .....
The substituted benzoic acids bumetanide, piretanide and furosemide can be
t0 selectively reduced to the corresponding bumetanide aldehyde, piretanide
aldehyde and
furosemide aldehyde using amine-substituted ammonium hydrides such as bis(4-
methylpiperazinyl)aluminum hydride by literature methods. See Muraki, M. and
Mukiayama, T., Chem. Letters, 1974, 1447; Muraki, M. and Mukiayama, T., Chem.
Letters, 1975, 215; and Hubert, T. et al., J. Org. Chem., 1984, 2279. It is
well known that
the more lipophilic benzaldehydes readily air-oxidize into the more
hydrophilic benzoic
28
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WO 2006/110187 PCT/US2005/043177
acids and that benzaldehydes are also metabolized into the corresponding
benzoic acids in
vivo, via the use of NADPH co-factor and with a number of oxidative P450
enzymes.
Scheme 6. Synthesis of Exemplary Benzaldehyde Analogs of Bumetanide,
Piretanide and Furosemide.
O OH O H
1) (alkyl)2NAIH
HZNOpS H CH3 2)H20 HZNOZS H CH3
O O \
\
bumetanide "bumetanide aldehyde"
R, = ---, R2 = H,
R3=0-aryI,R4=Rs=H
O OH \ O H
I
\ N I O 2j (alkyl)2NAIH kyl)2NAIH N O
/ 2NOZS
( H2NO2S H
CI CI
"furosemide aldehyde"
furosemide R, = --, R2 = H,
R3 = halide, R4 = R5 = H
O OH O H
1) (alkyl)2NAIH
2) H20
H2NO2S N1 H2NO2S N
O 1 /
~ v
piretanide "piretanide aldehyde"
R, = ---, RZ = H,
R3 = O-aryl, R4 = R5 = H
For reduction procedures used to convert benzoic acids to the corresponding
benzaldehydes, see:
Muraki, M. and Mukiayama, T., Chem. Letters, 1974, 1447; ibid, 1975, 215;
Hubert, T., D., Eyman, D. P. and Wiemer, D. F., J. Org. Chem., 1984, 2279.
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WO 2006/110187 PCT/US2005/043177
Scheme 7. Synthesis of Exemplary Polyethylene Glycol Esters of Bumetanide,
Furosemide and Piretanide.
O OH O O(CH2)m(OCH2CH2)11-Y
1) PEG-X, base
2) Hz0
H2NO2S H~~ CH3 H2NOZS HCH3
O \ O \
I14-1
bumetanide "bumetanide PEG esters"
Rz = OCH2CH2(OCH2CH2)11-Y
R3=0-aryI,R4=R5=H
m = 1 - 5, n = 1 - 100
O(CH2)m(OCH2CH241-Y
O OH 0
I \ 1) PEG-X, base I \
H z) Hz0 H
N N O
\ O \
H2NO2S H2NO2S /
CI Ci
"furosemide PEG esters"
R2 = OCH2CH2(OCH2CH2)~-i-Y
furosemide
R3 = chloride, R4 = R5 = H
m=1-5,n=1-100
O OH 0 O(CH2)m(OCH2CH2)n-i-Y
1) PEG-X, base
2) Hz0
-- - /
H2NO2S I N H2NO2S N
0 O
~ \~J
"piretanide PEG esters"
piretanide R2 = OCH2CH2(OCH2CH2),,.l-Y
R3 = O-aryi, R4 = R5 = H
m = 1 - 5, n = 1 - 100
PEG-X is X-(CH2)m(OCH2CH241-Y, where X is halo or other leaving group
(mesylate "OMs", tosylate "OTs") and Y
is OH or an alcohol protecting group such as an alkyl group, an aryl group, an
acyl group or an ester group, and
where m = 1 -5 and n = 1 -100.
CA 02604446 2007-10-09
WO 2006/110187 PCT/US2005/043177
Scheme 8. Synthesis of Exemplary Alkyl Polyethylene Glycol Ethers of
Azosemide and Torsemide.
N=N N=N
HN /N /N N H
\
H\\J~ 1) PEG-X, base R6 N
z) Zo
H2NO2S I / H2NO2S
CI CI
azosemide "azosemide methyl PEG ethers"
Rs = (CH2)mOCH2CH2(OCH2CH2)n.,-Y
R3 = chloride, R4 = R5 = H
m = 1 - 5, n = 1 - 100
R7
I X-
~
OZS N OZS N+
I /
HN' ~..~N C~( 1)PEG-X,base HN~ 1.{N CH3
\ 3 z) Hz0 _ I \
HN~O ( / HN'1~O
H3C'1~1 CH3 H3C'1~1 CH3
torsemide "torsemide methyl PEG ether
quaternary ammonium salts"
R7 = (CH2)mOCH2CH2(OCH2CH2)n_1-Y
X' = halide, mesylate, tosylate
m=1-5,n=1-100
PEG-X is X-(CH2)m(OCH2CH2)1_1-Y, where X is halo or other leaving group
(mesylate "OMs", tosylate "OTs") and Y
is OH or an alcohol protecting group such as an alkyl group, an aryl group, an
acyl group or an ester group, and
wherem=l -5andn=l-100.
Starting materials for synthesizing compounds of the present invention can
further
include compounds described in U.S. Patent No. 3,634,583 to Feit; U.S. Patent
No.
3,806,534 to Fiet; U.S. Patent No. 3,058,882 to Struem et al.; U.S. Patent No.
4,010,273
to Bormann; U.S. Patent No. 3,665,002 to Popelak; and U.S. Patent No.
3,665,002 to
Delarge, the disclosures of which are hereby incorporated by reference.
Compounds of the present invention can include isomers, tautomers,
zwitterions,
enantiomers, diastereomers, racemates or stereochemical mixtures thereof. The
term
"isomers" as used herein refers to compounds having the same number and kind
of atoms,
and hence the same molecular weight, but differing with respect to the
arrangement or
configuration of the atoms in space. Additionally, the term "isomers" includes
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WO 2006/110187 PCT/US2005/043177
stereoisomers and geometric isomers. The terms "stereoisomer" or "optical
isomer" as
used herein refer to a stable isomer that has at least one chiral atom or
restricted rotation
giving rise to perpendicular dissymmetric planes (e.g., certain biphenyls,
allenes, and
spiro compounds) and can rotate plane-polarized light. Because asymmetric
centers and
other chemical structures can exist in some of the compounds of the present
invention
which may give rise to stereoisomerism, the invention contemplates
stereoisomers and
mixtures thereof. The compounds of the present invention and their salts can
include
asymmetric carbon atoms and may therefore exist as single stereoisomers,
racemates, and
as mixtures of enantiomers and diastereomers. Typically, such compounds will
be
prepared as a racemic mixture. If desired, however, such compounds can be
prepared or
isolated as pure stereoisomers, i.e. as individual enantiomers or
diastereomers, or as
stereoisomer-enriched mixtures. Tautomers are readily interconvertible
constitutional
isomers and there is a change in connectivity of a ligand, as in the keto and
enol forms of
ethyl acetoacetate. The inventive methods and compositions may employ
tautomers of
any of said compounds. Zwitterions are inner salts or dipolar compounds
possessing
acidic and basic groups in the same molecule. At neutral pH, the cation and
anion of
most zwitterions are equally ionized.
The present invention further provides prodrugs comprising the compounds
described herein. The term "prodrug" is intended to refer to a compound that
is converted
under physiological conditions, by solvolysis or metabolically, to a specified
compound
that is pharmaceutically/pharmacologically active. The prodrug can be a
compound of
the present invention that has been chemically derivatized such that: (i) it
retains some, all
or none of the bioactivity of its parent drug compound, and (ii) it is
metabolized in a
subject to yield the parent drug compound. The prodrug of the present
invention may
also be a "partial prodrug" in that the compound has been chemically
derivatized such
that: (i) it retains some, all or none of the bioactivity of its parent drug
compound, and (ii)
it is metabolized in a subject to yield a biologically active derivative of
the compound.
The prodrugs can be formed utilizing a hydrolyzable coupling to the compounds
described herein. A further discussion of prodrugs can be found in Ettmayer et
al. J. Med.
Chem. 47(10):2394-2404 (2004).
Prodrugs of the present invention are capable of passage across the blood-
brain
barrier and may undergo hydrolysis by CNS esterases to provide the active
compound.
Further, the prodrugs provided herein may also exhibit improved
bioavailability,
improved aqueous solubility, improved passive intestinal absorption, improved
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WO 2006/110187 PCT/US2005/043177
transporter-mediated intestinal absorption, protection against accelerated
metabolism,
tissue-selective delivery and/or passive enrichment in the target tissue.
Prodrugs of the present invention can include compounds according to formula
I,
II, III, IV and/or V described herein. Prodrugs of the present invention can
further
include bumetanide, bumetanide dibenzylamide, bumetanide diethylamide,
bumetanide
morpholinoethyl ester, bumetanide 3-(dimethylaminopropyl) ester, bumetanide
N,N-
diethylglycolamide ester, bumetanide dimethylglycolamide ester, bumetanide
pivaxetil
ester, furosemide, furosemide ethyl ester, furosemide cyanomethyl ester,
furosemide
benzyl ester, furosemide morpholinoethyl ester, furosemide 3-
(dimethylaminopropyl)
ester, furosemide N,N-diethylglycolamide ester, furosemide dibenzylamide,
furosemide
benzyltrimethyl-ammonium salt, furosemide cetyltrimethylammonium salt,
furosemide
N,N-dimethylglycolamide ester, furosemide pivaxetil ester, furosemide
propaxetil ester,
piretanide, piretanide methyl ester, piretanide cyanomethyl ester, piretanide
benzyl ester,
piretanide morpholinoethyl ester, piretanide 3-(dimethylaminopropyl) ester,
piretanide
N,N-diethylglycolamide ester, piretanide diethylamide, piretanide
dibenzylamide,
piretanide benzylltrimethylammonium salt, piretanide cetylitrimethylammonium
salt,
piretanide N,N-dimethylglycolamide ester, piretanide pivaxetil ester,
piretanide propaxetil
ester, tetrazolyl-substituted azosemides, pyndinium-substituted torsemide
salts (also
termed pyridine-substituted torsemide quaternary ammonium salts), .as well as
similar
derivatives of 'indacrinone, and ozolinone. See previously presented schemes.
Moreover, as shown in the previously presented schemes, prodrugs can be formed
by attachment of biocompatible polymers, such as those previously described
including
polyethylene glycol (PEG), to compounds of the present invention using
linkages
degradable under physiological conditions. See also Schacht, E.H. et al.
Poly(ethylene
glycol) Chemistry and Biological Applications, American Chemical Society, San
Francisco, CA 297-315 (1997). Attachment of PEG to proteins can be employed to
reduce immunogenicity and/or extend the half-life of the compounds provided
herein.
Any conventional PEGylation method can be employed, provided that the
PEGylated
agent retains pharmaceutical activity.
Compositions of the subject invention are suitable for human and veterinary
applications and are preferably delivered as pharmaceutical compositions.
Pharmaceutical compositions comprise one or more treatment agents, or a
pharmaceutically acceptable salt thereof, and a physiologically acceptable
carrier. A
pharmaceutically acceptable salt, as used herein, refers to a salt form of a
compound
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CA 02604446 2007-10-09
WO 2006/110187 PCT/US2005/043177
permitting its use or formulation as a pharmaceutical and which retains tFie
biological
effectiveness of the free acid and base of the specified compound and is not
biologically
or otherwise undesirable. Examples of such salts are described in Handbook of
Pharinaceutical Salts: Properties, Selection, and Use, Wermuth, C.G. and
Stahl, P.H.
(eds.), Wiley-Verlag Helvetica Acta, Zurich, 2002 [ISBN 3-906390-26-8].
Examples of
such salts include alkali metal salts and addition salts of free acids and
bases. Examples
of pharmaceutically acceptable salts, include, but are not limited to,
sulfates, pyrosulfates,
bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates,
dihydrogen
phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides,
acetates,
propionates, decanoates, caprylates, acrylates, formates, isobutyrates,
caproates,
heptanoates, propiolates, oxalates, malonates, succinates, suberates,
sebacates, fumarates,
maleates, butyne- 1,4-dioates, hexyne- 1,6-dioates, benzoates,
chlorobenzoates,
methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates,
phthalates,
xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates,
citrates, lactates,
y-hydroxybutyrates, glycollates, tartrates, methanesulfonates, ethane
sulfonates,
propanesulfonates, toluenesulfonates, naphthalene-l-sulfonates, naphthalene-2-
sulfonates, and mandelates.
Pharmaceutical compositions of the present invention may also contain other
compounds, which may be biologically active or inactive. For example, one or
more
treatment agents of the present invention may be combined with another agent,
in a
treatment combination, and administered according to a treatment regimen of
the present
invention. Such combinations may be administered as separate compositions,
combined
for delivery in a complementary delivery system, or formulated in a combined
composition, such as a mixture or a fusion compound. Additionally, the
aforementioned
treatment combination may include a BBB permeability enhancer and/or a
hyperosmotic
agent.
The carriers and additives used for such pharmaceutical compositions can take
a
variety of forms depending on the anticipated mode of administration. Thus,
compositions for oral administration may be, for example, solid preparations
such as
tablets, sugar-coated tablets, hard capsules, soft capsules, granules, powders
and the like,
with suitable carriers and additives being starches, sugars, binders,
diluents, granulating
agents, lubricants, disintegrating agents and the like. Because of their ease
of use and
higher patient compliance, tablets and capsules represent advantageous oral
dosage forms
for many medical conditions.
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WO 2006/110187 PCT/US2005/043177
Similarly, compositions for liquid preparations include solutions, emulsions,
dispersions, suspensions, syrups, elixirs, and the like with suitable carriers
and additives
being water, alcohols, oils, glycols, preservatives, flavoring agents,
coloring agents,
suspending agents, and the like. Typical preparations for parenteral
administration
comprise the active ingredient with a carrier such as sterile water or
parenterally
acceptable oil including polyethylene glycol, polyvinyl pyrrolidone, lecithin,
arachis oil
or sesame oil, with other additives for aiding solubility or preservation may
also be
included. In the case of a solution, it can be lyophilized to a powder and
then
reconstituted immediately prior to use. For dispersions and suspensions,
appropriate
carriers and additives include aqueous gums, celluloses, silicates or oils.
The pharmaceutical compositions according to embodiments of the present
invention include those suitable for oral, rectal, topical, nasal, inhalation
(e.g., via an
aerosol) buccal (e.g., sub-lingual), vaginal, topical (i.e., both skin and
mucosal surfaces,
including airway surfaces), transdermal administration and pareriteral (e.g.,
subcutaneous,
intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal,
intrathecal,
intracerebral, intracranially, intraarterial, or intravenous), although the
most suitable route
in any given case will depend on the nature and severity of the condition
being treated
and on the nature of the particular active agent which is being used.
Pharmaceutical
compositions of the present invention are particularly suitable for oral,
sublingual,
parenteral, implantation, nasal and inhalational administration.
Compositions for injection will include the active ingredient together with
suitable
carriers including propylene glycol-alcohol-water, isotonic water, sterile
water for
injection (USP), emulPhorTM-alcohol-water, cremophor-ELTM or other suitable
carriers
known to those skilled in the art. These carriers may be used alone or in
combination
with other conventional solubilizing agents such as ethanol, a glycol, or
other agents
known to those skilled in the art.
Where the compounds of the present invention are to be applied in the form of
solutions or injections, the compounds may be used by dissolving or suspending
in any
conventional diluent. The diluents may include, for example, physiological
saline,
Ringer's solution, an aqueous glucose solution, an aqueous dextrose solution,
an alcohol,
a fatty acid ester, glycerol, a glycol, an oil derived from plant or animal
sources, a
paraffin and the like. These preparations may be prepared according to any
conventional
method known to those skilled in the art.
CA 02604446 2007-10-09
WO 2006/110187 PCT/US2005/043177
Compositions for nasal administration may be formulated as aerosols, drops,
powders and gels. Aerosol formulations typically comprise a solution or fine
suspension
of the. active ingredient in a physiologically acceptable aqueous or non-
aqueous solvent.
Such formulations are typically presented in single or multidose quantities in
a sterile
form in a sealed container. The sealed container can be a cartridge or refill
for use with
an atomizing device. Alternatively, the sealed container may be a unitary
dispensing
device such as a single use nasal inhaler, pump atomizer or an aerosol
dispenser fitted
with a metering valve set to deliver a therapeutically effective amount, which
is intended
for disposal once the contents have been completely used. When the dosage form
comprises an aerosol dispenser, it will contain a propellant such as a
compressed gas, air
as an example, or an organic propellant including a fluorochlorohydrocarbon or
fluorohydrocarbon.
Compositions suitable for buccal or sublingual administration include tablets,
lozenges and pastilles, wherein the active ingredient is formulated with a
carrier such as
sugar and acacia, tragacanth or gelatin and glycerin.
Compositions for rectal administration include suppositories containing a
conventional suppository base such as cocoa butter.
Compositions suitable for transdermal administration include ointments, gels
and
patches.
Other compositions known to those skilled in the art can also be applied for
percutaneous or subcutaneous administration, such as plasters.
Further, in preparing such pharmaceutical compositions comprising the active
ingredient or ingredients in admixture with components necessary for the
formulation of
the compositions, other conventional pharmacologically acceptable additives
may be
incorporated, for example, excipients, stabilizers, antiseptics, wetting
agents, emulsifying
agents, lubricants, sweetening agents, coloring agents, flavoring agents,
isotonicity
agents, buffering agents, antioxidants and the like. As the additives, there
may be
mentioned, for example, starch, sucrose, fructose, dextrose, lactose, glucose,
mannitol,
sorbitol, precipitated calcium carbonate, crystalline cellulose,
carboxymethylcellulose,
dextrin, gelatin, acacia, EDTA, magnesium stearate, talc,
hydroxypropylmethylcellulose,
sodium metabisulfite, and the like.
In further embodiments, the present invention provides kits including one or
more
containers comprising pharmaceutical dosage units comprising an effective
amount of
one or more compounds of the present invention.
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WO 2006/110187 PCT/US2005/043177
When aqueous suspensions or elixirs are desired for oral administration, the
essential active ingredient therein may be combined with various sweetening or
flavoring
agents, coloring matter or dyes and, if desired, emulsifying or suspending
agents, together
with diluents such as water, ethanol, propylene glycol, glycerin and
combinations thereof.
The compositions described herein may be administered as part of a sustained
release formulation. Such formulations may generally be prepared using well-
known
technology and administered by, for example, oral, rectal or transdermal
delivery
systems, or by implantation of a formulation or therapeutic device at one or
more desired
target site(s). Sustained-release formulations may contain a treatment
composition
comprising an inventive treatment agent alone, or in combination with a second
treatment
agent, dispersed in a carrier matrix and/or contained within a reservoir
surrounded by a
rate controlling membrane. Carriers for use within such formulations are
biocompatible,
and may also be biodegradable. According to one embodiment, the sustained
release
formulation provides a relatively constant level of active composition
release. According
to another embodiment, the sustained release formulation is contained in a
device that
may be actuated by the subject or medical personnel, upon onset of certain
symptoms, for
example, to deliver predetermined dosages of the treatment composition. The
amount of
the treatment composition contained within a sustained release formulation
depends upon
the site of implantation, the rate and expected duration of release, and the
nature of the
condition to be treated or prevented.
In certain embodiments, compositions of the present invention for treatment of
neuropathic pain and neuropsychiatric disorders are administered using a
formulation and
a route of administration that facilitates delivery of the treatment
composition(s) to the
central nervous system. Treatment compositions, such as NKCCI antagonists, may
be
formulated to facilitate crossing of the blood brain bamer as described above,
or may be
co-administered with an agent that crosses the blood brain barrier. Treatment
compositions may be delivered in liposome formulations, for example, that
cross the
blood brain barrier, or may be co-administered with other compounds, such as
bradykinins, bradykinin analogs or derivatives, or other compounds, such as
SERAPORTTM, that cross the blood brain barrier. Alternatively, treatment
compositions
of the present invention may be delivered using a spinal tap that places the
treatment
composition directly in the circulating cerebrospinal fluid. For some
treatment
conditions, there may be transient or permanent breakdowns of the blood brain
barrier
and specialized formulation of the treatment composition to cross the blood
brain barrier
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WO 2006/110187 PCT/US2005/043177
may not be necessary. We have determined, for example, that a bolus iv
injection of 20
mg furosemide reduces or abolishes both spontaneous interictal activity and
electrical
stimulation-evoked epileptiform activity in human patients who are refractory
to
antiepileptic drugs (AEDs) (Haglund & Hochman J. Neurophysiol. (Feb. 23, 2005)
doi:10.1152/ jn.00944.2004).
Routes and frequency of admiriistration of the therapeutic compositions
disclosed
herein, as well as dosages, vary according to the indication, and from
individual to
individual, and may be readily determined by a physician from information that
is
generally available, and by monitoring patients and adjusting the-dosages and
treatment
regimen aec:ordingly using standard teciiniques. In general, appropriate
dosr.~es -:nd
treatment regimen. provide the active cornposition(s) in an amount suff-c:ent
to provide
therapeutic anci/or prophylactic benefit. Dosages and treatment reQimen rnay
be
established by monitoring improved clinical outcomes in treated patients as
comparedl to
non-treated patients.
The term "effective amount" or "effective" is intended to de3igr:ate a dose
that
zo causes a rel;cf of sy-nptoins of a disease: or disorder as noted. through.
cli.ni.caL. testing and=
evaluatici., ;)atient observation, an.d/or: the like:: -"Effective amourrt"
or."effective ', fur!her.:.-.::..
can fi!rther, d:,sit;nate: a. dose that:.:cau,es. a detectable change. in. bic
logicai or chen.icr.l_:
activity: Th- detectable cha-ige5i may be (letected and/or:further. quantified
by.one skil',ed:- ..:. .
in. the art fer rhe. relevant mechaõisr.o: or.:.pr~,cess. -: . Mereover,.
'.'e.ft~; ctive. amour.t". c:
25:'' "effec'ive" can de~,ignate; an a-neunt,: thatrnaintains
~.a..desirecl:.physio logi.;a?.:.st%ate -: i.e.;,;
reduces or n:-,;vents.signifcant decline ;,nd,:or promotes;improvemeat in. the
cendition:.of.. ,. ._ .,., .
interest. Therapeutically effective dosages and treatment regirnen wili
tiepend orr the
condition, the severity of the condition, and the general state of the patient
being treated.
Since the pharmacokinetics and pliarmacodyr.amics of the treatment
compositions of the
30 present invention vary in different patients, a preferred method for
determining, a
therapeutically effective dosage i-:. a patient is to gradually escalate the
dosage and
moni!or -the clinical and laborat.ory indicia. For conlbination, tli erapy,
the.two or n14:=e
agents are coadministered such that- eac!) of the.agents is present in a
therapeutically
effective aniount for sufficient time to }.~roduce a therapeutic or
prophylactic effect. The
:35 term "coadniinistration" is intend--ci to encompass simultaneous or
sequeatial
administration of two or more agerrts in the same formulation or. unit dosage
form or in
separate formulations. Appropriate dosages and treatment regimen for treatment
of acute
38
CA 02604446 2007-10-09
WO 2006/110187 PCT/US2005/043177
episodic conditions, chronic conditions, or prophylaxis will necessarily vary
to
accommodate the condition of the patient.
By way of example, for the treatment to neuropathic pain, furosemide may be
administered orally to a patient in amounts of 10-40 mg at a frequency.of 1-3
times per
day, preferably in an amount of 40 mg three times per day. In an alternative
example,
bumetanide may be administered orally for the treatment of neuropathic pain in
amounts
of 1-10 mg at a frequency of 1-3 times per day. One of skill in the art will
appreciate that
smaller doses may be employed, for example, in pediatric applications.
In further embodiments, bumetanide analogs according to the present invention
may be administered in amounts of 1.5 to 6 mg daily, for example I tablet or
capsule
three times a day. In some embodiments, furosemide analogs according to the
present
invention may be administered in amounts of 60 to 240 mg/day, for example I
tablet or
capsule three times a day. In other embodiments, piretanide analogs according
to the
present invention may be administered in amounts of 10 to 20 mg daily, for
example 1
tablet or capsule once a day. In some embodiments, azosemide analogs according
to the
present invention may be administered in an amount of 60 mg per day. In other
embodiments, torsemide analogs according to the present invention may be
administered
in amounts of 10 to 20 mg daily, for example 1 tablet or capsule once a day.
It should be
noted that lower doses may be administered, particularly for IV
administration.
Methods and systems of the present invention may also be used to evaluate
candidate compounds and treatment regimen for the treatment and/or prophylaxis
of
neuropathic pain and neuropsychiatric disorders. Various techniques for
generating
candidate compounds potentially having the desired NKCC 1 cotransporter
antagonist
activity may be employed. Candidate compounds may be generated using
procedures
well known to those skilled in the art of synthetic organic chemistry.
Structure-activity
relationships and molecular modeling techniques are useful for the purpose of
modifying
known NKCC1 antagonists, such furosemide, bumetanide, ethacrinic acid and
related
compounds, to confer the desired activities and specificities. Methods for
screening
candidate compounds for desired activities are described in U.S. Patents
5,902,732,
5,976,825, 6,096,510 and 6,319,682, which are incorporated herein by reference
in their
entireties.
Candidate compounds may be screened for NKCCI antagonist activity using
screening methods of the present invention with various types of cells in
culture such as
glial cells, neuronal cells, renal cells, and the like, or in situ in animal
models. Screening
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WO 2006/110187 PCT/US2005/043177
techniques to identify chloride cotransporter antagonist activity, for
example, may involve
altering the ionic balance of the extracellular space in the tissue culture
sample, or in situ
in an animal model, by producing a higher than "normal" anionic chloride
concentration.
The geometrical and/or optical properties of the cell or tissue sample subject
to this
altered ionic balance are determined, and candidate agents are administered.
Following
l0 administration of the candidate agents, the corresponding geometrical
and/or optical
properties of the cell or tissue sample are monitored to determine whether the
ionic
imbalance remains, or whether the cells responded by altering the ionic
balances in the
extracellular and intracellular space. If the ionic imbalance remains, the
candidate agent
is likely a chloride cotransporter antagonist. By screening using various
types of cells or
tissues, candidate compounds having a high level of glial cell chloride
cotransporter
antagonist activity and having a reduced level of neuronal cell and renal cell
chloride
cotransporter antagonist activity may be identified. Similarly, effects on
different types
,
of cells and tissue systems may be assessed.
Additionally, the efficacy of candidate compounds may be assessed by
simulating
or inducing a condition, such as neuropathic pain, in situ in an animal model,
monitoring
the geometrical and/or optical properties of the cell or tissue sample during
stimulation of
the condition, administering the candidate compound, then monitoring the
geometrical
and/or optical properties of the cell or tissue sample following
administration of the
candidate compound, and comparing the geometrical and/or optical properties of
the cell
or tissue sample to determine the effect of the candidate compound. Testing
the efficacy
of treatment compositions for relief of neuropathic pain can be carried using
well known
methods and animal models, such as that described in Bennett, Hosp. Pract.
(Off Ed).
33:95-98, 1998.
As discussed above, compositions for use in the inventive methods may comprise
a treatment agent selected from the group consisting of: antibodies, or
antigen-binding
fragments thereof, that specifically bind to NKCC l; soluble ligands that bind
to NKCC 1;
anti-sense oligonucleotides to NKCCI; and small interfering RNA molecules
(siRNA or
RNAi) that are specific for NKCC I.
Antibodies that specifically bind to NKCC I are known in the art and include
those
available from Alpha Diagnostic International, Inc. (San Antonio, TX 78238).
An
"antigen-binding site," or "antigen-binding fragment" of an antibody refers to
the part of
the antibody that participates in antigen binding. The antigen binding site is
formed by
amino acid residues of the N-terminal variable ("V") regions of the heavy
("H") and light
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WO 2006/110187 PCT/US2005/043177
("L") chains. Three highly divergent stretches within the V regions of the
heavy and light
chains are referred to as "hypervariable regions" which are interposed between
more
conserved flanking stretches known as "framework regions," or "FRs". Thus the
term
"FR" refers to amino acid sequences which are naturally found between and
adjacent to
hypervariable regions in immunoglobulins. In an antibody molecule, the three
hypervariable regions of a light chain and the three hypervariable regions of
a heavy
chain are disposed relative to each other in three dimensional space to form
an antigen-
binding surface. The antigen-binding surface is complementary to the three-
dimensional
surface of a bound antigen, and the three hypervariable regions of each of the
heavy and
light chains are referred to as "complementarity-determining regions," or
"CDRs."
A number of molecules are known in the art that comprise antigen-binding sites
capable of exhibiting the binding properties of an antibody molecule. For
example, the
proteolytic enzyme papain preferentially cleaves IgG molecules to yield
several
fragments, two of which (the "F(ab)" fragments) each comprise a covalent
heterodimer
that includes an intact antigen-binding site. The enzyme pepsin is able to
cleave IgG
molecules to provide several fragments, including the "F(ab')2" fragment,
which
comprises both antigen-binding sites. An "Fv" fragment can be produced by
preferential
proteolytic cleavage of an IgM, IgG or IgA immunoglobulin molecule, but are
more
commonly derived using recombinant techniques known in the art. The Fv
fragment
includes a non-covalent VH::VL heterodimer including an antigen-binding site
which
retains much of the antigen recognition and binding capabilities of the native
antibody
molecule (Inbar et al. Proc. Natl. Acad. Sci. USA 69:2659-2662, 1972; Hochman
et al.
Biochem 15:2706-2710, 1976; and Ehrlich et al. Biochem 19:4091-4096, 1980).
Humanized antibodies that specifically bind to NKCCI may also be employed in
the inventive methods. A number of humanized antibody molecules comprising an
antigen-binding site derived from a non-human immunoglobulin have been
described,
including chimeric antibodies having rodent V regions and their associated
CDRs fused to
human constant domains (Winter et al. Nature 349:293-299, 1991; Lobuglio et
al. Proc.
Natl. Acad. Sci. USA 86:4220-4224, 1989; Shaw et al. J Immunol. 138:4534-4538,
1987;
and Brown et al. Cancer Res. 47:3577-3583, 1987); rodent CDRs grafted into a
human
supporting FR prior to fusion with an appropriate human antibody constant
domain
(Riechmann et al. Nature 332:323-327, 1988; Verhoeyen et al. Science 239:1534-
1536,
1988; and Jones et al. Nature 321:522-525, 1986); and rodent CDRs supported by
recombinantly veneered rodent FRs (European Patent Publication No. 519,596,
published
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WO 2006/110187 PCT/US2005/043177
Dec. 23, 1992). These "humanized" molecules are designed to minimize unwanted
immunological responses towards rodent antihuman antibody molecules which
limit the
duration and effectiveness of therapeutic applications of those moieties in
human
recipients.
Modulating the activity of NKCC 1 may alternatively be accomplished by
reducing or inhibiting expression of the polypeptide, which can be achieved by
interfering
with transcription and/or translation of the corresponding polynucleotide.
Polypeptide
expression may be inhibited, for example, by introducing anti-sense expression
vectors,
anti-sense oligodeoxyribonucleotides, anti-sense phosphorothioate oligodeoxy-
ribonucleotides, anti-sense oligoribonucleotides or anti-sense
phosphorothioate
oligoribonucleotides; or by other means well known in the art. All such anti-
sense
polynucleotides are referred to collectively herein as "anti-sense
oligonucleotides".
The anti-sense oligonucleotides for use in the inventive methods are
sufficiently
complementary to the NKCC1 polynucleotide to bind specifically to the
polynucleotide.
The sequence of an anti-sense oligonucleotide need not be 100% complementary
to the of
the polynucleotide in order for the anti-sense oligonucleotide to be effective
in the
inventive methods. Rather an anti-sense oligonucleotide is sufficiently
complementary
when binding of the anti-sense oligonucleotide to the polynucleotide
interferes with the
normal function of the polynucleotide to cause a loss of utility, and when non-
specific
binding of the oligonucleotide to other, non-target sequences is avoided. The
design of
appropriate anti-sense oligonucleotides is well known in the art.
Oligonucleotides that
are complementary to the 5' end of the message, for example the 5'
untranslated sequence
up to and including the AUG initiation codon, should work most efficiently at
inhibiting
translation. However, oligonucleotides complementary to either the 5'- or 3'-
non-
translated, non-coding, regions of the targeted polynucleotide may also be
employed.
Cell permeation and activity of anti-sense oligonucleotides can be enhanced by
appropriate chemical modifications, such as the use of phenoxazine-substituted
C-5
propynyl uracil oligonucleotides (Flanagan et al., Nat. Biotechnol. 17:48-52,
1999) or 2'-
O-(2-methoxy) ethyl (2'-MOE)-oligonucleotides (Zhang et al., Nat. Biotechnol.
18:862-
867, 2000). The use of techniques involving anti-sense oligonucleotides is
well known in
the art and is described, for example, in Robinson-Benion et al. (Methods in
Enzymol.
254:363-375, 1995) and Kawasaki et al. (Artific. Organs 20:836-848, 1996).
Expression of the NKCCI polypeptide may also be specifically suppressed by
methods such as RNA interference (RNAi). A review of this technique is found
in
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WO 2006/110187 PCT/US2005/043177
Science, 288:1370-1372, 2000. Briefly, traditional methods of gene
suppression,
employing anti-sense RNA or DNA, operate by binding to the reverse sequence of
a gene
of interest such that binding interferes with subsequent cellular processes
and therefore
blocks synthesis of the corresponding protein. RNAi also operates on a post-
translational
level and is sequence specific, but suppresses gene expression far more
efficiently.
Exemplary methods for controlling or modifying gene expression are provided in
WO
99/49029, WO 99/53050 and WO01/75164, the disclosures of which are hereby
incorporated by reference. In these methods, post-transcriptional gene
silencing is
brought about by a sequence-specific RNA degradation process which results in
the rapid
degradation of transcripts of sequence-related genes. Studies have shown that
double-
stranded RNA may act as a mediator of sequence-specific gene silencing (see,
for
example, Montgomery and Fire, Trends in Genetics, 14:255-258, 1998). Gene
constructs
that produce transcripts with self-complementary regions are particularly
efficient at gene
silencing.
It has been denionstrated that one or more ribonucleases specifically bind to
and
cleave double-stranded RNA into short fragments. The ribonuclease(s) remains
associated with these fragments, which in turn. specifically bind to
complementary
mRNA, i.e. specifically bind to the transcribed mRNA strand for the gene of
interest.
The mRNA for the gene is also degraded--by the ribonuclease(s) into short
fragments,
thereby obviating translation and expression of the gene. Additionally, an RNA-
:
polymerase may act to facilitate the synthesis of numerous copies of the
sliort fragments;.
which exponentially increases the efficiency of the system. A unique feature
of RNAi is.:.
that silencing is not limited to the cells where it is initiated. The gene-
silencing effects
may be disseminated to other parts of an organism.
The NKCC1 polynucleatide may thus be employed to generate gene silencing
constructs and/or gene-specific self-complementary, double-stranded RNA
sequences that
can be employed in the inventive methods using delivery methods known in the
art. A
gene construct may be employed to express the self-complementary RNA
sequences.
Alternatively, cells may be contacted with gene-specific double-stranded RNA
molecules,
such that the RNA molecules are internalized into the cell cytoplasm to exert
a gene
silencing effect. The double-stranded RNA must have sufficient homology to the
NKCCI gene to mediate RNAi without affecting expression of non-target genes.
The
double-stranded DNA is at least 20 nucleotides in length, and is preferably 21-
23
nucleotides in length. Preferably, the double-stranded RNA corresponds
specifically to a
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WO 2006/110187 PCT/US2005/043177
polynucleotide of the present invention. The use of small interfering RNA
(siRNA)
molecules of 21-23 nucleotides in length to suppress gene expression in
mammalian cells
is described in WO 01/75164. Tools for designing optimal inhibitory siRNAs
include
that available from DNAengine Inc. (Seattle, WA).
One RNAi technique employs genetic constructs within which sense and anti-
sense sequences are placed in regions flanking an intron sequence in proper
splicing
orientation with donor and acceptor splicing sites. Alternatively, spacer
sequences of
various lengths may be employed to separate self-complementary regions of
sequence in
the construct. During processing of the gene construct transcript, intron
sequences are
spliced-out, allowing sense and anti-sense sequences, as well as splice
junction
sequences, to bind forming double-stranded RNA. Select ribonucleases then bind
to and
cleave the double-stranded RNA, thereby initiating the cascade of events
leading to
degradation of specific mRNA gene sequences, and silencing specific genes.
For in vivo uses, a genetic construct, anti-sense oligonucleotide or RNA
molecule
may be administered by various art-recognized procedures (see, e.g., Rolland,
Crit. Rev.
Therap. Drug Carrier Systems 15:143-198, 1998, and cited references). Both
viral and
non-viral delivery methods have been used for gene therapy. Useful viral
vectors include,
for example, adenovirus, adeno-associated virus (AAV), retrovirus, vaccinia
virus and.
avian poxvirus. Improvements have been made in the efficiency of targeting
genes to
tumor cells with adenoviral vectors, for example, by coupling adenovirus to
DNA-
polylysine complexes and by strategies that exploit receptor-mediated
endocytosis for
selective targeting (see, e.g., Curiel et al., Hum. Gene Ther., 3:147-154,
1992; and
Cristiano & Curiel, Cancer Gene Ther. 3:49-57, 1996). Non-viral methods for
delivering
polynucleotides are reviewed in Chang & Seymour, (Eds) Curr. Opin. Mol. Ther.,
vol. 2,
2000. These methods include contacting cells with naked DNA, cationic
liposomes, or
polyplexes of polynucleotides with cationic polymers and dendrimers for
systemic
administration (Chang & Seymour, Ibid.). Liposomes can be modified by
incorporation
of ligands that recognize cell-surface receptors and allow targeting to
specific receptors
for uptake by receptor-mediated endocytosis (see, for example, Xu et al., Mol.
Genet.
Metab., 64:193-197; 1998; and Xu et al., Hum. Gene Ther., 10:2941-2952, 1999).
Tumor-targeting bacteria, such as Salmonella, are potentially useful for
delivering
genes to tumors following systemic administration (Low et al., Nat.
Biotechnol. 17:37-41,
1999). Bacteria can be engineered ex vivo to penetrate and to deliver DNA with
high
efficiency into, for example, mammalian epithelial cells in vivo (see, e.g.,
Grillot-
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Courvalin et al., Nat. Biotechnol. 16:862-866, 1998). Degradation-stabilized
oligonucleotides may be encapsulated into liposomes and delivered to patients
by
injection either intravenously or directly into a target site (for example,
the origin of
neuropathic pain). Alternatively, retroviral or adenoviral vectors, or naked
DNA
expressing anti-sense RNA for the inventive polypeptides, may be administered
to
patients. Suitable techniques for use in such methods are well known in the
art.
The treatment compositions and methods of the present invention have been
described, above, with respect to certain preferred embodiments. The Examples
set forth
below describe the results of specific experiments and are not intended to
limit the
invention in any fashion.
EXAMPLE 1
Methyl 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate
(Bumetanide Methyl Ester)
To a slurry of bumetanide (1.2g, 3.29mmol) in methanol (12mL) under nitrogen,
was added a mixture of thionyl chloride (70uL) in methanol (6mL) over 5
minutes. After
stirring for 5 minutes the reaction mixture became soluble. The reaction was
stirred for
an additional 30 minutes, at which time the reaction was complete as
determined by thin
layer chromatography (TLC). The methanol was removed under reduced pressure
and
the residue was brought up in ethyl acetate and washed with saturated sodium
bicarbonate, water and brine. The ethyl acetate was dried over anhydrous
magnesium
sulfate and concentrated to yield 1.1g (89%) of methyl 3-aminosulfonyl-5-
butylamino-4-
phenoxybenzoate as a white solid.
EXAMPLE 2
CYanomethyl 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate
(Bumetanide Cyanomethyl Ester)
Bumetanide (1.0g, 2.7mmol) was dissolved in dimethylformamide (DMF) and
chloroacetonitrile (195uL, 2.7mmol) was added followed by triethylamine
(465uL). The
reaction was heated to 100 C for 12 hours, at which time TLC and liquid
chromatography-coupled mass spectrometry (LC/MS) indicated the reaction was
complete. The reaction was cooled to room temperature, brought up in
dichloromethane
and washed with water, saturated with ammonium chloride and reduced to a
slurry. To
the slurry was added water (25mL) and crude product precipitated as an off
white solid.
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Pure cyanomethyl 3-3minosulfonyl-5-butylamino-4-phenoxybenzoate (850mg) was
obtained via recrystallization in acetonitrile.
EXAMPLE 3
Benzyl 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate
(Bumetanide Benzyl Ester)
Bumetanide (1.15g, 3.15mmo1) was dissolved in dimethylformamide (DMF,
IOmL) and benzyl chloride (400uL, 2.8mmol) was added followed by triethylamine
(480uL). The reaction was heated to 80 C for 12 hours, at which time TLC and
LC/MS
indicated the reaction was complete. The reaction was cooled to room
temperature,
brought up in dichloromethane and washed with water, saturated ammonium
chloride and
concentrated to a thick slurry. To the slurry was added water (25mL). The
resultant
solids were filtered and dried in a vacuum oven at 50 C for 12 hours to yield
I.Og (80%)
of benzyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.
EXAMPLE 4
2-(4-Morpholino)ethyl 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate
(Bumetanide Morpholinoethyl Ester)
Bumetanide (1.2g, 3.29mmol) was dissolved in dimethylformamide (DMF, 12mL)
and 4-(2-chloroethyl)morpholine hydrochloride (675mg, 3.62mmo1) was added
followed
by triethylamine (1mL) and sodium iodide (500mg 3.33mmol). The reaction was
heated
to 95 C for 8 hours, at which time TLC and LC/MS indicated the reaction was
complete.
The reaction was cooled to room temperature brought iup in dichloromethane and
washed
with water, saturated ammonium chloride and concentrated to dryness. After
purification
via biotage flash chromatography, the purified elute, on evaporation under
vacuum,
yielded 2-(4-morpholino)ethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate
as a
white solid (600mg, 62%).
EXAMPLE 5
3-(N,N-Dimethylaminopropyl) 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate
(Bumetanide 3-(Dimethylaminopropyl) Esterl
In a similar manner to Example 31, bumetanide can be reacted with 3-
(dimethylamino)propyl chloride hydrochloride, triethylamine and sodium iodide
in
dimethylformamide (DMF) to yield 3-(N,N-dimethylaminopropyl) 3-aminosulfonyl-5-
butylamino-4-phenoxybenzoate.
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EXAMPLE 6
N,N-Diethylaminocarbonylmethyl 3-Aminosulfonyl-5-butylamino-4-
phenoxybenzoate
(Bumetanide N,N-Diethylglycolamide Ester )
Bumetanide (1.2g, 3.29mmo1) was dissolved in dimethylformamide (12mL) and
2-chloro-N,N-diethylacetamide (500mg, 3.35mmol) was added followed by
triethylamine
(0.68mL) and sodium iodide (500mg 3.33mmol). The reaction was heated to 95 C
for 8
hours, at which time TLC and LC/MS indicated the reaction was complete. The
reaction
was cooled to room temperature brought up in dichloromethane and washed with
water,
saturated ammonium chloride and reduced to a thick slurry. To the slurry was
added
water (25mL), and the resultant solids precipitated from the solution. The
product was
filtered and dried in a vacuum oven at 50 C for 12 hours to yield 1.Og of N,N-
diethylaminocarbonylmethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.
EXAMPLE 7
N,N-Diethyl 3-Aminosulfonyl-5-butylamino-4-phenoxybenzamide
(Bumetanide Diethylamide)
Bumetanide (1.16g, 3.2mmol) was dissolved in dichloromethane (lOmL) and 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 690mg, 3.6mmo1) was added.
After 5 minutes N-hydroxybenzotriazole (HOBt, 498mg, 3.6mmol) was added and
the
solution was stirred for an additional 5 minutes. Diethylamine (332uL,
3.2mmol) was
added and the reaction was stirred for 2 hours. The reaction was washed with
saturated
sodium bicarbonate, water and brine, and dried with magnesium sulfate. The
dichloromethane was removed under reduced pressure to yield 860mg (65%) of
pure
N,N-diethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzamide.
EXAMPLE 8
N,N-Dibenzyl 3-Aminosulfonyf-5-butylamino-4-phenoxybenzamide
(Bumetanide Dibenzylamide)
Bumetanide (960mg, 2.6mmo1) was dissolved in dimethylformamide (DMF,
lOmL) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 560mg, 3.6mmol)
was added. After 10 minutes 1-hydroxybenzotriazole (HOBt, 392mg, 2.9mmol) was
added and the solution was stirred for an additional 10 minutes. Dibenzylamine
(1mL,
5.2mmol) was added and the reaction was stirred for 2 hours, at which time the
reaction
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was complete by LC/MS. The reaction was poured into saturated ammonium
chloride
(20mL) and extracted with ethyl acetate (2xlOOmL). The ethyl acetate was
washed with
saturated sodium bicarbonate, water and brine, and dried over anhydrous
magnesium
sulfate. The ethyl acetate was removed under reduced pressure to yield 1.Og
(75%) of
N,N-dibenzyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzamide as white solid.
EXAMPLE 9
Benzyltrimethylammonium 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate
(Bumetanide Benzylltrimethylammonium Salt)
To a solution of benzyltrimethylammonium hydroxide (451mg, 2.7mmol) in water
(10mL) was added bumetanide (I g, 2.7 mmol) over a period of 5 minutes. The
reaction
mixture became clear after 10 minutes of stirring. The water was removed under
reduced
pressure to yield a crude colorless oil. Pure product was obtained from
recrystallization
of the oil with water and heptane to yield 690mg of benzyltrimethylammonium 3-
aminosulfonyl-5-butylamino-4-phenoxybenzoate as light pink crystals.
EXAMPLE 10
Cetyltrimethylammonium 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate
(Bumetanide Cetylltrimethylammonium Salt)
In a similar manner to Example 9, bumetanide can be reacted with
cetyltrimethylammonium hydroxide in water to yield cetyltrimethylammonium 3-
aminosulfonyl-5-butylamino-4-phenoxybenzoate.
EXAMPLE 11
N,N-Dimethylaminocarbonylmethyl 3-Aminosulfonyl-5-butylamino-4-
phenoxybenzoate
(Bumetanide N,N-Dimethylglycolamide Ester)
Bumetanide (1.2g, 3.29mmol) was dissolved in dimethylformamide (DMF, IOmL)
and 2-chloro-N,N-dimethylamide (410uL, 3.9mmol) was added, followed by
triethylamine (.70mL) and sodium iodide (545mg, 3.6mmol). The reaction was
heated to
50 C for 10 hours, at which time TLC and LC/MS indicated the reaction was
complete.
The solvent was removed under reduced pressure and the residue was dissolved
in ethyl
acetate and washed with saturated sodium bicarbonate, water and brine, and
dried over
anhydrous magnesium sulfate. The ethyl acetate was removed under reduced
pressure
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and the product was purified via flash chromatography to yield 685mg (60%) of
pure
N,N-dimethylaminocarbonylmethyl3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.
EXAMPLE 12
t-Butylcarbonyloxymethyl 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate
(Bumetanide Pivaxetil Ester)
Bumetanide (1.2g, 3.29mmol) was dissolved in dimethylformamide (DMF, lOmL)
and chloromethyl pivalate (575uL, 3.9mmol) was added followed by triethylamine
(0.70mL) and sodium iodide (545mg, 3.6mmol). The reaction was heated to 50 C
for 10
hours, at which time TLC and LC/MS indicated the reaction was complete. The
solvent
was removed under reduced pressure and the residue was dissolved in ethyl
acetate and
washed with saturated sodium bicarbonate, water and brine, and dried over
anhydrous
magnesium sulfate. The ethyl acetate was removed under reduced pressure and
the
product was purified via flash chromatography to yield 653mg (60%) of pure t-
butylcarbonyloxymethyl 3 -aminosulfonyl-5-butylamino-4-phenoxybenzoate.
EXAMPLE 13
Ethylcarbonyloxymethyl 3-Aminosulfonyl-5-butylamino-4-phenoxybenzoate
(Bumetanide Propaxetil Ester)
In a similar manner to Example 12, bumetanide can be reacted with chloromethyl
propionate, triethylamine and sodium iodide in dimethylformamide (DMF) to
yield
ethylcarbonyloxymethyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzoate.
EXAMPLE 14
Methyl 3-Aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate
(Piretanide Methyl Ester)
In a similar manner to Example 1, piretanide can be reacted with thionyl
chloride
and methanol to yield methyl 3-aminosulfonyl-4-phenoxy-5-(1-
pyrrolidinyl)benzoate.
EXAMPLE 15
CVanomethyl3-Aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate
(Piretanide Cyanomethyl Ester)
In a similar manner to Example 2, piretanide can be reacted with
chloroacetonitrile in DMF to yield cyanomethyl 3-aminosulfonyl-4-phenoxy-5-(1-
pyrrolidinyl)benzoate.
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EXAMPLE 16
Benzyl 3-Aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate
(Piretanide Benzyl Ester)
In a similar manner to Example 3, piretanide can be reacted with benzyl
chloride
in DMF to yield benzyl3-aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate.
EXAMPLE 17
2-(4-Morpholino)ethyl 3-Aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate
(Piretanide Morpholinoethyl Ester)
In a similar manner to Example 4, piretanide can be reacted with 4-(2-
chloroethvl)morpholine hydrochloride, triethylamine and sodium iodide in DMF
to yield
2-(4-morpholino)ethyl3-aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate.
EXAMPLE 18
3-(N,N-Dimethylaminopropyl 3-Aminosulfonyl-4-phenoxy-5-(1-
pyrrolidinyl)benzoate
(Piretanide 3-(Dimethylaminopropyl) Esterl
In a similar manner to Exaniple 31, piretanide can be reacted with 3-
(dimethylanlino)propyl chloride hydrochloride, triethylamine and sodium iodide
in
dimethylfonnamide (DMF) to yield 3-(N,N-dimethylaminopropyl 3-aminosulfonyl-4-
phenoxy-5-(1-pyrroli dinyl)benzoate.
EXAMPLE 19
N,N-Diethylaminocarbonylmethyl 3-Aminosulfonyl-4-phenoxy-5-
JU (1-pyrrolidinyl)benzoate
(Piretanide N,N-Diethylglycolamide Ester)
In a similar manner to Example 6, piretanide can be reacted with 2-chloro-N,N-
diethylacetamide, triethylamine and sodium iodide in dimethylformamide (DMF)
to yield
N,N-diethylaminocarbonylmethyl 3-aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)-
benzoate.
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EXAMPLE 20
N,N-Diethyl 3-Aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate
(Piretanide Diethylamide)
In a similar manner to Example 7, piretanide can be reacted with EDC, HOBt and
diethylamine in DMF to yield N,N-diethyl 3-aminosulfonyl-4-phenoxy-5-(1-
pyrrolidinyl)-benzamide.
EXAMPLE 21
N,N-Dibenzyl 3-Aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate
(Piretanide Dibenzylamide)
In a similar manner to Example 8, piretanide can be reacted with EDC, HOBt and
dibenzylamine in DMF to yield N,N-dibenzyl 3-aminosulfonyl-4-phenoxy-5-(1-
pyrrolidinyl) benzamide.
EXAMPLE 22
Benzyltrimethylammonium 3-Aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate
(Piretanide Benzylltrimethylammonium Salt)
In a similar manner to Example 9, piretanide can be reacted with
benzyltrimethylammonium hydroxide to yield benzyltrimethylammonium 3-
aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate.
EXAMPLE 23
Cetyltrimethylammonium 3-Aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate
(Piretanide Cetylltrimethylammonium Salt)
In a similar manner to Example 10, piretanide can be reacted with
cetyltrimethylammonium hydroxide in water to yield cetyltrimethylammonium 3-
aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate.
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EXAMPLE 24
N,N-Dimethylaminocarbonylmethvl 3-Aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)-
benzoate
(Piretanide N,N-Dimethylglycolamide Ester)
In a similar manner to Example 11, piretanide can be reacted with 2-chloro-N,N
dimethylacetamide, triethylamine and sodium iodide in DMF to yield N,N-
dimethylaminocarbonylmethyl 3-aminosulfonyl-4-phenoxy-5-(1-
pyrrolidinyl)benzoate.
EXAMPLE 25
t-Butylcarbonyloxymethyl 3-Aminosulfonvl-4-phenoxy-5-(1-pyrrolidinyl)benzoate
(Piretanide Pivaxetil Ester)
In a similar manner to Example 12, piretanide can be reacted with chloromethyl
pivalate, triethylamine and sodium iodide in DMF to yield-t-
butylcarbonyloxymethyl 3-
aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate.
EXAMPLE 26
Ethylcarbonvloxymethyl 3-Aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate
(Piretanide Propaxetil Ester)
In a similar manner to Example 13, piretanide can be reacted with chloromethyl
propionate, triethylamine and sodium iodide in DMF to yield
ethylcarbonyloxymethyl 3-
aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzoate.
EXAMPLE 27
Ethyl 5-Aminosulfonyl-4-chloro-2-1(2-furanylmethyl)aminolbenzoate
(Furosemide Ethyl Ester)
The method of Bundgaard, H., Norgaard, T. and Nielsen, N. M., Int. J.
Pharmaceutics, 1988, 42, 217-224, can be employed to prepare ethyl 5-
aminosulfonyl-4-
chloro-2-[(2-furanylmethyl)amino]benzoate, m.p. 163 - 165 .
EXAMPLE 28
Cyanomethyl5-Aminosulfonyl-4-chloro-2-[(2-furanylmethyl)aminolbenzoate
(Furosemide Cyanomethyl Ester)
In a similar manner to Example 2, furosemide can be reacted with
chloroacetonitrile in DMF to yield cyanomethyl 5-aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)amino]-benzoate.
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EXAMPLE 29
Benzyl 5-Aminosulfonyl-4-chloro-2-1(2-furanylmethyl)aminolbenzoate
(Furosemide Benzyl Ester)
In a similar manner to Example 3, furosemide can be reacted with benzyl
chloride
in DMF to yield benzyl 5-aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)amino]benzoate.
EXAMPLE 30
2-(4-Morpholino)ethyl 5-Aminosulfonyl-4-chloro-2-f (2-
furanylmethyl)aminolbenzoate
(Furosemide Morpholinoethyl Ester)
The method of Mork, N., Bundgaard, H., Shalmi, M. and Christensen, S., Int. J.
Pharmaceutics, 1990, 60, 163-169, can be employed to prepare 2-(4-
morpholino)ethyl 5-
aminosulfonyl-4-chloro-2-[(2-furanylmethyl)amino]benzoate, m.p. 134 - 135 .
EXAMPLE 31
3-(N,N-Dimethylaminopropyl 5-Aminosulfonyl-4-chloro-2-[(2-
fu ranylmethyl)aminol benzoate
(Furosemide 3-(Dimethylaminopropyl) Ester)
The method of Mork, N., Bundgaard, H., Shalmi, M. and Christensen, S., Int. J.
Pharmaceutics, 1990, 60, 163-169, can be employed to prepare 3-(N,N-
dimethylaminopropyl 5-aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)amino]benzoate,
m.p.212-213 .
EXAMPLE 32
N,N-Diethylaminocarbonylmethyl 5-Aminosulfonyl-4-chloro-2-f (2-
furanylmethyl)aminolbenzoate
(Furosemide N,N-Diethylglycolamide Ester)
The method of Mork, N., Bundgaard, H., Shalmi, M. and Christensen, S., Int. J.
Pharmaceutics, 1990, 60, 163-169, can be employed to prepare N,N-diethyl-
aminocarbonylmethyl 5-aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)amino]benzoate,
m.p. 135 - 136 .
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EXAMPLE 33
N,N-Diethyl 5-Aminosulfonyl-4-chloro-2-[(2-furanylmethyl)aminolbenzamide
(Furosemide Diethylamide)
In a similar manner to Example 7, furosemide can be reacted with EDC, HOBt
and diethylamine in DMF to yield N,N-diethyl 5-aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)-amino]benzamide.
EXAMPLE 34
N,N-Dibenzyl 5-Aminosulfonyl-4-chloro-2-1(2-furanylmethyl)aminolbenzamide
(Furosemide Dibenzylamide)
In similar manner to Example 8, furosemide can be reacted with EDC, HOBt and
dibenzylamine in DMF to yield N,N-dibenzyl 5-aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)amino] benzamide.
EXAMPLE 35
Benzyltrimethylammonium 5-Aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)aminolbenzoate
(Furosemide Benzyltrimethylammonium Salt)
In similar manner to Example 9, furosemide can be reacted with
benzyltrimethylammonium hydroxide to yield benzyltrimethylammonium 5-
aminosulfonyl-4-chloro-2-[(2-furanylmethyl)amino]benzoate.
EXAMPLE 36
Cetyltrimethylammonium 5-Aminosulfonyl-4-chloro-2-1(2-
furanylmethyl)aminolbenzoate
(Furosemide Cetyltrimethylammonium Salt)
In similar manner to Example 10, furosemide can be reacted with
cetyltrimethylammonium hydroxide in water to yield cetyltrimethylammonium 5-
aminosulfonyl-4-chloro-2-[(2-furanylmethyl)amino]benzoate.
EXAMPLE 37
N,N-Dimethylaminocarbonylmethyl 5-Aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)aminolbenzoate
(Furosemide N,N-Dimethylplycolamide Ester)
The method of Bundgaard, H., Norgaard, T. and Nielsen, N. M., Int. .I.
Pharmaceutics, 1988, 42, 217-224, can be employed to prepare N,N-
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dimethylaminocarbonylmethyl 5-aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)amino]-
benzoate, m.p. 193 - 194 .
EXAMPLE 38
t-Butylcarbonyloxymethyl 5-Aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)aminolbenzoate
(Furosemide Pivaxetil Ester)
The method of Mork, N., Bundgaard, H., Shalmi, M. and Christensen, S., Int. J.
Pharmaceutics, 1990, 60, 163-169, can be employed to prepare t-
butylcarbonyloxymethyl
5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl)amino]benzoate.
EXAMPLE 39
Ethylcarbonyloxymethyl 5-Aminosulfonyl-4-chloro-2-1(2-
furanylmethyl)aminolbenzoate
(Furosemide Propaxetil Ester)
The method of Mork, N., Bundgaard, H., Shalmi, M. and Christensen, S., Int. J.
Pharmaceutics, 1990, 60, 163-169, can be employed to prepare
ethylcarbonyloxymethyl
5-aminosulfonyl-4-chloro-2-[(2-furanylmethyl)amino]benzoate, m.p. 141 - 142 .
EXAMPLE 40
5-(1-(t-Butylcarbonyloxymethyl)-1H-tetrazol-5-yll-2-chloro-4-((2-
thienylmethyl)aminolbenzenesulfonamide
(Tetrazolyl-Substituted Azosemide)
In a similar manner to Example 12, azosemide can be reacted with chloromethyl
pivalate, triethylamine and sodium iodide in DMF to yield 5-[1-(t-
Butylcarbonyloxymethyl)-1 H-tetrazol-5-yl]-2-chloro--4-[(2-
thienylmethyl)amino]benzenesulfonamide.
EXAMPLE 41
2-Chloro-5-[ 1-(ethylcarbonyloxymethyl)-1 H-tetrazol-5-yl1-4-[(2-
thienylmethyl)aminolbenzenesulfonamide
(Tetrazolyl-Substituted Azosemide)
In similar manner to Example 12, azosemide can be reacted with chloromethyl
propionate, triethylamine and sodium iodide in DMF to yield 2-chloro-5-[1-
(ethylcarbonyloxymethyl)-1 H-tetrazol-5-yl]-4-[(2-
thienylmethyl)amino]benzenesulfonamide.
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EXAMPLE 42
2-Chloro-5-f 1-(hvdroxymethyl)-1H-tetrazol-5-yll-4-f (2-
thienylmethyl)aminolbenzenesulfonamide
(Tetrazolyl-Substituted Azosemide)
Azosemide can be reacted with formaldehyde in methylene chloride, methylene
chloride-DMF mixtures or DMF to yield 2-chloro-5-[1-(hydroxymethyl)-1H-
tetrazol-5-
yl]-4-[(2-thienylmethyl)amino]benzenesulfonamide.
EXAMPLE 43
1'5 2-Chloro-5- f 1-(methoxymethyl)-1H-tetrazol-5-yll-4-f (2-
thienylmethyl)aminolbenzenesulfonamide
(Tetrazolyl-Substituted Azosemide)
Azosemide can be reacted with ' formaldehyde and methanol in methylene
chloride, methylene chloride-DMF mixtures or DMF to yield 2-chloro-5-[1-
(methoxymethyl)-1 H-tetrazol-5-yl]-4-[(2-
thienylmethyl)amino]benzenesulfonamide.
EXAMPLE 44
2-Chloro-5-11-(methylthiomethyl)-iH-tetrazol-5-yll-4-f (2-
thienylmethyl)aminolbenzenesulfonamide
(Tetrazolyl-Substituted Azosemide)
Azosemide can be reacted with formaldehyde and methanethiol in methylene
chloride, methylene chloride-DMF mixtures or DMF to yield 2-chloro-5-[1-
(methylthiomethyl)-1 H-tetrazol-5-yl]-4-[(2-
thienylmethyl)amino]benzenesulfonamide.
EXAMPLE 45
5- f 1-(Benzyloxymethyl)-1 H-tetrazol-5-yll- 2-chloro-4- f(2-
thienylmethyl)aminolbenzenesulfonamide
(Tetrazolyl-Substituted Azosemide)
Azosemide can be reacted with benzyl chloromethyl ether, triethylamine and
sodium iodide in DMF to yield 5-[1-(benzyloxymethyl)-1H-tetrazol-5-yl]- 2-
chloro-4-[(2-
thienylmethyl)amino]benzenesulfonamide.
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EXAMPLE 46
Benzyltrimethylammonium Salt of 2-Chloro-5-(1H-tetrazol-5-yl)-4-[(2-
thienylmethyl)aminolbenzenesulfonamide
(Azosemide Benzyltrimethylammonium Salt)
In similar manner to Example 9, azosemide can be reacted with
benzyltrimethylammonium hydroxide in water to yield the
benzyltrimethylammonium
salt of 2-chloro-5-(1 H-tetrazol-5-yl)-4-[(2-
thienylmethyl)amino]benzenesulfonamide.
EXAMPLE 47
Cetyltrimethylammonium Salt of 2-Chloro-5-(1H-tetrazol-5-yl)-4-[(2-
thienylmethyl)aminolbenzenesulfonamide
(Azosemide Cetyltrimethylammonium Salt)
In similar manner to Example 9, azosemide can be reacted with
cetyltrimethylammonium hydroxide in water to yield the cetyltrimethylammonium
salt of
2-chloro-5-(1 H-tetrazol-5-yl)-4-[(2-thienylmethyl)amino]benzenesulfonamide.
EXAMPLE 48
3-Isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium t-
Butylcarbonyloxymethochloride
(Pyridinium-Substituted Torsemide Salt)
In similar manner to Example 12, torsemide can be reacted with chloromethyl
pivalate, triethylamine and sodium iodide in DMF to yield 3-
isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium t-
butylcarbonyloxymethochloride and some 3-isopropylcarbamylsulfonamido-4-(3'-
methylphenyl)aminopyridinium t-butylcarbonyloxy-methoiodide.
EXAMPLE 49
3-Isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
Ethylcarbonyloxymethochloride
(Pyridinium-Substituted Torsemide Salt)
In similar manner to Example 12, torsemide can be reacted with chloromethyl
propionate, triethylamine and sodium iodide in DMF to yield 3-
isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
ethylcarbonyloxy-
methochloride and some 3-isopropylcarbamylsulfonamido-4-(3'-methylphenyl)-
aminopyridinium ethylcarbonyloxymethoiodide.
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EXAMPLE 50
3-Isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
benzyloxymethochloride
(Pyridinium-Substituted Torsemide Salt)
In a similar manner to Example 3, torsemide can be reacted with benzyl
chloromethyl ether and triethylamine in DMF to yield 3-
isopropylcarbamylsulfonamido-
4-(3'-methylphenyl)aminopyridinium benzyloxymethochloride.
EXAMPLE 51
3-Isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
methoxymethochloride
(Pyridinium-Substituted Torsemide Salt)
In a similar manner to Example 3, torsemide can be reacted with methyl
chloromethyl ether and triethylamine and in DMF to yield 3-
isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
methoxymethochloride.
EXAMPLE 52
3-Isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
phenylmethochloride
(Pyridinium-Substituted Torsemide Salt)
In a similar manner to Example 3, torsemide can be reacted with benzyl
chloride
and triethylamine in DMF to yield 3-isopropylcarbamylsulfonamido-4-(3'-
methylphenyl)-aminopyridinium phenylmethochloride.
EXAMPLE 53
3-Isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
Benzylthiomethochloride
(Pyridinium-Substituted Torsemide Salt)
In a similar manner to Example 3, torsemide can be reacted with benzyl
chloromethyl thioether and triethylamine in DMF to yield 3-
isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyri dinium
benzylthiamethochloride.
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EXAMPLE 54
3-Isopropvlcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
Methylthiomethochloride
(Pyridinium-Substituted Torsemide Salt)
In a similar manner to Example 3, torsemide can be reacted with methyl
chloromethyl thioether and triethylamine and in DMF to yield 3-
isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
methylthiametho-
chloride.
EXAMPLE 55
Methoxy(polyethvleneoxy),,_i-ethyl 3-Aminosulfonyl-5-butylamino-4-
phenoxybenzoate (Bumetanide mPEG350 Esters)
In a manner similar to Example 3, bumetanide can be reacted with MeO-PEG350-
Cl (Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in
DMF to
yield methoxy(polyethyleneoxy)õ_i-ethyl 3-aminosulfonyl-5-butylamino-4-
phenoxybenzoate where n is in the 7-8 range.
EXAMPLE 56
Methoxy(polyethyleneoxy)õ_~-ethyl 3-Aminosulfonyl-5-butylamino-4-
phenoxybenzoate (Bumetanide mPEG1000 Esters)
In a manner similar to Example 3, bumetanide can be reacted with MeO-
PEG1000-OTs (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and
triethylamine
in DMF to yield methoxy(polyethyleneoxy)õ_i-ethyl 3-aminosulfonyl-5-butylamino-
4-
phenoxybenzoate where n is in the 19-24 range.
EXAMPLE 57
Methoxy(polyethyleneoxy)õ_i-ethyl 3-Aminosulfonyl-4-phenoxy-5-(1-
pyrrolidinyl)benzoate (Piretanide mPEG350 Esters)
In similar manner to Example 3, piretanide can be reacted with MeO-PEG350-Cl
(Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF
to yield
methoxy(polyethyleneoxy),_i-ethyl 3-aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)-
benzoate where n is in the 7-8 range.
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EXAMPLE 58
Methoxv(polyethyleneoxy)õ_1 -ethyl 3-Aminosulfonyl-4-phenoxy-5-(1-
pyrrolidinyl)benzoate (Piretanide mPEGl000 Esters)
In similar manner to Example 3, piretanide can be reacted with MeO-PEG1000-
OTs (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in
DMF to
yield methoxy(polyethyleneoxy)õ_ i-ethyl 3-aminosulfonyl-4-phenoxy-5-(I-
pyrrolidinyl)-
benzoate where n is in the 19-24 range.
EXAMPLE 59
Methoxy(polyethyleneoxy)õ_~-ethyl 5-Aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)aminolbenzoate (Furosemide mPEG350 Esters)
In similar manner to Example 3, furosemide can be reacted with MeO-PEG350-Cl
(Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF
to yield
methoxy(polyethyleneoxy)õ_ i -ethyl 5-aminosul fonyl-4-chloro-2-[(2-
furanylmethyl)-
amino]-benzoate where n is in the 7-8 range.
EXAMPLE 60
Methoxy(po1yethy1eneoxy)õ_i-ethyl5-Aminosulfonyl-4-chloro-2- f (2-
furanylmethyl)aminolbenzoate (Furosemide mPEG1000 Esters)
In similar manner to Example 3, furosemide can be reacted with MeO-PEG1000-
OTs (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in
DMF to
yield methoxy(polyethyleneoxy)õ_i-ethyl 5-aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)-
amino]-benzoate where n is in the 19-24 range.
EXAMPLE 61
541-[Methoxv(polyethyleneoxy)õ-, -ethyl] -lH-tetrazol-5-yll-2-chloro-4-1(2-
thienylmethyl)aminolbenzenesulfonamides
(N-mPEG350-Tetrazolyl-Substituted Azosemides)
In similar manner to Example 3, azosemide can be reacted with MeO-PEG350-Cl
(Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF
to yield
5-[1-[methoxy(polyethyleneoxy),,.i-ethyl]-1H-tetrazol-5-yl]-2-chloro-4-[(2-
thienylmethyl)-amino]benzenesulfonamides where n is in the 7-8 range.
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EXAMPLE 62
5-(1-[Methoxy(polyethyleneoxv)õ_~ -ethyll-1 H-tetrazol-5-yl] -2-chloro-4-[(2-
thienylmethyl)aminolbenzenesulfonamides
(N-mPEG1000-Tetrazolyl-Substituted Azosemides)
In similar manner to Example 3, azosemide can be reacted with MeO-PEG1000-
OTs (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in
DMF to
yield 5-[1-[methoxy(polyethyleneoxy)õ_1 -ethyl]-1H-tetrazol-5-yl]-2-chloro-4-
[(2-
thienylmethyl)-amino]benzenesulfonamides where n is in the 19-24 range.
EXAMPLE 63
3-Isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
Methoxy(polyethyleneoxy)õ_j -ethochlorides
(N-mPEG350-Pyridinium Torsemide Salts)
In similar manner to Example 3, torsemide can be reacted with MeO-PEG350-Cl
(Biolink Life Sciences, Inc., Cary, NC, BLS-106-350) and triethylamine in DMF
to yield
3-isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
methoxy(polyethyleneoxy)õ_I-ethochlorides where n is in the 7-8 range.
EXAMPLE 64
3-Isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
Methoxy(polyethyleneoxy)õ-,-ethochlorides
(N-mPEG1000-Pyridinium Torsemide Salts)
In similar manner to Example 3, torsemide can be reacted with MeO-PEG1000-
OTs (Biolink Life Sciences, Inc., Cary, NC, BLS-107-1000) and triethylamine in
DMF to
yield 3-isopropylcarbamylsulfonamido-4-(3'-methylphenyl)aminopyridinium
methoxy-
(polyethyleneoxy),,_i-ethochlorides where n is in the 19-24 range.
EXAMPLE 65
3-Aminosulfonyl-5-butylamino-4-phenoxybenzaldehyde (Bumetanide Aldehyde)
By the method of Muraki and Mukiayama (Chem. Letters, 1974, 1447 and Chem.
Letters, 1975, 215), bumetanide can be reacted with bis(4-
methylpiperazinyl)aluminum
hydride to yield 3-aminosulfonyl-5-butylamino-4-phenoxybenzaldehyde.
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EXAMPLE 66
3-Aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzaldehyde
(Piretanide Aldehyde)
By the method of Muraki and Mukiayama (Chem. Letters, 1974, 1447 and Chem.
Letters, 1975, 215), piretanide can be reacted with bis(4-
methylpiperazinyl)aluminum
hydride to yield 3-aminosulfonyl-4-phenoxy-5-(1-pyrrolidinyl)benzaldehyde.
EXAMPLE 67
5-Aminosulfonyl-4-chloro-2-1(2-furanylmethyl)aminolbenzaldehyde
(Furosemide Aldehyde)
By the method of Muraki and Mukiayama (Chem. Letters, 1974, 1447 and Chem.
Letters, 1975, 215), furosemide can be reacted with bis(4-
methylpiperazinyl)aluminum
hydride to yield 5-aminosulfonyl-4-chloro-2-[(2-
furanylmethyl)amino]benzaldehyde.
EXAMPLE 68
The Effects of Furosemide on Epileptiform Discharjles in Hippocampal Slices
During these studies, spontaneous epileptiform activity was elicited by a
variety of
treatments. Sprague-Dawley rats (males and females; 25-35 days old) were
decapitated,
the top of the skull was rapidly removed, and the brain chilled with ice-cold
oxygenated
slicing medium. The slicing medium was a sucrose-based artificial
cerebrospinal fluid
(sACSF) consisting of 220 mM sucrose, 3 mM KCI, 1.25 mM NaHZPO4, 2 mM MgSO4,
26 mM NaHCO3, 2 mM CaClz, and 10 mM dextrose (295-305 mOsm). A hemisphere of
brain containing hippocampus was blocked and glued (cyanoacrylic adhesive) to
the stage
of a Vibroslicer (Frederick Haer, Brunsick, ME). Horizontal or transverse
slices 400 m
thick were cut in 4 C, oxygenated (95% 02; 5% C02) slicing medium. The slices
were
immediately transferred to a holding chamber where they remained submerged in
oxygenated bathing medium (ACSF) consisting of 124 mM NaCl, 3 mM KCI, 1.25 mM
NaH2PO4, 2 mM MgSO4, 26 mM NaHCO3, 2 mM CaClz, and 10 mM dextrose (295-305
mOsm). The slices were held at room temperature for at least 45 minutes before
being
transferred to a submersion-style recording chamber (all other experiments).
In the
recording chamber, the slices were perfused with oxygenated recording medium
at 34-35
C. All animal procedures were conducted in accordance with NIH and University
of
Washington animal care guidelines.
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In most slice experiments, simultaneous extracellular field electrode
recordings
were obtained from CA 1 and CA3 areas. A bipolar tungsten stimulating
electrode was
placed on the Schaffer collaterals to evoke synaptically-driven field
responses in CA 1.
Stimuli consisted of 100-300 sec duration pulses at an intensity of four
times the
population-spike threshold. After discharges were evoked by a 2 second train
of such
stimuli delivered at 60 Hz. Spontaneous interictal-like bursts were observed
in slices
treated by the following modifications or additions to the bathing medium: 10
mM
potassium (6 slices; 4 animals; average - 81 bursts/min.); 200-300 M 4-
aminopyridine
(4 slices; 2 animals; average - 33 burst/min.); 50-100 M bicuculline (4
slices; 3 animals;
average - 14 bursts/min); M Mg++ (1 hour of perfusion - 3 slices; 2 animals;
average -
20 bursts/min. or 3 hours of perfusion - 2 slices; 2 animals); zero calcium/6
mM KCI and
2 mM EGTA (4 slices; 3 animals). In all treatments, furosemide was added to
the
recording medium once a consistent level of bursting was established.
In the first of these procedures, episodes of after discharges were evoked by
electrical stimulation of the Schaffer collaterals (Stasheff et al., Brain
Res. 344:296, 1985)
and the extracellular field response was monitored in the CAl pyramidal cell
region (13
slices; 8 animals). The concentration of Mg++ in the bathing medium was
reduced to 0.9
mM and after discharges were evoked by stimulation at 60 Hz for 2 seconds at
an
intensity 4 times the population spike threshold (population spike threshold
intensity
varied between 20-150 A at 100-300 sec pulse duration). The tissue was
allowed to
recover for 10 minutes between stimulation trials. In each experiment, the
initial
response of CA 1 to synaptic input was first tested by recording the field
potential evoked
by a single stimulus pulse. In the control condition, Schaffer collateral
stimulation
evoked a single population spike (Fig. 1A, inset). Tetanic stimulation evoked
approximately 30 seconds after discharge (Fig. 1 A, left) associated with a
large change in
intrinsic signal (Fig. lA, right).
For imaging of intrinsic optical signals, the tissue was placed in a perfusion
chamber located on the stage of an upright microscope and illuminated with a
beam of
white light (tungsten filament lighi and lens system; Dedo Inc.) directed
through the
microscope condenser. The light was controlled and regulated (power supply -
Lamda
Inc.) to minimize fluctuations and filtered (695 nm longpass) so that the
slice was
transilluminated with long wavelengths (red). Field of view and magnification
were
determined by the choice of microscope objectives (4X for monitoring the
entire slice).
Image-frames were acquired with a charge-coupled device (CCD) camera (Dage MTI
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Inc.) at 30 HZ and were digitized at 8 bits with a spatial resolution of 512 x
480 pixels
using an Imaging Technology Inc. Series 151 imaging system; gains and offsets
of the
camera-control box and the A/D board were adjusted to optimize the sensitivity
of the
system. Imaging hardware was controlled by a 486-PC compatible computer. To
increase signal/noise, an averaged-image was composed from 16 individual image-
frames, integrated over 0.5 sec and averaged together. An experimental series
typically
involved the continuous acquisition of a series of averaged-images over a
several minute
time period; at least 10 of these averaged-images were acquired as control-
images prior o
stimulation. Pseudocolored images were calculated by subtracting the first
control-image
from subsequently acquired images and assigning a color lookup table to the
pixel values.
For these images, usually a linear low-pass filter was used to remove high
frequency
noise and a linear-histogram stretch was used to map the pixel values over the
dynamic
range of the system. All operations on these images were linear so that
quantitative
information was preserved. Noise was defined as the maximum standard deviation
of
fluctuations of AR/R of the sequence of control images within a given
acquisition series,
where AR/R represented the magnitude of the change in light-transmission
through the
tissue. Delta R/R was calculated by taking all the difference-images and
dividing by the
first control image: (subsequent image - first-control-image)/first-control-
image. The
noise was always <0.01 for each of the chosen image sequences. The absolute
change in
light transmission through the tissue was estimated during some experiments by
acquiring
images after placing neutral density filters between the camera and the light
source. On
average, the camera electronics and imaging system electronics amplified the
signal 10-
fold prior to digitization so that the peak absolute changes in light
transmission through
the tissue were usually between 1 /a and 2%.
The gray-scale photo shown in Fig. 1 D is a video image of a typical
hippocampal
slice in the recording chamber. The fine gold-wire mesh that was used to hold
the tissue
in place can be seen as dark lines running diagonally across the slice. A
stimulating
electrode can be seen in the upper right on the stratum radiatum of CAI. The
recording
electrode (too thin to be seen in the photo) was inserted at the point
indicated by the white
arrow. Fig. 1 A illustrates that two seconds of stimulation at 60 Hz elicited
after discharge
activity and shows a typical after discharge episode recorded by the
extracellular
electrode. The inset of Fig. lA shows the CA1 field response to a single 200
sec test
pulse (artifact at arrow) delivered to the Schaffer collaterals. Fig. lAl
shows a map of
the peak change in optical transmission through the tissue evoked by Schaffer
collateral
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stimulation. The region of maximum optical change corresponds to the apical
and basal
dendritic regions of CA1 on either side of the stimulating electrode. Fig. 1B
illustrates
sample traces showing responses to stimulation after 20 minutes of perfusion
with
medium containing 2.5 mM furosemide. Both the electrical after discharge
activity
(shown in Fig. IB) and the stimulation-evoked optical changes (shown in Fig.
IB1) were
blocked. However, there was a hyper-excitable field response (multiple
population
spikes) to the test pulse (inset). Figs IC and 1C1 illustrate that restoration
of initial
response patterns was seen after 45 minutes of perfusion with normal bathing
medium.
The opposing effects of furosemide-blockade of the stimulation-evoked after
discharges and a concomitant increase of the synaptic response to a test-pulse
illustrate
the two key results: (1) furosemide blocked epileptiform activity, and (2)
synchronization
(as reflected by spontaneous epileptiform activity) and excitability (as
reflected by the
response to a single synaptic input) were dissociated. Experiments in which
the dose-
dependency of furosemide was examined determined that a minimum concentration
of
1.25 mM was required to block both the after discharges and optical changes.
EXAMPLE 69
The effects of furosemide on epileptiform discharges in hippocampal slices
perfused with high-K+ (10 mM) bathing medium
Rat hippocampal slices, prepared as described above, were perfused with a high-
K+ solution until extended periods of spontaneous interictal-like bursting
were recorded
simultaneously in CA3 (top traces) and CA1 (lower traces) pyramidal cell
regions (Figs.
2A and 2B). After 15 minutes of perfusion with furosemide-containing medium
(2.5 mM
furosemide), the burst discharges increased in magnitude (Figs. 2C and 2D).
However,
after 45 minutes of furosemide perfusion, the bursts were blocked in a
reversible manner
(Figs 2E, 2F, 2G and 2H). During this entire sequence of furosemide perfusion,
the
synaptic response to a single test pulse delivered to the Schaffer colalterals
was either
unchanged or enhanced (data not shown). It is possible that the initial
increase in
discharge amplitude reflected a furosemide-induced decrease in inhibition
(Misgeld et al.,
Science 232:1413, 1986; Thompson et al., J. Neurophysiol. 60:105, 1988;
Thompson and
Gdhwiler, J. Neuropysiol. 61:512, 1989; and Pearce, Neuron 10:189, 1993). It
has
previously been reported (Pearce, Neuron 10:189, 1993) that furosemide blocks
a
component of the inhibitory currents in hippocampal slices with a latency (<15
min.)
similar to the time to onset of the increased excitability observed here. The
longer latency
CA 02604446 2007-10-09
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required for the furosemide-block of the spontaneous bursting might correspond
to
additional time required for a sufficient block of the furosemide-sensitive
cellular volume
regulation mechanisms under high-K+ conditions.
After testing the effects of furosemide on slices perfused with high-K+,
similar
studies were performed with a variety of other commonly studied in vitro
models of
epileptiform discharge (Galvan et al., Brain Res. 241:75, 1982; Schwartzkroin
and Prince,
Brain Res.183:61, 1980; Anderson et al., Brain Res. 398:215, 1986; and Zhang
et al.,
Epilepsy Res. 20:105, 1995). After prolonged exposure (2-3 hours) to magnesium-
free
medium (0-Mg++), slices have been shown to develop epileptiform discharges
that are
resistant to common clinically used anticonvulsant drugs (Zhang et al.,
Epilepsy Res.
20:105, 1995). Recordings from entorhinal cortex (Fig. 21) and subiculum (not
shown)
showed that after 3 hours of perfusion with 0-Mg++ medium, slices developed
bursting
patterns that appeared similar to these previously described "anticonvulsant
resistant"
bursts. One hour after the addition of furosemide to the bathing medium, these
bursts
were blocked (Fig. 2J). Furosemide also blocked spontaneous burst discharges
observed
with the following additions/modifications to the bathing medium: (1) addition
of 200-
300 M 4-aminopyridine (4-AP; a potassium channel blocker) (Figs. 2K and 2L);
(2)
addition of the GABA antagonist, bicuculline, at 50-100 gM (Figs. 2M ad 2N);
(3)
removal of magnesium (0-Mg++) - I hours perfusion (Figs. 20 and 2P); and (4)
removal
of calcium plus extracellular chelation (0-Ca++) (Figs. 2Q and 2R). With each
of these
manipulations, spontaneous interictal-like patterns were simultaneously
recorded from
CA 1 and CA3 subfields (Figs. 2K, 2L, 2M and 2N show only the CA3 trace and
Figs. 20,
2P, 2Q, and 2R show only the CA1 trace). In the 0-Ca++ experiments, 5 mM
furosemide
blocked the bursting with a latency of 15-20 minutes. For all other protocols,
bursting
was blocked by 2.5 mM furosemide with a latency of 20-60 minutes. Furosemide
reversibly blocked the spontaneous bursting activity in both CA 1 and CA3 in
all
experiments (Figs 2L, 2N, 2P and 2R).
EXAMPLE 70
The effects of furosemide on epileptiform activity induced by i.v. iniection
of kainic
acid in anesthetized rats
This example illustrates an in vivo model in which epileptiform activity was
induced by i.v. injection of kainic acid (KA) into anesthetized rats (Lothman
et al.,
Neurology 31:806, 1981). The results are illustrated in Figs. 3A - 3H. Sprague-
Dawley
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rats (4 animals; weights 250-270 g) were anesthetized with urethane (1.25 g/kg
i.p.) and
anesthesia maintained by additional urethane injections (0.25 g/kg i.p.) as
needed. Body
temperature was monitored using a rectal temperature probe and maintained at
35-37 C
with a heating pad; heart rate (EKG) was continuously monitored. The jugular
vein was
cannulated on one side for intravenous drug administration. Rats were placed
in a Kopf
stereotaxic device (with the top of the skull level), and a bipolar stainless-
steel
microelectrode insulated to 0.5 mm of the tip was inserted to a depth of 0.5-
1.2 mm from
the cortical surface to record electroencephalographic (EEG) activity in the
fronto-parietal
cortex. In some experiments, a 2M NaCI-containing pipette was lowered to a
depth of
2.5-3.0 mm to record hippocampal EEG. Data were stored on VHS videotape and
analyzed off-line.
Following the surgical preparation and electrode placement, animals were
allowed
to recover for 30 minutes before the experiments were initiated with an
injection of kainic
acid (10-12 mg/kg i.v.). Intense seizure activity, an increased heart rate,
and rapid
movements of the vibrissae were induced with a latency of about 30 minutes.
Once stable
electrical seizure was evident, furosemide was delivered in 20 mg/kg boluses
every 30
minutes to a total of 3 injections. Experiments were terminated with the
intravenous
administration of urethane. Animal care was in accordance with NIH guidelines
and
approved by the University of Washington Animal Care Committee.
Figs. 3A-3H show furosemide blockade of kainic acid-evoked electrical "status
epilepticus" in urethane-anesthetized rats. EKG recordings are shown as the
top traces
and EEG recordings are shown as the bottom traces. In this model, intense
electrical
discharge (electrical "status epilepticus") was recorded from the cortex (or
from depth
hippocampal electrodes) 30-60 minutes after KA injection (10-12 mg/kg) (Figs.
3C and
3D). Control experiments (and previous reports, Lothman et al., Neurology,
31:806,
1981) showed that this status-like activity was maintained for well over 3
hours.
Subsequent intravenous injections of furosemide (cumulative dose: 40-60 mg/kg)
blocked
seizure activity with a latency of 30-45 minutes, often producing a relatively
flat EEG
(Figs. 3E, 3F, 3G and 3H). Even 90 minutes after the furosemide injection,
cortical
activity remained near normal baseline levels (i.e., that observed prior to
the KA and
furosemide injections). Studies on the pharmacokinetics of furosemide in the
rat indicate
that the dosages used in this example were well below toxic levels (Hammarlund
and
Paalzow, Biopharmaceutics Drug Disposition, 3:345, 1982).
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Experimental methods for Examples 71 - 74
Hippocampal slices were prepared from Sprague-Dawley adult rats as described
previously. Transverse hippocampal slices 100 m thick were cut with a
vibrating cutter.
Slices typically contained the entire hippocampus and subiculum. After
cutting, slices
were stored in an oxygenated holding chamber at room temperature for at least
one hour
before recording. All recordings were acquired in an interface type chamber
with
oxygenated (95% 02, 5%COZ) artificial cerebral spinal fluid (ACSF) at 34 -35
C.
Normal ACSF contained (in mmol/1): 124 NaCI, 3 KCI, 1.25 NaH2PO4, 1.2 MgSO4,
26
NaHCO3, 2 CaC1z, and 10 dextrose.
Sharp-electrodes for intracellular recordings from CA1 and CA3 pyramidal cells
were filled with 4 M potassium acetate. Field recordings from the CA 1 and CA3
cell
body layers were acquired with low-resistance glass electrodes filled with 2 M
NaCI. For
stimulation of the Schaffer collateral or hilar pathways, a small monopolar
tungsten
electrode was placed on the surface of the slice. Spontaneous and stimulation-
evoked
activities from field and intracellular recordings were digitized
(Neurocorder, Neurodata
Instruments,. New York, NY) and stored. on videotape. AxoScope softwa.re
(Axon:
InstrR:ments) on a per:;onal cumputer was: used~for off-line analysis of data.
In some experimerits, normal. or low-chloride mediunr was iised containing-
bicuculline (20 M), 4-amino pyridine-(4-AP) (100 M), or high-Kr (7.5 or 12
mM). In.
all experirnents, low-chloride solutions (7, and.2.1 ni1V1 [Cl-]o) were
prepared by equimolar
replacement of NaCI' witli 'Na+-gluconate -(Sigma).. All solutious were
prepared so that_
they had a- pH.. of approximateiy 7.4 and...an osmotarity of 290-300 mOsm at
35 C and at
equilibrium from carboxygenation with 95%02 / 5%CO2.
Atter placement in the interface chamber, slices were superfused at
approximately
1 ml%min. At this flow-rate, it took 87 10 rninutes for changes in the
perfusion media to be
completed. All of the times reported here have taken this delay into account
and have an
error of approximately 2 minutes.
EXAMPLE 71
Timinp, of cessation of spontaneous epileptiform burstinp, in areas in CA1 and
CA3
The relative contributions of the factors that modulate synchronized activity
vary
between areas CA 1 and. CA3. These factors include differences in the local
circuitry and
region-speciflc differences in ce11 packing and volume fraction of the
extracellular.spaces.
If the anti-epileptic effects of anion or chloride-cotransport antagonism are
due to a
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desynchronization in the timing of neuronal discharge, chloride-cotransport
blockade
might be expected to differentially affect areas CAl and CA3. To test this, a
series of
experiments was performed to characterize differences in the timing of the
blockade of
spontaneous epileptiform activity in areas CA1 and CA3.
Field activity was recorded simultaneously in areas CA1 and CA3 (approximately
midway between the most proximal and distal extent the CA3 region), and
spontaneous
bursting was induced by treatment with high-[K+]o (12 M; n = 12), bicuculline
(20 mM;
n = 12), or 4-AP (100 M; n = 5). Single electrical stimuli were delivered to
the Schaffer
collaterals, midway between areas CA1 and CA3, every 30 seconds so that the
field
responses in areas CA1 and CA3 could be monitored throughout the duration of
each
experiment. In all experiments, at least 20 minutes of continuous spontaneous
epileptiform bursting was observed prior to switching to low [C1-]o (21 mM) or
furosemide-containing (2.5 mM) medium.
In all cases, after 30-40 minutes exposure to furosemide or low-chloride
medium,
spontaneous bursting ceased in area CA 1 before the bursting ceased in area
CA3. The
temporal sequence of events typically observed included an initial increase in
burst
frequency and amplitude of the spontaneous field events, . then a reduction in
the
amplitude of the burst discharges which was more rapid in CA1 than in CA3.
After CA1
became silent, CA3 continued to discharge for 5-10 minutes, until it too no
longer
exhibited spontaneous epileptiform events.
This temporal pattern of burst cessation was observed with all epileptiform-
inducing treatments tested, regardless of whether the agent used for blockade
of
spontaneous bursting was furosemide or low-[Cl"]o medium. Throughout all
stages of
these experiments, stimulation of the Schaffer collaterals evoked hyperexcited
field
responses in both the CA 1 and CA3 cell body layers. Immediately after
spontaneous
bursting was blocked in both areas CA 1 and CA3, hyperexcited population
spikes could
still be evoked.
We considered the possibility that the observed cessation of bursting in CA1
prior
to CA3 was an artifact of the organization of synaptic contacts between these
areas
relative to our choice of recording sites. It is known that the projections of
the various
subregions of CA3 terminate in an organized fashion in CA l; CA3 cells closer
to the
dentate gyrus (proximal CA3) tend to project most heavily to the distal
portions of CA1
(near the subicular border), whereas CA3 projections arising from cells
located more
distally in CA3 terminate more heavily in portions of CA1 located closer to
the CA2
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border. If the cessation of bursting occurs in the different subregions of CA3
at different
times, the results of the above set of experiments might arise not as a
difference between
CA 1 and CA3, but rather as a function of variability in bursting activity
across CA3
subregions. We tested this possibility in three experiments. Immediately after
the
spontaneous bursting ceased in CA 1, we surveyed the CA3 field with a
recording
electrode. Recordings from several different CA3 locations (from the most
proximal to
the most distal portions of CA3), showed that all subregions of area CA3 were
spontaneously bursting during the time that CA1 was silent.
The observation that CA3 continued to discharge spontaneously after CA1
became silent was unexpected since population discharges in CA3 are generally
thought
to evoke discharges in CA1 through excitatory synaptic transmission. As
previously
described, single-pulse stimuli delivered to the Schaffer collaterals still
evoked multiple
population spikes in CAl even after the blockade of spontaneous bursting;
thus,
hyperexcited excitatory synaptic transmissions in CA3-to-CA 1 synapse was
intact. Given
this maintained efficacy of synaptic transmission, and the continued
spontaneous field
discharges in CA3, we postulated that the loss of spontaneous bursting in CA 1
was due to
a decrease in synchronization of incoming excitatory drive. Further, since
spontaneous
epileptiform discharge in CA3 also eventually ceased, perhaps this
desynchronization
process occurred at different times in the two hippocampal subfields.
EXAMPLE 72
Effect of chloride-cotransport antagonism on the synchronization of CA1 and
CA3
field population dischar2es
The observation from Example 4 suggested a temporal relationship between the
exposure time to low-[CI"]o or furosemide-containing medium and the
characteristics of
the spontaneous burst activity. Further, this relationship was different
between areas CA 1
and CA3. In order to better characterize the temporal relationships, we
compared the
occurrences of CA1 action potentials and the population spike events in the
field
responses of CA 1 and CA3 subfields during spontaneous and stimulation-evoked
burst
discharge.
Intracellular recordings were obtained from CA 1 pyramidal cells, with the
intracellular electrode placed close (<100 M) to the CA1 field electrode. The
slice was
stimulated every 20 seconds with single stimuli delivered to the Schaffer
collaterals.
After continuous spontaneous bursting was established for at least 20 minutes,
the bathing
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medium was switched to bicuculline-containing low-[C1"]o (21 mM) medium. After
approximately 20 minutes, the burst frequency and amplitude was at its
greatest.
Simultaneous field and intracellular recordings during this time showed that
the CA1 field
and intracellular recordings were closely synchronized with the CA3 field
discharges.
During each spontaneous discharge, the CA3 field response preceded the CA 1
discharge
by several milliseconds. During stimulation-evoked events, action potential
discharges of
the CA1 pyramidal cell were closely synchronized to both CA3 and CA1 field
discharges.
With continued exposure to low-[Cl-]o medium, the latency between the
spontaneous discharges of areas CA1 and CA3 increased, with a maximum latency
of 30-
40 milliseconds occurring after 30-40 minutes exposure to the bicuculline-
containing
low-chloride medium. During this time, the amplitude of both the CA1 and CA3
spontaneous field discharges decreased. Stimulation-evoked discharges during
this time
closely mimicked the spontaneously occurring discharges in morphology and
relative
latency. However, the initial stimulus-evoked depolarization of the neuron
(presumably,
the monosynaptic EPSP) began without any significant increase in latency. The
time
interval during which these data were acquired corresponds to the time
immediately prior
to the cessation of spontaneous bursting in CA1.
After 40-50 minutes perfusion with low-[Cl"]o medium, the spontaneous bursts
were nearly abolished in CA1 but were unaffected in CA3. Schaffer collateral
stimulation during this time showed that monosynaptically-triggered responses
of CA1
pyramidal cells occurred without any significant increase in latency, but that
stimulation-
evoked field responses were almost abolished. The time interval during which
these data
were acquired corresponds to the moments immediately prior to the cessation of
spontaneous bursting in CA3.
After prolonged exposure to low-[CI-]o medium, large increases (>30
milliseconds) developed in the latency between Schaffer collateral stimulation
and the
consequent CA3 field discharge. Eventually, no field responses could be evoked
by
Schaffer collateral stimulation in either areas CA1 and CA3. However, action
potential
discharge from CA 1 pyramidal cells in response to Schaffer collateral
stimulation could
be evoked with little change in response latency. Indeed, for the entire
duration of the
experiments (greater than two hours), action potential discharges form CA1
pyramidal
cells could be evoked at short latency by Schaffer collateral stimulation.
Further,
although stimulation-evoked hyperexcited discharges of CA3 were eventually
blocked
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after prolonged exposure to low-[Cl-]o medium, the antidromic response in CA3
appeared
to be preserved.
EXAMPLE 73
Effects of chloride-cotransport antagonism on the synchronization of burst
discharges in CAl pyramidal cells
The foregoing data suggest the disappearance of the field responses may be due
to
a desynchronization of the occurrence of action potentials among neurons. That
is,
although synaptically-driven excitation of CA 1 pyramidal cells was not
preserved, action
potential synchrony among the CA 1 neuronal population was not sufficient to
summate
into a measurable DC field response. In order to test this, paired
intracellular recordings
of CA 1 pyramidal cells were acquired simultaneously with CA 1 field
responses. In these
experiments, both the intracellular electrodes and the field recording
electrodes were
placed within 200 m of one another.
During the period of maximum spontaneous activity induced by bicuculline-
containing low-[CI-]o medium, recordings showed that action potentials between
pairs of
CA1 neurons and the CA1 field discharges were tightly synchronized both during
spontaneous and stimulation-evoked discharges. After continued exposure to low-
[Cl-]o
medium, when the amplitude of the CA1 field discharge began to broaden and
diminish,
both spontaneous and stimulation-evoked discharges showed a desynchronization
in the
timing of the occurrences of action potentials between pairs of CA1 neurons,
and between
the action potentials and the field responses. This desynchronization was
coincident with
the suppression of CA1 field amplitude. By the time that spontaneous bursting
in CA1
ceased, a significant increase in latency had developed between Schaffer
collateral
stimulation and CA 1 field discharge. At this time, paired intracellular
recordings showed
a dramatic desynchronization in the timing of action potential discharge
between pairs of
neurons and between the occurrence of action potentials and the field
discharges evoked
by Schaffer collateral stimulation.
It is possible that the observed desynchronization of CAl action potential
discharge is due to the randomization of mechanisms necessary for synaptically-
driven
action potential generation, such as a disruption in the timing of synaptic
release or
random conduction failures at neuronal processes. If this were the case, then
one would
expect that the occurrence of action potentials between a given pair of
neurons would
vary randomly with respect to one another, from stimulation to stimulation. We
tested
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this by comparing the patterns of action potential discharge of pairs of
neurons between
multiple consecutive stimuli of the Schaffer collaterals. During each
stimulation event,
the action potentials occurred at nearly identical times with respect to one
another, and
showed an almost identical burst morphology from stimulation to stimulation.
We also
checked to see whether the occurrence of action potentials between a given
pair of
neurons during spontaneous field discharges was fixed in time. The patterns of
action
potential discharges from a given pair of CA t neurons was compared between
consecutive spontaneous field bursts during the time when the occurrence of
action
potentials was clearly desynchronized. Just as in the case of stimulation-
evoked action
potential discharge described above, the action potentials generated during a
spontaneous
population discharge occurred at nearly identical times with respect to one
another, and
showed a nearly identical burst morphology from one spontaneous discharge to
the next.
EXAMPLE 74
Effects of low-chloride treatment on spontaneous synaptic activity
It is possible that the anti-epileptic effects associated with chloride-
cotransport
antagonism are mediated by some action on transmitter release. Blockade of
chloride-
cotransport could alter the amount or timing of transmitter released from
terminals, thus
affecting neuronal synchronization. To test whether low-[CI"]o exposure
affected
mechanisms associated with transmitter release, intracellular CA1 responses
were
recorded simultaneously with CA 1 and CA3 field responses during a treatment
which
dramatically increases spontaneous synaptic release of transmitter from
presynaptic
terminals.
Increased spontaneous release of transmitter was induced by treatment with 4-
AP
(100 M). After 40 minutes exposure to 4-AP-containing medium, spontaneous
synchronized burst discharges were recorded in area CA 1 and CA3. Switching to
4-AP-
containing low-[Cl"]o medium led initially, as was shown previously, to
enhanced
spontaneous bursting. High-grain intracellular recordings showed that high-
amplitude
spontaneous synaptic activity was elicited by 4-AP treatment. Further exposure
to low-
chloride medium blocked spontaneous burst discharge in CA1, although CA3
continued
to discharge spontaneously. At this time, CA 1 intracellular recordings showed
that
spontaneous synaptic noise was further increased, and remained so for
prolonged
exposure times to 4-AP-containing low-chloride medium. These data suggest that
mechanisms responsible for synaptic release from terminals are not adversely
affected by
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low-chloride exposure in a manner that could explain the blockade of 4-AP-
induced
spontaneous bursting in CA1. These results also eliminate the possibility that
the effects
of low-[CI-]o exposure are due to alterations in CA1 dendritic properties
which would
compromise their efficiency in conducting PSPs to the soma.
Experimental Methods for Examples 75 to 79
In all of the following experiments, [Cl']o was reduced by equimolar
replacement
of NaCI with Na+-gluconate. Gluconate was used rather than other anion
replacements
for several reasons. First, patch-clamp studies have demonstrated that
gluconate appears
to be virtually impermeant to chloride channels, whereas other anions
(including sulfate,
isethionate, and acetate) are permeable to varying degrees. Second, transport
of
extracellular potassium through glial NKCCI cotransport is blocked when
extracellular
chloride is replaced by gluconate but is not completely blocked when replaced
by
isethionate. Since this furosemide-sensitive cotransporter plays a significant
role in cell
swelling and volume changes of the extracellular space (ECS), we wished to use
the
appropriate anion replacement so that the effects of our treatment would be
comparable to
previous furosemide experiments (Hochman et al. Science, 270:99-102, 1995; US
Patent
No. 5,902,732). Third, formate, acetate, and proprionate generate weak acids
when
employed as Cl- substitutes and lead to a prompt fall in intracellular pH;
gluconate
remains extracellular and has not been reported to induce intracellular pH
shifts. Fourth,
for purposes of comparison we wished to use the same anion replacement that
had been
used in previous studies examining the effects of low-[Cl]o on activity-evoked
changes of
the ECS.
There is some suggestion that certain anion-replacements might chelate
calcium.
Although subsequent work has failed to demonstrate any significant ability of
anion-
substitutes to chelate calcium, there is still some concern in the literature
regarding this
issue. Calcium chelation did not appear to be an issue in the following
experiments, since
resting membrane potentials remained normal and synaptic responses (indeed,
hyperexcitable synaptic responses) could be elicited even after several hours
of exposure
to medium in which [CI']o had been reduced by gluconate substitution. Further,
we
confirmed that calcium concentration in our low-[Cl"]o -medium was identical
to that in
our control-medium by measurements made with Caz+ - selective microelectrodes.
Sprague-Dawley adult rats were prepared as previously described. Briefly,
transverse hippocampal slices, 400 m thick, were cut using a vibrating
cutter. Slices
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CA 02604446 2007-10-09
WO 2006/110187 PCT/US2005/043177
typically contained the entire hippocampus and subiculum. After cutting,
slices were
stored in an oxygenated holding chamber for at least one hour prior to
recording. All
recordings were acquired in an interface type chamber with oxygenated (95%
02/5%
CO2) artificial cerebral spinal fluid (ACSF) at 34 -35 C. Normal ACSF
contained (in
mmol/1): 124 NaCI, 3 KCI, 1.25 NaH2PO4, 1.2 MgS04, 26 NaHCO3, 2 CaC12, and 10
dextrose. In some experiments, normal or low-chloride niediunt was used
containing
bicuculline (20 M), 4-AP (100 M), or high-K+ (12 rnM). Low-chloride
solutions (7,
16, and 21 mM [Cl-]o) were prepared by equimolar replacement of NaCl with Na+-
gluconate (Sigma Chemical Co., St. Louis, MO). All solutions were prepared so
that they
had a pH of.' approximately 7.4 and an osmolarity of 290-300 mOsm at 35 C and
at
equilibrium from carboxygenation with 95% 02 / 5% COz.
Sharp-electrodes filled with 4 M potassium acetate were used for intracellular
recordings fronl CAl pyramidal cells. Field recordings from. the CAl or CA3
cell body
layers were acquired with low-resistance glass electrodes filled with NaCI (2
M). For
stimulation of the Schaffer collateral pathway, a small monopolar electrode
was ptac:d on
20,,... the .trfh:;e of tl;e slice. rnidwny. between. areas .CA1. and..CA3.
stirra:lation-: voked activities fi=om . fl.e;cl and intracellular. recrn-
dings. were (ligitized
i Neurocorder;: .i'Icurodata lnntrunients,: New;. ;York, NY), and : stored on
video ta,)e.:.: -
AxoScope sottware (Axon: Instruments Inc.). on:: a PC-computer was used.,for
off-line. .. :
analvses of.data. .
Ion-se:!ective microelectro(les were fabricated according-to standard.-
.m.;thods well
knuwn in. tbe-ai-t: . Doti ble-barreled. }:ipcttes.were pulled. and broken.
to. a- tip diameter of.:
anproxiniately 3.0 m. The reference barrel was filled with ACSF and the other
barrel
was sylanized artd the til) bacl:-f.illed with a resin selective for K+
(rorning 4773 t7). '11e
remainder ofthe sylanized barrel was filled with KC] (140 mM). Each barrel was
le;l, via
Ag/A.gCI N:-:res, to a hish imp:,dance dua! -differential amplifier (WP=l.
M223). Eacl; ic n- .
selective niicroelectrcde was calibr.ated by the use of solutions of knov,m
ionic
composition., nd was considered ;;i,ritable if it was charac.terized by a near-
Ne.rnstiap-slopz~
response and if it remained stable throughout the duration of the experiment.
After placement in the interface chamber, slices were superfused at
approxinlately
1 mi/minute.. At this flow-rate, it took approximately 8-10 minutes for
changes in
perftision media to be completed_ All of the times reported here have taken
this time-
delay into account and. have an error of approximately f 2 minutes.
CA 02604446 2007-10-09
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EXAMPLE 75
Effects of low-[Cl-lo on CAl field recordinEs
Other studies have shown that prolonged exposure of cortical and hippocampal
slices to low-[CI-]o does not affect basic intrinsic and synaptic properties
such as input
resistance, resting membrane potential, depolarization-induced action-
potential
generation, or excitatory synaptic transmission. A previous study has also
partly
characterized the epileptogenic properties of low-[CI-]o exposure to the CA1
area of
hippocampus. The following studies were performed to observe the times of
onset and
possible cessation of low-[Cl"]o-induced hyperexcitability and
hypersynchronization.
Slices (n = 6) were initially perfused with normal medium until stable
intracellular and
field recordings were established in a CA 1 pyramidal cell and the CA 1 cell
body layer,
respectively. In two experiments, the same cell was held throughout the entire
length of
the experiment (greater than 2 hours). In the remaining experiments (n = 4),
the initial
intracellular recording was lost during the sequence of medium changes and
additional
recordings were acquired from different cells. Patterns of neuronal activity
in these
experiments were identical to those seen when a single cell was observed.
The field and intracellular electrodes were always placed in close proximity
to one
another (< 200 m). In each case, after approximately 15-20 minutes exposure
to the
low-[Cl-]o-medium (7 mM), spontaneous bursting developed, first at the
cellular level,
and then in the field. This spontaneous field activity, representing
synchronized burst
discharge in a large population of neurons, lasted from 5 - 10 minutes, after
which time
the field recording became silent. When the field first became silent, the
cell continued to
discharge spontaneously. This result suggests that population activity has
been
"desynchronized" while the ability of individual cells to discharge has not
been impaired.
After approximately 30 minutes exposure to low-[Cl-]o-medium, intracellular
recording
showed that cells continued to discharge spontaneously even though the field
remained
silent. The response of the cell to intracellular current injection at two
time points
demonstrated that the cell's ability to generate action potentials had not
been impaired by
low-[CI-]o exposure. Further, electrical stimulation in CA 1 stratum radiatum
elicited
burst discharges, indicating that a hyperexcitable state was maintained in the
tissue.
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EXAMPLE 76
Effects of low-[C1-lo on high-1K+lo-induced epileptiform activity in CA1
The previous set of experiments showed that tissue exposure to low-[CI-]o
medium induced a brief period of spontaneous field potential bursting which
ceased
within 10 minutes. If a reduction of [Cl-]o is indeed eventually capable of
blocking
spontaneous epileptiform (i.e. synchronized) bursting, then these results
suggest that anti-
epileptic effects would likely be observable only after this initial period of
bursting
activity has ceased. We therefore examined the temporal effects of low-[Cl-]o-
treatment
on high-[K+]o-induced bursting activity. Slices (n = 12) were exposed to
medium in
which [K+]o had been increased to 12 mM, and field potentials were recorded
with a field
electrode in the CA1 cell body layer. Spontaneous field potential bursting was
observed
for at least 20 minutes, and then the slices were exposed to medium in which
[K+]o was
maintained at 12 mM, but [Cl-]o was reduced to 21 mM. Within 15-20 minutes
after the
tissue was exposed to the low-[Cl-]o/high-[K+]o-medium, the burst amplitude
increased
and each field event had a longer duration. After a brief period of this
facilitated field
activity (lasting 5-10 minutes), the btirsting stopped. To test whether this
blockade was.
reversible, after at least 10 minutes of field potential silence, we switched
back to high-
[KT]o-medium . with normal [Cl"]o. The bursting returned within 20-40 minutes.
Throughout each experiment, the CA1 field response to Schaffer collateral
stimulation.
was monitored. The largest field responses were recorded just before the
cessation of
spontaneous bursting, during the period when the spontaneous bursts had the
largest
anlplitude. Even after the blockade of spontaneous bursting, however, multiple
popttlation spikes were elicited by Schaffer collateral stimulation,
indicating that synaptic
transtnission was intact, and that the tissue remained hyperexcitable.
In four slices, intracellular recordings from CAl pyramidal cells were
acqaired
3o along with the CA1 field recording. During the period of high-[Ky]o-induced
spontaneous bursting, hyperpolarizing current was injected into the cell so
that
postsynaptic potentials (PSPs) could be better observed. After low-[Cl-]o-
blockade of
spontaneous bursting, spontaneously occurring action potentials and PSPs were
still
observed. These observations further support the view that synaptic activity,
per se, was
not blocked by the low-[CI-]o treatment.
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EXAMPLE 77
Low-(Cl-lo - blockade of epileptiform activity induced by 4-AP, hi2h-[K+lo,
and
bicuculline in CAl and CA3
We next tested whether low-[Cl"]o treatment could block epileptiform activity
in
areas CAl =and CA3, which was elicited by different pharmacological
treatments, as we
had shown for furosemide treatment. For this set of experiments, we chose to
test the
effects of low-[CI-]o treatment on spontaneous bursting which had been induced
by high-
[K+]o (12 mM) (n = 5), 4-AP (100 M) (n = 4), and bicuculline (20 and 100 M)
(n = 5).
In each set of experiments, field responses were recorded simultaneously from
areas CA1
and CA3, and in each case, the spontaneous epileptiform activity in both areas
CA1 and
CA3, was reversibly blocked within 30 minutes after [Cl-]o in the perfusion
medium had
been reduced to 21 mM. These data suggest that, like furosemide, low-[CI-]o
reversibly
blocks spontaneous bursting in several of the most commonly studied in vitro
models of
epileptiform activity.
EXAMPLE 78
Comparison between low-[CI"loand furosemide on blockade of high-[K+lo-induced
epileptiform activity
The data from the previous sets of experiments are consistent with the
hypothesis
that the anti-epileptic effects of both low-[CI-]o and furosemide are mediated
by their
actions on the same physiological mechanisms. To further test this hypothesis,
we
compared the temporal sequence of effects of low-[CI-]o (n = 12) and
furosemide (2.5 and
5 mM) (n = 4) on high-[K+]o-induced bursting, as recorded with a field
electrode in CA1.
We found that both low-[CI-]o and furosemide treatment induced a similar
temporal
sequence of effects: an initial brief period of increased amplitude of field
activity, and
then blockade (reversible) of spontaneous field activity. In both cases,
electrical
stimulation of the Schaffer collaterals elicited hyperexcited responses even
after the
spontaneous bursting had been blocked.
EXAMPLE 79
Conseguences of prolonged exposure to low-[Cl-lo medium with varied [K+lo
In the preceding experiments, we monitored field activity in some slices for >
1
hour after the spontaneous bursting had been blocked by low-[CI-]o exposure.
After such
prolonged low-[CI-]o exposure, spontaneous, long-lasting, depolarizing shifts
developed.
The morphology and frequency of these late-occurring field events appeared to
be related
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to the extracellular potassium and chloride concentrations. Motivated by these
observations, we performed a set of experiments in which we systematically
varied [C1-]o
and [K+]o and observed the effects of these ion changes on the late-occurring
spontaneous
field events.
In our first set of experiments, slices were exposed to medium containing low-
[Cl]o (7 mM) and normal-[K+]o (3 mM) (n = 6). After 50-70 minutes exposure to
this
medium, spontaneous events were recorded in area CA1; these events appeared as
5-10
mV negative shifts in the DC field, with the first episode lasting for 30-60
seconds. Each
subsequent episode was longer than the previous one. This observation
suggested that
ion-homeostatic mechanisms were diminished over time as a result of the ion
concentrations in the bathing medium. In some experiments (n = 2) in which
these
negative DC field shifts had been induced, intracellular recordings from CA 1
pyramidal
cells were acquired simultaneously with the CA1 field recordings.
For these experiments, the intracellular and field recordings were acquired
close to
one another (< 200 m). Prior to each negative field shift (10-20 seconds),
the neuron
began to depolarize. Cellular depolarization was indicated by a decrease in
resting
membrane potential, an increase in spontaneous firing frequency, and a
reduction of
action potential amplitude. Coincident with the onset of the negative field
shifts, the cells
became sufficiently depolarized so that they were unable to fire spontaneous
or current-
elicited (not shown) action potentials. Since neuronal depolarization began 10-
20
seconds prior to the field shift, it may be that a gradual increase in
extracellular potassium
resulted in the depolarization of a neuronal population, thus initiating these
field events.
Such an increase in [K+]o might be due to alterations of the chloride-
dependent glial
cotransport mechanisms that normally move potassium from extracellular to
intracellular
spaces. To test whether increases in [K+]o preceded these negative field
shifts (and
paralleled cellular depolarization), experiments (n=2) were performed in which
a K+-
selective microelectrode was used to record changes in [K+]o.
In each experiment, the K+-selective microelectrode and a field electrode were
placed in the CA 1 pyramidal layer close to one another (< 200 m), and a
stimulation
pulse was delivered to the Schaffer collaterals every 20 seconds so that the
magnitude of
the population spike could be monitored. Multiple spontaneously occurring
negative field
shifts were evoked by perfusion with low-[Cl-o] (7 mM) medium. Each event was
associated with a significant increase in [K+]o, with the [K+]o increase
starting several
seconds prior to the onset of negative field shift. A slow 1.5-2.0 mM increase
in [K+]o
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WO 2006/110187 PCT/US2005/043177
occurred over a time interval of approximately 1-2 minute seconds prior to the
onset of
each event. The stimulation-evoked field responses slowly increased in
amplitude over
time, along with the increasing [K+]o, until just before the negative field
shift.
In a second set of experiments (n = 4), [K+]o was increased to 12 mM and [Cl-
]o
was increased to 16 mM. After 50-90 minutes exposure to this medium, slow
oscillations
were recorded in area CA1. These oscillations were characterized by 5-10 mV
negative
DC shifts in the field potential and had a periodicity of approximately I
cycle/40 seconds.
Initially, these oscillations occurred intermittently and had an irregular
morphology.
Over time, these oscillations became continuous and developed a regular
waveform.
Upon exposure to furosemide (2.5 mM), the amplitude of the oscillations was
gradually
decreased and the frequency increased until the oscillations were completely
blocked.
Such low-[Cl]o - induced oscillations in tissue slices have not been
previously reported.
However, the temporal characteristics of the oscillatory events bear a
striking
resemblance to the low-[Cl]o - induced [K+]o oscillations which were
previously
described in a purely axonal preparation.
. In a third set of experiments (n = 5) [Cl]o was further increased to 21 mM
and
[K+]o was reduced back to 3 mM. In these experiments, single, infrequently
occurring
negative shifts of the field potential developed within 40 - 70 minutes (data
not shown).
These events (5-10 mV) lasting 40-60 seconds, occurred at random intervals,
and
maintained a relatively constant duration throughout the experiment. These
events could
sometimes be elicited by a single electrical stimulus delivered to the
Schaffer collaterals.
I Finally, in a final set of experiments (n = 5), [Cl]o was kept at 21 mM and
[K+]o
was raised to 12 mM. In these experiments, late-occurring spontaneous field
events were
not observed during the course of the experiments (2-3 hours).
30.- EXAMPLE 80
Chanties in [K+L during low-chloride exposure
Sprague-Dawley adult rats were prepared as previously described. Transverse
hippocampal slices, 400 m thick, were cut with a vibrating cuter and stored
in an
oxygenated holding chamber for 1 hour before recording. A submersion-type
chamber
was used for K+-selective microelectrode recordings. Slices were perfused with
oxygenated (95% 02/5% C02) artificial cerebrospinal fluid (ACSF) at 34-35 C.
Normal
ACSF contained 10 mM dextrose, 124 mM NaC1, 3 mM KCI, 1.25 mM NaH2PO4, 1.2
mM MgSO4, 26 mM NaHCO3 and 2 mM CaCIZ. In some experiments, normal or low-
CA 02604446 2007-10-09
WO 2006/110187 PCT/US2005/043177
chloride medium was used containing 4-aminopyridine (4-AP) at 100gM. Low-
chloride
solutions (21 mM [Cl]o) were prepared by equimolar replacement of NaCI with
Na+-
gluconate (Sigma Chemical Co.).
Field recordings from the CAI or CA3 cell body layers were acquired with low-
resistance glass electrodes filled with NaCl (2M). For stimulation of the
Schaffer
collateral pathway, a monopolar stainless-steel electrode was placed on the
surface of the
slide midway between areas CA 1 and CA3. All recordings were digitized
(Neurorocorder, Neurodata Instruments, New York, NY) and stored on videotape.
K+ selective microelectrodes were fabricated according to standard methods.
Briefly, the reference barrel of a doublc-barreled pipette was filled with
ACSF, and the
other barrel was sylanized and the tip back-filled with KCI with K+-selective
resin
(Corning 4773.17). Ion-selective micro .lectrodes were calibrated and
cc,nsidered suitable
if they haci a Nernstian slope response and remained stable throughout the
duration of the
experiment.
Exposure of hippocampal slices to 1ow-[C1-]o medium has been shown to include
20a temporally-dependent sequence of changes on the. activity of CA 1
pyramidal cells, with=
three characteristics phases,,as described.above. In brief; exposure to low-
[Ci-lo medium
results in a brief period. of., increase~t- hyperexcitability and.
spor.tancous- epileptifurm :.
discharge. With fuu-th.er>exposure.to iow-[Cl]o medium, spontaneous
epileptiform activity:,.
is blocked, hut cellula.r,... hyperex.citability. remains, .:and action
potent.iai. firing .times:
become less synchronized witli ot;e- auother:.: L-astly,.with prolonged
exposure; the, actiorr.
potential fiving times beconic sufficiently desyrichronized:so that
stiraulatien-.evoked_fieldL:;_,:
responses completely disappe=.tr; yet individual cells continue to show
menosyr.apticlly-
evoked responses io Schaffer collateral stimulation. The following results
demonstrate
that the antiepileptic effects of furosemide on chloride-cotransport
antagonism are
independent of direct actions on excitatory synaptic transmission, and are a
consequence
of a desynchronization of population activity with our any associated decrease
in
excitabili!v.
In six hippocampal slices, K+-selective and field microelectrodes were placed
in
the CA I cell body layer, and a stimulating electrode was placed on the
Schaf.fer coll;:*.eral
pathway, and single-pulse stimuli (300 s) were delivered every 20 seconds.
After stable
baseline 'K+jo was observeil for.at least 20 minutes, the perfiision
wasswitched to low-
[C1]~, medium. Within 1-2 minutes of exposure to low-[C1-]o medium, the field
respr,nses
became hvperexcitable as the [K+]o began to rise. After approximately 4-5
niinutes of
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WO 2006/110187 PCT/US2005/043177
exposure to low-[Cl"]o medium, the magnitude of the field response diminished
until it
was completely abolished. The corresponding recording of [K+]o showed that
potassium
began to rise immediately after exposure to low-[CI-]o medium, and that the
peak of this
[K+]o rise corresponded in time to the maximally hyperexcitable CA 1 field
response.
Coincident with the reduction of the magnitude of the field response, the
[K+]o began to
diminish until after 8-10 minutes exposure to low-[CI-]o medium, it became
constant for
the remainder of the experiment at 1.8-2.5 mM above control levels. Four
slices were
switched back to control medium and allowed to fully recover. The experiment
was then
repeated with the K+-selective microelectrode placed in the stratum radiatum.
A similar
sequence of changes in [K+]o was observed in the dendritic layer, with the
values of [K+]o
being 0.2-0.3 mM less than those observed in the cell body layers.
In four hippocampal slices, the responses of stimulation-evoked changes in
[K+]o
between control conditions and after the CA1 field response was completely
abolished by
low-[CI-]o exposure were compared. In each slice, the [K+]o-selective
measurements were
acquired first in the cell body layer, and then after allowance for complete
recovery in
control medium, the experiment was repeated with the K+-selective electrode
moved to
the stratum radiatum. Each stimulation trial consisted of a 10 Hz volley
delivered to the
Schaffer collateral for 5 seconds. The peak rises in [K+]o'were similar
between control
conditions an after prolonged exposure to low-[CI-]o medium, and between the
cell body
and dendritic layers. However, the recovery times observed after prolonged
exposure to
low-[CI-]o were significantly longer than those observed during control
conditions.
These results demonstrate that the administration of furosemide resulted in
increased [K+]o in the extracellular spaces. Exposure of the brain tissue to
low-[CI-]o
medium immediately induced a rise in [K+]o by 1-2 mM, which remained
throughout the
duration of exposure, and was coincident with the initial increase in
excitability and the
eventual abolishment of the CA 1 field response. This loss of CA 1 field
response during
low-[CI-]o exposure is most likely due to the desynchronization of neuronal
firing times.
Significantly, the stimulation-evoked increases in [K+]o, in both the cell
body and
dendritic layers were nearly identical before and after the complete low-[Cl-
]o blockade of
the CA 1 field response. This data suggests that comparable stimulation-evoked
synaptic
drive and action potential generation occurred under control conditions and
after low [Cl"
]o blockade of the field. Together these data demonstrate that the
antiepileptic and
desynchronizing effects of the chloride-cotransport antagonist, furosemide,
are
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WO 2006/110187 PCT/US2005/043177
independent of direct actions on excitatory synaptic transmission and are a
consequence
of a desynchronization of population activity without decrease in
excitability.
EXAMPLE 81
Changes in extracellular pH during low-chloride exposure
Antagonists of the anion/chloride-dependent cotransporter, such as furosemide
and low-[CI-]o, may affect extracellular pH transients that might contribute
to the
maintenance of synchronized population activity. Rat hippocampal brain slices
were
prepared as described in Example 80, except the NaHCO3 was substituted by
equimolar
amount of HEPES (26 nM) and an interface-type chamber was used.
In four hippocampal brain slices continuous spontaneous bursting was elicited
by
exposure to medium containing 100 M 4-AP, as described in Example 13. Field
recordings were acquired simultaneously from the cell body layers in areas CA
1 and
CA3. A stimulus delivered every 30 seconds to the Schaffer collaterals
throughout the
duration of the experiments. After at least 20 minutes of continuous bursting
was
observed, the slices were exposed to nominally bicarbonate free, 4-AP-
containing HEPES
medium. There were no significant changes observed in the spontaneous or
stimulation-
evoked field responses resulting from prolonged exposure (0.2 hours) to HEPES
medium.
After the slices had been exposed for at least 2 hours to the HEPES medium,
the
perfusion was switched to 4-AP-containing HEPES medium in which the [Cl]o had
been
reduced to 21 mM. Exposure to the low-[CI"]o HEPES medium induced the
identical
sequences of events, and at the same time course, as had previously been
observed with
low-[Cl-]o NaHCO3-containing medium. After complete blockade of spontaneous
bursting, the perfusion medium was switched back to HEPES medium with normal
[Cl"]o.
Within 20-40 minutes, spontaneous bursting resumed. At the time the
spontaneous
~ bursting had resumed, the slices had been perfused with nominally
bicarbonate-free
HEPES medium for greater than 3 hours.
This data suggests that the actions of chloride-cotransport antagonism on
synchronization and excitability are independent of affects on the dynamics of
extracellular pH.
Figure 4 illustrates a schematic model of ion cotransport under conditions of
reduced [C]"]. Fig. 4A, left panel, shows that the chloride gradient necessary
for the
generation of IPSPs in neurons is maintained by efflux of ions through a
furosemide-
sensitive K+,CI- cotransporter. Under normal conditions, a high concentration
of
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WO 2006/110187 PCT/US2005/043177
intracellular potassium (maintained by the 3Na+, 2K+-ATPase pump) serves as
the driving
force for the extrusion of Cl" against its concentration gradient. In glial
cells, as shown in
the right panel of Fig. 4A, the moveinent of ions through the furosemide-
sensitive NKCC
co-transporter is from extracellular to intracellular spaces. The ion-
gradients necessary
for this cotransport are maintained, in part, by the "transmembrane sodium
cycle":
sodium ions taken into glial cells through NKCC cotransport are continuously
extruded
by the 3Na+,2K+,-ATPase pump so that a low intracellular sodium concentration
is
maintained. The rate and direction of ion-flux through the furosemide-
dependent
cotransporters are functionally proportional to their ion-product differences
written as
[K+]; x[Cl j i -[Klo x[Cl] o) for neuror..al Ky, Cl" cotransport and as [Na+]
i x [K ]i x [Cl"
~-[Aia+] ;, x[K+]o x[Cl-]? o) for giial NKCC cotransport. The sign of these
ion-product
differences show the direction of ion transport with positive being from it-
ttracellular to
extracellu?ar spaces.
Figure 4B shows a schematic phenonienological model that explains the
emergence of the late-occurring spontaneous field events that arise as a
result of
Z0... prolonged '.ow -[C i]o exposure. Wa denote the ion-product. diff:;re-
ices-.for neurons and
glia as QN and Qc:; respectively. L'nder control conditions (1), the-
differences of the ion-
p rodu.:.ts: for: neure.asi.are::such Lhat.I"}-..and,Cl- are cotransportett:
from intracetlutar, to
extraeel.lnlar:spaCes:((~1N >.0); the dif.ferenr.es. in ion-products.for glial
cells are such that
Na". Ky at id Cf- are. cotransported frorn: the..~CS. to. intracellular
coni~~~artt~i:,nts~. (i~,G:0).
When [%'11"]o is reduce(I (2), th;, ion-pr.oiluct.differences are:altered so
that neuronal e"floix:
of KCI is increased; hrtwev-er, the l;iial, icon cotransport.is.reversed
>.,(;); sc; tiiat;_there.
is a net efflux of KCI and NaCI froin intracelltilar to extracellular spaces.
These changes
result in buildup of extracellular potassium o.ver.time. Eventually, [K+]o
reaches..a.level
that induces the - depolarization of neuronal populations, resulting in an
even larger
accumiilatipn of [K+]o. - This' large accumulation of extracellular ions then
serves to
reverse- rlte ien-product ditferences so that KCI is moved from extrzcellular
to
intra,:ellular spaces (Qc4 < 0, Q(; < 0) (3). Ft:rther clearance of the
extrace'=lular potassiurri
eventually resets the transmembrane ion gradients to initial conditions. By
cycling
through this process, repetitive negative field events are generated.
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EXAMPLE 82
Therapeutic Efficacy of Furosemide in the Alleviation of Pain Symptoms in an
Animal Model of Neuropathic Pain
The ability of furosemide to alleviate pain will be examined in rodents using
the
Chung model of neuropathic pain (see, for example, Walker et al. Mol. Med.
Today
5:319-321, 1999). Sixteen adult male Long-Evans rats will be used in this
study. All rats
will receive spinal ligation of the L5 nerve as detailed below. Eight of the
sixteen rats
will receive an injection (intravenous) of furosemide and the remaining eight
will receive
intravenous injection of vehicle only. Pain threshold will be assessed
immediately using
the mechanical paw withdrawal test. Differences in pain thresholds between the
two
groups will be compared. If furosemide alleviates pain, the group with the
furosemide
treatment will exhibit a higher pain threshold than the group that received
vehicle.
Chung model of neuropathy
Spinal nerve ligation is performed under isoflourane anesthesia with animals
placed in the prone position to access the left L4-L6 spinal nerves. Under
magnification,
approximately one-third of the transverse process is removed. The L5 spinal
nerve is
identified and carefully dissected free from the adjacent L4 spinal nerve and
then tightly
ligated using a 6-0 "silk suture. The wound is treated with an antiseptic
solution, the
muscle layer is sutured, and the incision is closed with wound clips.
Behavioral testing of
mechanical paw withdrawal threshold takes place within a 3 - 7 day period
following the
incision. Briefly, animals are placed within a Plexiglas chamber (20 x 10.5 x
40.5 cm)
and allowed to habituate for 15 min. The chamber is positioned on top of a
mesh screen
so that mechanical stimuli can be administered to the plantar surface of both
hindpaws.
Mechanical threshold measurements for each hindpaw are obtained using an
up/down
method with eight von Frey monofilaments (5, 7, 13, 26, 43, 64, 106, and 202
mN). Each
trial begins with a von Frey force of 13 mN delivered to the right hindpaw for
approximately I sec, and then the left hindpaw. If there is no withdrawal
response, the
next higher force is delivered. If there is a response, the next lower force
is delivered.
This procedure is performed until no response is made at the highest force
(202 mN) or
until four stimuli are administered following the initial response. The 50%
paw
withdrawal threshold for each paw is calculated using the following formula:
[Xth]log =
[vFr]log + ky where [vFr] is the force of the last von Frey used, k = 0.2268
which is the
average interval (in log units) between the von Frey monofilaments, and y is a
value that
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depends upon the pattern of withdrawal responses. If an animal does not
respond to the
highest von Frey hair (202 mN), then y = 1.00 and the 50% mechanical paw
withdrawal
response for that paw is calculated to be 340.5 mN. Mechanical paw withdrawal
threshold testing is performed three times and the 50% withdrawal values are
averaged
over the three trials to determine the mean mechanical paw withdrawal
threshold for the
right and left paw for each animal.
EXAMPLE 83
Therapeutic Efficacy of Furosemide and Bumetanide in Alleviating the Symptoms
of Intense Anxiety or Post Traumatic Stress Disorder
The therapeutic usefulness of furosemide and bumetanide in the treatment of
post
traumatic stress disorder is examined by determining the ability of these
compounds to
alleviate intense anxiety in contextual fear conditioning in rats.
Contextual fear conditioning involves pairing an aversive event, in this case
moderate foot shock, with a distinctive environment. The strength of the fear
memory is
assessed using freezing, a species-typical defensive reaction in rats, marked
by complete
immobility, except for breathing. If rats are placed into a distinctive
environment and are
immediately shocked they do not learn to fear the context. However, if they
are allowed.
to explore the distinctive environment sometime before the immediate shock,
they show
intense anxiety and fear when placed back into the same environment. We can
take
advantage of this fact and, by procedurally dividing contextual fear
conditioning into two
phases, we can separately study effects of treatments on memory for the
context
(specifically a hippocampus based process) from learning the association
between context
and shock or experiencing the aversiveness of the shock (which depend upon
emotional
response circuitry including amygdala). Post traumatic stress syndrome (PTSD)
in
humans has been shown to be related to emotional response circuitry in the
amygdala,
and for this reason contextual memory conditioning is a widely accepted model
for
PTSD.
The experiment employed 24 rats. Each rat received a single 5 min episode of
exploration of a small, novel environment. Seventy-two hours later they were
placed into
the same environment and immediately received two moderate foot-shocks (1
milliamp)
separated by 53 sec. Twenty-four hours later, 8 of the rats received an
injection (I.V.) of
furosemide (100 mg/kg) in vehicle (DMSO), and 8 of the rats were injected I.V.
with
bumetanide (50 mg/kg) in vehicle (DMSO). The remaining 8 rats received an
injection of
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DMSO alone. Each rat was again placed into the same environment for 8 min
during
which time freezing was measured, as an index of Pavlovian conditioned fear.
Four identical chambers (20 X 20 X 15 cm) were used. All aspects of the timing
and control of events were under microcomputer control (MedPC, MedAssociates
Inc.,
Vermont, USA). Measurement of freezing was accomplished through an overhead
video
camera connected to the microcomputer and was automatically scored using a
specialty
piece of software, FreezeFrameTM (OER Inc., Reston, VA). Total freezing time
was
analyzed in a one-way analysis of variance (ANOVA) test, with drug dose as the
within-
groups factor.
As shown in Fig. 5 significantly less freezing was observed in animals treated
with either bumetanide or furosemide than in animals receiving vehicle alone,
indicating
that bumetanide and furosemide may be effectively employed in the treatment of
post
traumatic stress disorder.
EXAMPLE 84
Therapeutic Efficacy of Furosemide and Bumetanide in Alleviating Anxiety
The therapeutic efficacy of furosemide and bumetanide in alleviating anxiety
was
examined by evaluating the effects of these compounds in fear potentiated
startle (FPS)
test in rats. This test is commonly used to distinguish anxiolytic drug
effects from non-
specific effects, such as sedation/muscle relaxation
Twenty-four rats received a 30 min period of habituation to the FPS apparatus.
Twenty-four hours later, baseline startle amplitudes were collected. The rats
were then
divided into three matched groups based on baseline startle amplitudes. One of
the rats
exhibited a significantly higher baseline startle than the others and was
excluded from the
experiments. Groups 1 and 2 therefore consisted of 8 rats each, with Group 3
consisting
of 7 rats. Following baseline startle amplitude collection, 20 light/shock
pairings were
delivered on two sessions over two consecutive days (i.e., 10 light/shock
pairings per
day). On the final day (day 5), Groups 2 and 3 received an injection (i.v.) of
either
furosemide (100 mg/kg) or bumetanide (70 mg/kg) in vehicle (DMSO) and Group 1
received vehicle alone. Immediately following injections, startle amplitudes
were
assessed during startle alone trials and startle plus fear (light followed by
startle) trials.
Fear potentiated startle (light + startle amplitudes minus startle alone
amplitudes) was
compared between the treatment groups.
Animals were trained and tested in four identical stabilimeter devices (Med-
Associates). Briefly, each rat was placed in a small Plexiglas cylinder. The
floor of each
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stabilimeter consisted of four 6-mm-diameter stainless steel bars spaced 18 mm
apart
through which shock can be delivered. Cylinder movements result in
displacement of an
accelerometer _where the resultant voltage is proportional to the velocity of
the cage
displacement. Startle amplitude is defined as the maximum accelerometer
voltage that
occurs during the first 0.25 sec after the startle stimulus is delivered. The
analog output
of the accelerometer was amplified, digitized on a scale of 0-4096 units and
stored on a
microcomputer. Each stabilimeter was enclosed in a ventilated, light-, and
sound-
attenuating box. All sound level measurements were made with a Precision Sound
Level
Meter. The noise of a ventilating fan attached to a sidewall of each wooden
box produced
an overall background noise level of 64 dB. The startle stimulus was a 50 ms
burst of
white noise (5 ms rise-decay time) generated by a white noise generator. The
visual
conditioned stimulus employed was illumination of a light bulb adjacent to the
white
noise source. The unconditioned stimulus was a 0.6 mA foot shock with duration
of 0.5
see, generated by four constant-current shockers located outside the chamber.
The
presentation and sequencing of all stimuli were under the control of the
microcomputer.
FPS procedures consisted of 5 days of testing; during days 1 and 2 baseline
startle
responses were collected, days 3 and 4 light/shock pairings were delivered,
day 5 testing
for fear potentiated startle was conducted.
Matching: On the. first two days all rats were placed in the Plexiglas
cylinders and
3 min later presented with 30 startle stimuli at a 30 sec interstimulus
interval. An
intensity of 105 dB was used. The mean startle amplitude across the 30 startle
stimuli on
the second day was used to assign rats into treatment groups with similar
means.
Training: On the following 2 days, rats were placed in the Plexiglas
cylinders.
Each day following 3 min after entry, 10 CS-shock pairings were delivered. The
shock
was delivered during the last 0.5 sec of the 3.7 sec CSs at an average
intertrial interval of
4 min (range, 3-5 min).
Testing: Rats were placed in the same startle boxes where they were trained
and
after 3 min were presented with 18 startle-eliciting stimuli (all at 105 dB).
These initial
startle stimuli were used to again habituate the rats to the acoustic startle
stimuli. Thirty
seconds after the last of these stimuli, each animal received 60 startle
stimuli with half of
the stimuli presented alone (startle alone trials) and the other half
presented 3.2 sec after
the onset of the 3.7 sec CS (CS-startle trials). All startle stimuli are
presented at a mean
30 sec interstimulus interval, randomly varying between 20 and 40 sec.
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Measures: The treatment groups were compared on the difference in startle
amplitude between CS-startle and startle-alone trials (fear potentiation).
Fig. 6 shows the baseline startle amplitudes for each group of rats determined
prior to the test day. Fig. 7 shows the amplitude of response on startle alone
trials
determined on the test day immediately following injection of either DMSO
alone,
bumetanide or furosemide, with Fig. 8 showing the difference score (startle
alone - fear
potentiated startle) on the test day. As shown in the figures, a statistically
significantly
lower difference score was observed in rats treated with either furosemide or
bumetanide
than in rats treated with vehicle alone, indicating that both furosemide and
bumetanide are
effective in reducing anxiety.
Figs. 9 and 10 show the startle alone amplitude and the difference score,
respectively, one week after treatment with either furosemide or bumetanide.
Animals
treated with either furosemide or bumetanide were found to have a higher
difference
score than animals treated with vehicle alone. However, as the error bars are
so large for
the vehicle-treated animals, the data does not imply any statistically
significant difference
between vehicle and bumetanide, with possibly a small difference between
vehicle and
furosemide.
Fig. 12 shows the difference score for animals treated with either vehicle
alone
(n=13), or 10 mg (n=13), 30 mg (n=13), 40 mg (n=5), 50 mg (n=13) or 70 mg
(n=13) of
bumetanide. Doses of 40 mg, 50 mg and 70 mg were found to be significantly
more
effective in reducing anxiety than doses of 10 mg or 30 mg.
EXAMPLE 85
Therapeutic Efficacy of Bumetanide Analogs in Alleviating Anxiety
The therapeutic efficacy of several bumetanide analogs in alleviating anxiety
was
examined using the fear potentiated startle (FPS) test in rats as described
above.
Fig. 11 shows the difference score (startle alone - fear potentiated startle)
on the
test day in rats treated with one of the following bumetanide analogs:
bumetanide N,N-
diethylglycolamide ester (referred to as 2MIK053); bumetanide methyl ester
(referred to
as 3MIK054); bumetanide N,N-dimethylglycolamide ester (referred to as 3MIK069-
11);
bumetanide morpholinodethyl ester (referred to as 3MIK066-4); bumetanide
pivaxetil
ester (referred to as 3MIK069-12); bumetanide cyanomethyl ester (referred to
as
3MIK047); bumetanide dibenzylamide (referred to as 3MIK065); or bumetanide 3-
(dimethylaminoproply) ester (referred to as 3MIK066-5). The vehicle was DMSO.
As
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can be seen from Fig. 11, the difference score obtained after administration
of most of the
bumetanide analogs was significantly lower than that observed following
administration
of.vehicle alone, demonstrating that these analogs may be effectively employed
to reduce
anxiety.
In further studies, rats were injected with either bumetanide, saline or a
bumetanide analog, and their urine output was measured. As shown in Fig. 13,
bumetanide N,N-diethylglycolamide ester (column 2), bumetanide pivaxetil ester
(column
3) and bumetanide cyanomethyl ester (column 4) showed significantly lower
diuretic
effects than bumetanide obtained from two different sources (columns 1 and
13). Column
6 represents the results obtained following administration of saline.
While the present invention has been described with reference to the specific
embodiments thereof, it will be understood by those skilled in the art that
various changes
may be made and equivalents may be substituted without departing from the true
spirit
and scope of the invention. In addition, many modifications may be made to
adapt a
particular situation, material, composition of matter, method, method step or
steps, for use
in practicing the present invention. All such modifications are intended to be
within the
scope of the claims appended hereto.
All patents and publications cited herein and PCT Application WO 00/37616,
published June 29, 2000, are specifically incorporated by reference herein in
their
entireties.
SEQ ID NO: 1-2 are set out in the attached Sequence Listing. The codes for
polynucleotide and polypeptide sequences used in the attached Sequence Listing
conform
to WIPO Standard ST.25 (1988), Appendix 2.
CA 02604446 2007-10-09
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SEQUENCE LISTING
<110> Hochman, Daryl W.
Partridge, John J.
<120> Methods And Compositions For The
Treatment Of Anxiety Disorders
<130> 48000.1003c3PCT
<150> 11/251,724
<151> 2005-10-17
<150> 11/130,945
<151> 2005-05-17
<150> 11/101,000
<151> 2005-04-07
<160> 2
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Ala Lys Gly Ser Glu Glu Ala Lys Gly Arg Phe Arg Val Asn Phe Val
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Asp Pro Ala Ala Ser Ser Ser Ala Glu Asp Ser Leu Ser Asp Ala Ala
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Ser Gly His His Gln His Tyr Tyr Tyr Asp Thr His Thr Asn Thr Tyr
1
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Tyr Leu Arg Thr Phe Gly His Asn Thr Met Asp Ala Val Pro Arg Ile
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ttcaggctct atgtaaggac aacatctacc cagctttcca gatgtttgct aaaggttatg 2100
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4
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tcttaattgc tgaactgaat gttattgcac caattatctc aaacttcttc cttgcatcat 2220
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taatgttcgt cattaactgg tgggctgcat tgctaacata tgtgatagtc cttgggctgt 2400
atatttatgt tacctacaaa aaaccagatg tgaattgggg atcctctaca caagccctga 2460
cttacctgaa tgcactgcag cattcaattc gtctttctgg agtggaagac cacgtgaaaa 2520
actttaggcc acagtgtctt gttatgacag gtgctccaaa ctcacgtcca gctttacttc 2580
atcttgttca tgatttcaca aaaaatgttg gtttgatgat ctgtggccat gtacatatgg 2640
gtcctcgaag acaagccatg aaagagatgt ccatcgatca agccaaatat cagcgatggc 2700
ttattaagaa caaaatgaag gcattttatg ctccagtaca tgcagatgac ttgagagaag 2760
gtgcacagta tttgatgcag gctgctggtc ttggtcgtat gaagccaaac acacttgtcc 2820
ttggatttaa gaaagattgg ttgcaagcag atatgaggga tgtggatatg tatataaact 2880
tatttcatga tgcttttgac atacaatatg gagtagtggt tattcgccta aaagaaggtc 2940
tggatatatc tcatcttcaa ggacaagaag aattattgtc atcacaagag aaatctcctg 3000
gcaccaagga tgtggtagta agtgtggaat atagtaaaaa gtccgattta gatacttcca 3060
aaccactcag tgaaaaacca attacacaca aagttgagga agaggatggc aagactgcaa 3120
ctcaaccact gttgaaaaaa gaatccaaag gccctattgt gcctttaaat gtagctgacc 3180
aaaagcttct tgaagctagt acacagtttc agaaaaaaca aggaaagaat actattgatg 3240
tctggtggct ttttgatgat ggaggtttga ccttattgat accttacctt ctgacgacca 3300
agaaaaaatg gaaagactgt aagatcagag tattcattgg tggaaagata aacagaatag 3360
accatgaccg gagagcgatg gctactttgc ttagcaagtt ccggatagac ttttctgata 3420
tcatggttct aggagatatc aataccaaac caaagaaaga aaatattata gcttttgagg 3480
aaatcattga gccatacaga cttcatgaag atgataaaga gcaagatatt gcagataaaa 3540
tgaaagaaga tgaaccatgg cgaataacag ataatgagct tgaactttat aagaccaaga 3600
cataccggca gatcaggtta aatgagttat taaaggaaca ttcaagcaca gctaatatta 3660
ttgtcatgag tctcccagtt gcacgaaaag gtgctgtgtc tagtgctctc tacatggcat 3720
ggttagaagc tctatctaag gacctaccac caatcctcct agttcgtggg aatcatcaga 3780
gtgtccttac cttctattca taaatgttct atacagtgga cagccctcca gaatggtact 3840
tcagtgccta gtgtagtaac tgaaatcttc aatgacacat taacatcaca atggcgaatg 3900
gtgacttttc tttcacgatt tcattaattt gaaagcacac aggaaagttg ctccattgat 3960
aacgtgtatg gagacttcgg ttttagtcaa ttccatatct caatcttaat ggtgattctt 4020
ctctgttgaa ctgaagtttg tgagagtagt tttcctttgc tacttgaata gcaataaaag 4080
cgtgttaact ttttgattga tgaaagaagt acaaaaagcc tttagccttg aggtgccttc 4140
tgaaattaac caaatttcat ccatatatcc tcttttataa acttatagaa tgtcaaactt 4200
tgccttcaac tgtttttatt tctagtctct tccactttaa aacaaaatga acactgcttg 4260
tcttcttcca ttgaccattt agtgttgagt actgtatgtg ttttgttaat tctataaagg 4320
tatctgttag atattaaagg tgagaattag ggcaggttaa tcaaaaatgg ggaaggggaa 4380
atggtaacca aaaagtaacc ccatggtaag gtttatatga gtatatgtga atatagagct 4440
aggaaaaaaa gcccccccaa ataccttttt aacccctctg attggctatt attactatat 4500
ttattattat ttattgaaac cttagggaag attgaagatt catcccatac ttctatatac 4560
catgcttaaa aatcacgtca ttctttaaac aaaaatactc aagatcattt atatttattt 4620
ggagagaaaa ctgtcctaat ttagaatttc cctcaaatct gagggacttt taagaaatgc 4680
taacagattt ttctggagga aatttagaca aaacaatgtc atttagtaga atatttcagt 4740
atttaagtgg aatttcagta tactgtacta tcctttataa gtcattaaaa taatgtttca 4800
tcaaatggtt aaatggacca ctggtttctt agagaaatgt ttttaggctt aattcattca 4860
attgtcaagt acacttagtc ttaatacact caggtttgaa cagattattc tgaatattaa 4920
aatttaatcc attcttaata ttttaaaact tttgttaaga aaaactgcca gtttgtgctt 4980
ttgaaatgtc tgttttgaca tcatagtcta gtaaaatttt gacagtgcat atgtactgtt 5040
actaaaagct ttatatgaaa ttattaatgt gaagtttttc atttataatt caaggaagga 5100
tttcctgaaa acatttcaag ggatttatgt ctacatattt gtgtgtgtgt gtgtatatat 5160
atgtaatatg catacacaga tgcatatgtg tatatataat gaaatttatg ttgctggtat 5220
tttgcatttt aaagtgatca agattcatta ggcaaacttt ggtttaagta aacatatgtt 5280
caaaatcaga ttaacagata caggtttcat agagaacaaa ggtgatcatt tgaagggcat 5340
gctgtaattt cacacaattt tccagttcaa aaatggagaa tacttcgcct aaaatactgt 5400
taagtgggtt aattgataca agtttctgtg gtggaaaatt tatgcaggtt ttcacgaatc 5460
cttttttttt tttttttttt tttttgagac ggagtcttgc tctgttgcca cgctggaatg 5520
cagtaacgtg atcttggctc actgcgacct ccacctcccc agttcaagcg attctcctgc 5580
CA 02604446 2007-10-09
WO 2006/110187 PCT/US2005/043177
ctcagcctcc ctagtagctg ggactacggg tgcacgccac catgcccagc taatttttgt 5640
attttgagta gagacagggt ttcaccgtgt tggctaggat ggtgtctatc tcttgacctt 5700
gtgatccacc cgcctcagcc tcccagagtg ctgggattac aggtgcgagc cactgcgcct 5760
ggctggtttt catgaatctt gatagacatc tataacgtta ttattttcag tggtgtgcag 5820
catttttgct tcatgagtat gacctaggta tagagatctg ataacttgaa ttcagaatat 5880
taagaaaatg aagtaactga ttttctaaaa aaaaaaaaaa aaaaaatttc tacattataa 5940
ctcacagcat tgttccattg caggttttgc aatgtttggg ggtaaagaca gtagaaatat 6000
tattcagtaa acaataatgt gtgaactttt aagatggata atagggcatg gactgagtgc 6060
tgctatcttg aaatgtgcac aggtacactt accttttttt tttttttttt taagtttttc 6120
ccattcagga aaacaacatt gtgatctgta ctacaggaac caaatgtcat gcgtcataca 6180
tgtgggtata aagtacataa aatatatcta actattcata atgtggggtg ggtaatactg 6240
tctgtgaaat aatgtaagaa gcttttcact taaaaaaaat gcattacttt cacttaacac 6300
tagacaccag gtcgaaaatt ttcaaggtta tagtacttat ttcaacaatt cttagagatg 6360
ctagctagtg ttgaagctaa aaatagcttt atttatgctg aattgtgatt tttttatgcc 6420
aaattttttt tagttctaat cattgatgat agcttggaaa taaataatta tgccatggca 6480
tttgacagtt cattattcct ataagaatta aattgagttt agagagaatg gtggtgttga 6540
gctgattatt aacagttact gaaatcaaat atttatttgt tacattattc catttgtatt 6600
ttaggtttcc ttttacattc tttttatatg cattctgaca ttacatattt tttaagacta 6660
tggaaataat ttaaagattt aagctctggt ggatgattat ctgctaagta agtctgaaaa 6720
tgtaatattt tgataatact gtaatatacc tgtcacacaa atgcttttct aatgttttaa 6780
ccttgagtat tgcagttgct gctttgtaca gaggttactg caataaagga agtggattca 6840
ttaaacctat ttaatgtcca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 6891
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