U.S. patent application number 12/114719 was filed with the patent office on 2008-08-28 for methods of preventing and reducing the severity of stress-associated conditions.
Invention is credited to Mitchell F. Brin, John E. Donello, Daniel W. Gil, Scott Whitcup.
Application Number | 20080207627 12/114719 |
Document ID | / |
Family ID | 33540268 |
Filed Date | 2008-08-28 |
United States Patent
Application |
20080207627 |
Kind Code |
A1 |
Gil; Daniel W. ; et
al. |
August 28, 2008 |
METHODS OF PREVENTING AND REDUCING THE SEVERITY OF
STRESS-ASSOCIATED CONDITIONS
Abstract
The present invention provides a method of preventing or
reducing the severity of a stress-associated condition in a subject
by systemically administering to the subject an effective amount of
brimonidine or a pharmaceutically acceptable salt, ester, amide,
sterioisomer or racemic mixture thereof. Stress-associated
conditions that can be treated according to a method of the
invention include, but are not limited to, dyspepsia, tachycardias
other than tachycardia associated with myocardial ischemia, panic
attack, non-inflammatory dermatogical conditions, disorders of
muscle contraction, sensory hypersensitivity associated with
migraine, and behavioral disorders.
Inventors: |
Gil; Daniel W.; (Corona Del
Mar, CA) ; Whitcup; Scott; (Laguna Hills, CA)
; Brin; Mitchell F.; (Newport Beach, CA) ;
Donello; John E.; (Dana Point, CA) |
Correspondence
Address: |
ALLERGAN, INC.
2525 DUPONT DRIVE, T2-7H
IRVINE
CA
92612-1599
US
|
Family ID: |
33540268 |
Appl. No.: |
12/114719 |
Filed: |
May 2, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10607439 |
Jun 25, 2003 |
|
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12114719 |
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Current U.S.
Class: |
514/249 |
Current CPC
Class: |
A61P 3/10 20180101; A61P
17/00 20180101; A61P 25/00 20180101; A61P 25/14 20180101; A61P
13/10 20180101; A61P 37/08 20180101; A61P 1/00 20180101; A61P 1/04
20180101; A61K 31/498 20130101; A61P 25/06 20180101; A61P 29/00
20180101; A61P 1/14 20180101; A61P 21/00 20180101; A61P 11/00
20180101 |
Class at
Publication: |
514/249 |
International
Class: |
A61K 31/498 20060101
A61K031/498; A61P 29/00 20060101 A61P029/00 |
Claims
1-22. (canceled)
23. A method of reducing the severity of an inflammatory
dermatological condition in a subject, the method comprising
topically administering to a subject in need of such reduction an
effective amount of brimonidine or a pharmaceutically acceptable
salt, ester, amide, sterioisomer or racemic mixture thereof.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 10/607,439, the contents of which are incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates generally to the sympathetic nervous
system and various stress-associated conditions and, in particular,
to the .alpha.-2 adrenergic agonist, brimonidine.
[0004] 2. Background Information
[0005] Conditions that are associated with or exacerbated by stress
can be mediated, at least in part, by the sympathetic nervous
system. Such stress-associated conditions include, without
limitation, gastrointestinal disease; irritable bowel syndrome;
dyspepsia; tachycardia; panic attack; insulin-resistance; type II
diabetes; dermatogical conditions; disorders of muscle contraction
such as tension type headache; sensory hypersensitivity associated
with migraine such as nausea, photophobia and phonophobia; and
stress-associated behavioral disorders such as overeating and drug
dependence.
[0006] Unfortunately, treatments for such stress-associated
conditions have generally been ineffective or unsatisfactory, for
example, due to unwanted side-effects such as sedation. Thus, there
is a need for novel methods of preventing or reducing the severity
of stress-associated conditions. The present invention satisfies
this need and provides related advantages as well.
SUMMARY OF THE INVENTION
[0007] The present invention provides a method of preventing or
reducing the severity of a stress-associated condition in a subject
by systemically administering to the subject an effective amount of
brimonidine or a pharmaceutically acceptable salt, ester, amide,
sterioisomer or racemic mixture thereof, where the
stress-associated condition is one of the following:
gastrointestinal disease; irritable bowel syndrome; dyspepsia;
tachycardia; panic attack; insulin-resistance; type II diabetes; a
non-inflammatory dermatogical condition; a disorder of muscle
contraction; sensory hypersensitivity associated with migraine; or
a stress-associated behavioral disorder.
[0008] In one embodiment, a method of the invention prevents or
reduces the severity of gastrointestinal disease. In other
embodiments, a method of the invention prevents or reduces the
severity of irritable bowel syndrome or dyspepsia. In another
embodiment, a-method of the invention prevents or reduces the
severity of tachycardia other than tachycardia associated with
myocardial ischemia, for example, tachycardia associated with a
pulmonary disorder. In a further embodiment, a method of the
invention prevents or reduces the severity of panic attack. In
still further embodiments, a method of the invention prevents or
reduces the severity of insulin-resistance, or prevents or reduces
the severity of type II diabetes. In yet a further embodiment, a
method of the invention prevents or reduces the severity of a
non-inflammatory dermatological condition. In other embodiments, a
method of the invention prevents or reduces the severity of a
disorder of muscle contraction such as a disorder of skeletal
muscle contraction or a disorder of smooth muscle contraction, for
example, a disorder of smooth muscle contraction associated with
cystitis or associated with non-bacterial prostatitis or a disorder
of muscle contraction associated with tension type headache. In
another embodiment, a method of the invention prevents or reduces
the severity of sensory hypersensitivity associated with migraine.
In a further embodiment, a method of the invention prevents or
reduces the severity of sensory hypersensitivity associated with a
stress-associated behavioral disorder. In a method of the
invention, an effective amount of brimonidine can be administered
by any of a variety of methods including, but not limited to,
orally, topically, intravenously or via a patch.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows the tactile hypersensitivity observed with
several distinct chemical models. Each experimental group included
5-6 wildtype mice. Tactile hypersensitivity was assessed as
described below; sensitization scores determined every 5 minutes
during the 35 minute measurement period were summed and calculated
as the mean +/-SEM. Each group was compared to a vehicle control
using an unpaired two-tailed t-test (* p<0.01, ** p<0.001).
(a) Spinal injection of the .alpha.-1 agonist, phenylephrine,
induces tactile hypersensitivity in a dose dependent fashion.
Phenylephrine (filled circle) was injected intrathecally at various
doses. The .alpha.-1 antagonist, 5-MU (30 ug/kg i.p.; filled
square) was administered 15 minutes prior to intrathecal
administration of 30 ng phenylephrine. (b) Systemic phenylephrine
induces tactile hypersensitivity in a dose dependent fashion.
Phenylephrine (filled circle) was injected intraperitoneally at
various doses. The .alpha.-1 antagonist, 5-MU (30 ug/kg i.p.;
filled square) was administered 15 minutes prior to administration
of 30 ng/kg phenylephrine. (c) Spinal sulprostone, a selective
EP.sub.1/EP.sub.3 agonist, induces chemical tactile
hypersensitivity in a dose responsive fashion. Increasing doses of
sulprostone (filled circle) were injected intrathecally. An
EP.sub.1 antagonist (100 ng i.t.; filled square) was injected 15
minutes prior to administration of 200 ng sulprostone. (d) Spinal
administration of NMDA induces tactile hypersensitivity in a dose
responsive fashion. NMDA (filled circle) was injected intrathecally
at various doses. The NMDA antagonist, memantine (1 ug i.t.;
(filled square), was injected 15 minutes prior to administration of
100 ng NMDA.
[0010] FIG. 2 shows that the increased sympathetic tone of
.alpha.-2A and .alpha.-2C knockout mice enhances induction of
tactile hypersensitivity by .alpha.-1 receptor activation. Wildtype
(filled circle), .alpha.-2A knockout (filled square), and
.alpha.-2C knockout (filled triangle) mice were-injected
intraperitoneally with increasing doses of phenylephrine and
assayed for tactile hypersensitivity. .alpha.-2A knockout mice were
pretreated with 50 mg/kg i.p. guanethidine to cause a temporary
chemical sympathectomy 24-30 hours prior to an i.p. injection with
phenylephrine (open square). Each group of mice consisted of 5-6
animals. The mean sensitization score and SEM were calculated and
compared to a vehicle control group using an unpaired two-tailed
t-test (* p<0.01, ** p<0.001).
[0011] FIG. 3 shows that the sympathetic nervous system enhances
sulprostone-induced tactile hypersensitivity. Wildtype (filled
circle), .alpha.-2A (filled square), and .alpha.-2C (filled
triangle) knockout mice were injected intrathecally with increasing
doses of sulprostone and assayed for tactile hypersensitivity.
.alpha.-2A knockout mice were pretreated with guanethidine (50
mg/kg i.p.) to cause a temporary chemical sympathectomy 24 hours
prior to an intrathecal sulprostone injection (open square). Each
group of mice consisted of 5-6 animals. The mean sensitization
score and SEM were calculated and compared to a vehicle control
group using an unpaired two-tailed t-test (* p<0.01, **
p<0.001).
[0012] FIG. 4 shows that .alpha.-2 knockout mice do not exhibit
altered NMDA-induced tactile hypersensitivity. Wildtype (filled
circle), .alpha.-2A (filled square), and .alpha.-2C (filled
triangle) knockout mice were injected intrathecally with increasing
doses of NMDA. Each group of 5-6 mice was scored for tactile
hypersensitivity. The mean response and SEM were calculated and
compared to a vehicle control group using an unpaired two-tailed
t-test (* p<0.01, ** p<0.001).
[0013] FIG. 5 shows that .alpha.-adrenergic agonists differ in
alleviation of sympathetically-enhanced sensory hypersensitivity.
The response of 5-6 mice per group was scored; the mean response
and SEM were calculated as described above. Each drug-treated group
was compared to a vehicle control group using an unpaired
two-tailed t-test (* p<0.01, ** p<0.001). (a) Spinal
brimonidine and clonidine alleviate NMDA-induced tactile
hypersensitivity in wildtype mice. Mice were injected intrathecally
with DMSO vehicle or co-injected intrathecally with 100 ng NMDA and
saline, 0.4 .mu.g brimonidine (UK14304) or 1 .mu.g clonidine. (b)
Spinal brimonidine and clonidine alleviate sulprostone-induced
tactile hypersensitivity in wildtype mice. Mice were injected
intrathecally with DMSO vehicle or co-injected intrathecally with
200 ng sulprostone and saline, 0.4 .mu.g brimonidine (UK14304) or
0.4 .mu.g clonidine. (c) Spinal brimonidine and clonidine alleviate
NMDA-induced tactile hypersensitivity in the .alpha.-2C knockout
mice, but not in the .alpha.-2A knockout mice. Mice were injected
intrathecally with DMSO vehicle or coinjected intrathecally with
100 ng NMDA and saline, 0.4 .mu.g brimonidine (UK14304) or 1 ug
clonidine. (d) Spinal brimonidine and clonidine differ in their
ability to alleviate sulprostone-induced tactile hypersensitivity
in the .alpha.-2C knockout mice. Mice were injected with DMSO
vehicle or co-injected intrathecally with 200 ng (.alpha.-2C
knockout) or 30 ng (.alpha.-2A knockout) sulprostone and saline,
0.4 .mu.g brimonidine (UK14304) or 0.4 .mu.g clonidine. .alpha.-2
agonist analgesia is absent in the .alpha.-2A knockout mice;
clonidine analgesia is also lost in the .alpha.-2C knockout
mice.
[0014] FIG. 6 shows that brimonidine, but not clonidine or
tizanidine, alleviates sulprostone-induced tactile hypersensitivity
in the absence of sedation. The dose-responsive anti-hypersensitive
and sedative effects of three .alpha.-2 agonists (tizanidine,
triangle; clonidine, square; and brimonidine, circle) were compared
in models of sulprostone-induced tactile hypersensitivity and
locomotor activity, respectively. The mean total sensitivity scare
and standard error of the mean was calculated and indicated as a
solid line (left axis). Locomotor activity relative to
vehicle-treated animals was expressed as a percentage, and the
percent sedation calculated as 100% minus the percent locomotor
activity and indicated as a hatched line (right axis).
[0015] FIG. 7 shows variable .alpha.-2 vs. .alpha.-1 agonist
selectivity in .alpha.-adrenergic agonists clonidine and
brimonidine. Increasing concentrations of phenylephrine (filled
square), clonidine (filled diamond), tizanidine (filled circle),
dexmeditomidine (filled triangle) and brimonidine (filled inverted
triangle) were tested for .alpha.-1 and .alpha.-2 agonist activity
using in vitro cell-based functional assays. (a, b) .alpha.-1A and
.alpha.-1B agonist activity of .alpha.-adrenergic agonists. The
increase in intracellular calcium in HEK cells stably expressing
the bovine .alpha.-1A receptor (a) or the hamster .alpha.-1B
receptor (b) following addition of various concentrations of
.alpha.-adrenergic agonists was determined by measuring the change
in fluorescence of a calcium-sensitive dye. Agonists were tested
6-15 times in triplicate, and the mean fluorescence and SEM
calculated at each concentration. Results from a typical experiment
are shown. (c, d) .alpha.-2A and .alpha.-2C agonist activity of
.alpha.-adrenergic agonists. Inhibition of forskolin-induced cAMP
accumulation in PC12 cells stably expressing the human .alpha.-2A
receptor (c) or the human .alpha.-2C receptor (d) following
addition of various-concentrations of .alpha.-adrenergic agonists.
Agonists were tested 3-5 times in triplicate, and the mean %
inhibition and SEM calculated at each concentration. Results from a
typical experiment are shown. (e) Co-administration of prazosin
with clonidine restores clonidine-mediated analgesia in .alpha.-2C
knockout mice. Wildtype (open bars) and .alpha.-2C knockout
(hatched bars) mice were injected with vehicle, prazosin (100 ng/kg
i.p.), sulprostone (200 ng i.t.), clonidine (400 ng i.t.) or
various combinations as indicated. The tactile hypersensitivity of
5-6 mice per group was scored, and the mean response and SEM was
calculated. Each drug-treated group was compared to a vehicle
control group using an unpaired two-tailed t-test (* p<0.01, **
p<0.001).
DETAILED DESCRIPTION OF THE INVENTION
[0016] Adrenergic receptors mediate physiological responses to the
catecholamines, norephinephrine and epinephrine, and are members of
the superfamily of G protein-coupled receptors having seven
transmembrane domains. These receptors, which are divided
pharmacologically into .alpha.-1, .alpha.-2 and .beta.-adrenergic
receptor types, are involved in diverse physiological functions
including functions of the cardiovascular and central nervous
systems. The .alpha.-adrenergic receptors mediate most excitatory
functions: .alpha.-1 adrenergic receptors generally mediate
responses in the effector organ, while .alpha.-2 adrenergic
receptors are located postsynaptically as well as presynaptically,
where they regulate release of neurotransmitters. Agonists of
.alpha.-2 adrenergic receptors currently are used clinically in the
treatment of hypertension, glaucoma, spasticity, and
attention-deficit disorder, in the suppression of opiate
withdrawal, and as adjuncts to general anesthesia.
[0017] .alpha.-2 adrenergic receptors are presently classified into
three subtypes based on their pharmacological and molecular
characterization: .alpha.-2A/D (.alpha.-2A in human and .alpha.-2D
in rat); .alpha.-2B; and .alpha.-2C (Bylund et al., Pharmacol. Rev.
46:121-136 (1994); and Hein and Kobilka, Neuropharmacol. 34:357-366
(1995)). The .alpha.-2A and .alpha.-2B subtypes can regulate
arterial contraction in some vascular beds, and the .alpha.-2A and
.alpha.-2C subtypes mediate feedback inhibition of norepinephrine
release from sympathetic nerve endings. The .alpha.-2A subtype also
mediates many of the central effects of .alpha.-2 adrenergic
agonists (Calzada and Artinano, Pharmacol. Res. 44: 195-208 (2001);
Hein et al., Ann. NY Acad. Science 881:265-271 (1999); and Ruffolo
(Ed.), .alpha.-Adrenoreceptors: Molecular Biology, Biochemistry and
Pharmacology S. Karger Publisher's Inc. Farmington, Conn.
(1991)).
[0018] Previous studies have shown that norepinephrine has a higher
affinity for the .alpha.-2C receptor (K.sub.i=650 nM) than the
.alpha.-2A receptor (K.sub.i=5800 nM; Link et al., Mol. Pharm.
42:16-27 (1992)). Thus, the autoinhibitory action on norepinephrine
release is mediated through the .alpha.-2C receptor at low
concentrations of norepinephrine, and through the .alpha.-2A
receptor at high concentrations of norepinephrine (Altman et al.,
Mol. Pharm. 56:154-161 (1999)). As a result, feedback inhibition of
basal norepinephrine release is mediated by the .alpha.-2C
receptor, while the .alpha.-2A receptor mediates feedback
inhibition of release under conditions of high frequency
stimulation (Hein et al., Ann. N.Y. Acad. Sci. 881:265-271 (1999)).
As disclosed herein in Example II, the .alpha.-2C knockout mice,
which have a decreased presynaptic inhibition of sympathetic
outflow under basal (or low frequency stimulation) conditions, are
more sensitive to augmentation of .alpha.-1 receptor activity
through phenylephrine treatment (see FIG. 2). Furthermore, as shown
herein in FIG. 3, .alpha.-2A knockout mice are more sensitive to
sulprostone-induced tactile hypersensitivity, while in .alpha.-2C
knockout mice, the sulprostone sensitivity is the same as that of
wildtype mice. These results demonstrate that sulprostone treatment
results in high frequency sympathetic nerve stimulation, as
evidenced by the fact that only .alpha.-2A knockout mice, which
lack presynaptic inhibition of high frequency sympathetic outflow,
exhibit a decreased threshold to sulprostone-induced tactile
hypersensitivity.
[0019] As further disclosed herein in Example III, brimonidine was
analgesic in both wild type and .alpha.-2C knockout mice with
sulprostone-induced tactile hypersensitivity. In contrast,
clonidine was analgesic in wild type mice but not in .alpha.-2C
knockout mice (compare FIGS. 5b and d). As expected, neither
clonidine nor brimonidine were analgesic in .alpha.-2A knockout
mice, which lack the spinal .alpha.-2A adrenergic receptor which
mediates analgesic activity. Thus, in .alpha.-2C knockout mice
treated with sulprostone, which serve as a model for
sympathetically-enhanced conditions, the pan-agonists brimonidine
and clonidine have strikingly different activities. Additional
results disclosed herein demonstrate that, in wild type mice,
brimonidine, but not other pan-agonists such as tizanidine or
clonidine, had analgesic activity without concomitant sedation (see
FIG. 6). Furthermore, brimonidine was highly selective (more than
1000-fold) for .alpha.-2 adrenergic receptors as compared to
.alpha.-1 receptors in functional assays as compared to other
pan-agonists such as clonidine and tizanidine, which exhibited less
than 10-fold selectivity (see FIG. 7 and Table 2). These results
demonstrate the differential functional activity of the
pan-agonists brimonidine and clonidine and indicate that .alpha.-2
versus .alpha.-1 functional selectivity can be advantageous in
treating sympathetically-enhanced conditions such as
stress-associated conditions without concomitant sedation.
[0020] Dyspepsia has been described as a biopsychosocial disorder
and is generally characterized, in part, by epigastric discomfort
following meals. In addition to postprandial upper abdominal
discomfort or pain, dyspepsia can be characterized by early
satiety, nausea, vomiting, abdominal distension, bloating, or
anorexia in the absence of organic disease (Thumshirn, Gut 51
Suppl. 1: i63-66 (2002; Anderson, Dorland's Illustrated Medical
Dictionary 28.sup.th Edition, W.B. Saunder's Company, Philadelphia
(1994)).
[0021] The methods of the invention can be useful for preventing or
reducing the severity of dyspepsia, which, as used herein, is a
term which means impaired digestion. Any of a variety of types of
dyspepsia can be treated according to a method of the invention.
The term dyspepsia includes, without limitation, acid dyspepsia,
which is associated with excessive acidity of the stomach;
appendicular dyspepsia, also known as appendix dyspepsia, in which
dyspeptic symptoms accompany chronic appendicitis; catarrhal
dyspepsia, which is accompanied by gastric inflammation; chichiko
dyspepsia, a condition of farinaceous malnutrition found in poorly
nourished infants; cholelithic dyspepsia, which involves sudden
dyspeptic attacks associated with gallbladder disturbance; colonic
dyspepsia, which involves a functional disturbance of the large
intestine; fermentive dyspepsia, which is characterized by
fermentation of ingested food; flatulent dyspepsia, which is
associated with the formation of gas in the stomach and often
involves upper abdominal discomfort accompanied by frequent
belching; gastric dyspepsia, which originates in the stomach; and
intestinal dyspepsia, which originates in the intestines. It is
understood that these and other mildly or acutely symptomatic forms
of the condition are included in the definition of "dyspepsia" as
used herein. In one embodiment, the methods of the invention are
used to prevent or reduce the severity of dyspepsia other than
dyspepsia associated with gastric inflammation.
[0022] In another embodiment, the invention relates to treating
gastrointestinal disease. Inflammatory bowel disease (IBD) or
irritable bowel syndrome (IBS) are gastrointestinal diseases which
affect one-half of all Americans during their lifetime, at a cost
of greater than $2.6 billion dollars for IBD and greater than $8
billion dollars for IBS. The frequency or severity of visceral
hypersensitivity associated with IBD, IBS and other
gastrointestinal diseases including inflammatory gastrointestinal
diseases is exacerbated by stress. As disclosed herein, the methods
of the invention can be useful for preventing or reducing the
severity of visceral hypersensitivity associated with a
stress-associated gastrointestinal disease such as, without
limitation, ulcerative colitis (UC), Crohn's disease (CD), or
irritable bowel syndrome (IBS). Thus, the present invention
provides a method of preventing or reducing the severity of
visceral hypersensitivity associated with a stress-associated
gastrointestinal disease in a subject by systemically administering
to the subject an effective amount of brimonidine or a
pharmaceutically acceptable salt, ester, amide, sterioisomer or
racemic mixture thereof.
[0023] The methods of the invention also can be useful for
preventing or reducing the severity of tachycardia which is not
associated with myocardial ischemia. As used herein, the term
"tachycardia" means excessive rapidity of heart rate and includes
tachyarrhymthias. In adults, the term tachycardia generally refers
to a heart rate of greater than 100 beats per minute. The term
tachycardia encompasses tachycardias secondary to a variety of
disorders other than myocardial ischemia including, without
limitation, paroxysmal tachycardia, in which the tachycardia is of
sudden onset and cessation and either ventricular or
supraventricular, and nonparoxysmal tachycardia, which is a
tachycardia of slow onset, generally with a heart rate of 70 to 130
beats per minute. In one embodiment, a method of the invention
prevents or reduces the severity of an automatic tachycardia which
is not associated with myocardial ischemia. In another embodiment,
a method of the invention prevents or reduces the severity of
tachycardia in an adult subject. In a further embodiment, a method
of the invention prevents or reduces the severity of tachycardia in
a subject who is a child.
[0024] Tachycardias to be treated according to a method of the
invention include those originating from any part of the heart such
as ventricular tachycardias and supraventricular tachycardias,
which can be classified, for example, into atrial and junctional
(nodal) tachycardias. Thus, the methods of the invention can be
useful for preventing or reducing the severity of, for example,
ventricular tachycardias, which are abnormally rapid ventricular
rhythms with aberrant ventricular excitation, often in excess of
150 beats per minutes, generated within the ventricle and sometimes
occurring in conjunction with atrioventricular dissociation. The
methods of the invention further can be useful for preventing or
reducing the severity of supraventricular tachycardias (SVT), which
are regular tachycardias in which the point of stimulation is
located above the bundle branches such as in the sinus node, atria
or atrioventricular junction or which arise from a large reentrant
circuit including both atrial and ventricular sites. In one
embodiment, a method of the invention is used to prevent or reduce
the severity of an atrial tachycardia, which is characterized by a
rapid cardiac rate generally between 160 and 190 beats per minutes
and which originates from an atrial locus; such tachycardias
include, but are not limited to, paroxysmal atrial tachycardias. In
another embodiment, a method of the invention is used to prevent or
reduce the severity of a junctional tachycardia, which is a
tachycardia arising in response to impulses originating in the
atrioventricular junction and which is generally characterized by a
heart rate greater than 75 beats per minute. Junctional
tachycardias include nonparoxysmal and paroxysmal junctional
tachycardias, such as junctional tachycardias resulting from
reentry or enhanced automaticity. It is understood that the methods
also can be used to prevent or reduce the severity of, without
limitation, double tachycardias, in which two types of ectopic
tachycardia are involved; sinus tachycardias, which originate in
the sinus node and can be associated with shock, hypotension,
congestive heart failure or fever; orthostatic tachycardia, which
is characterized by a disproportionate rapidity of heart rate upon
rising from a reclining to a standing position; and chaotic atrial
tachycardia, which is characterized by atrial rates of 100 to 130
beats per minute, markedly variable P wave morphology and irregular
P-P intervals (Anderson, supra, 1994).
[0025] Tachycardias to be treated according to a method of the
invention can be associated with one or more disorders such as
pulmonary disease, diabetes, or surgical trauma and can occur, for
example, in the elderly. As an example, chaotic atrial tachycardia
(multifocal atrial tachycardia) can be present, for example, in
patients with chronic obstructive pulmonary disease, in patients
with diabetes, and in the elderly. As a further example,
nonparoxysmal junctional tachycardia can be associated, for
example, with surgical trauma. It is understood that these and a
variety of well known automatic and other tachycardias which are
not associated with myocardial ischemia can be prevented or reduced
in severity according to the methods of the invention. In another
embodiment, the invention provides a method of preventing or
reducing the severity of tachycardias of all types including those
associated with myocardial ischemia.
[0026] The methods of the invention also can be useful for
preventing or reducing the severity of panic attack, a common
disorder with a prevalence of around 3% in the general population
(Potokar and Nutt, Int. J. Clin. Pract. 54: 110-114 (2000)). Panic
disorder involving recurrent panic attacks is typically observed in
young adults, with an average age of onset of 24 years, and is more
common in females than in males. The term "panic attack," as used
herein, means a discrete period of intense fear or discomfort
accompanied by one or more of the following symptoms: accelerated
heart rate or palpitation; chest pain; chills or hot flushes;
derealization or depersonalization; fear of dying; fear of losing
control or going crazy; dizziness or faintness; feelings of
choking; nausea or abdominal distress; paraesthesia; sensations of
shortness of breath or smothering; sweating; or trembling or
shaking. A panic attack typically begins with the sudden onset of
intense apprehension or fear and generally has a duration of about
5 to 20 minutes. The term panic attack encompasses both full-blown
and limited-symptom attacks; full-blown attacks involve four or
more of the above symptoms while limited-symptom attacks involve
fewer than four symptoms. A method of the invention can entirely
prevent a panic attack, or can prevent or reduce the severity of
one or any combination of the attendant symptoms described
above.
[0027] Some patients with panic attacks develop "panic disorder,"
which also can be prevented or reduced in severity using
brimonidine according to a method of the invention. The term panic
attack, as used herein, encompasses panic disorder, which is
defined as recurrent panic attacks in conjunction with persistent
concern over additional episodes or the consequences of the attacks
or a notable change in behavior experienced for at least one month
following one or more panic attacks. Panic disorder can be
associated with other psychiatric conditions such as
depression.
[0028] The central sympathetic nervous system can play a critical
role in the development of insulin-resistance and hypertension
which characterize type II diabetes (Rocchini et al., Hypertension
33-[part II]:548-553 (1999)). Further provided herein is a method
of preventing or reducing the severity of type II diabetes, a
disorder characterized by hypertension, hyperlipidemia and
insulin-resistance and which is exacerbated by stress. As disclosed
herein, brimonidine or a pharmaceuticallly acceptable salt, ester,
amide, sterioisomer or racemic mixture thereof, can be systemically
administered to a subject in order to prevent or reduce the
severity of type II diabetes in the subject.
[0029] The methods of the invention also can be useful for
preventing or reducing the severity of a non-inflammatory
dermatological condition. Such methods can be useful, for example,
for preventing or reducing the severity of one or more symptoms
such as itching or other discomfort associated with a
non-inflammatory dermatological condition. As used herein, the term
"non-inflammatory dermatological condition" means any dermatosis or
other skin disease or condition that is not caused or accompanied
by inflammation. A non-inflammatory dermatological condition to be
treated according to a method of the invention can originate or be
exacerbated under stressful conditions. Non-inflammatory
dermatological conditions encompass, without limitation,
non-inflammatory dermatoses including non-inflammatory blistering
diseases such as epidermolysis bullosa and porphyria; ichthyosis;
keratosis pilaris; juvenile plantar dermatosis (JPD); lichen
plantus dermatosis; and xerosis. One skilled in the art understands
that these and other non-inflammatory dermatological conditions
known in the art can be treated by a method disclosed herein.
[0030] In a separate embodiment, the invention provides a method of
preventing or reducing the severity of a stress-associated
inflammatory dermatological condition in a subject by systemically
administering to the subject an effective amount of brimonidine or
a pharmaceutically acceptable salt, ester, amide, sterioisomer or
racemic mixture thereof. Such methods can be useful, for example,
in preventing or reducing the severity of one or more symptoms such
as itching or other discomfort associated with the inflammatory
dermatological condition. Any of a variety of inflammatory
dermatological conditions are encompassed by the methods of the
invention including, without limitation, any of a variety of forms
of acute or chronic dermatitis such as psoriasis, allergic
dermatitis such as allergic contact dermatitis, atopic dermatitis,
dermatitis calorica, contact dermatitis, cosmetic dermatitis,
eczema, exfoliative dermatitis, factitial dermatitis, irritant
dermatitis, lichen simplex chronicus, marine dermatitis,
neurodermatitis, perioral dermatitis, phototoxic dermatitis,
seborrheic dermatitis, stasis dermatitis and dermatitis
vegetans.
[0031] The methods of the invention can be useful for preventing or
reducing the severity of a variety of disorders of muscle
contraction, which are conditions that result, at least in part,
from inappropriate muscle contraction. Disorders of muscle
contraction to be treated according to a method of the invention
include, without limitation, disorders of skeletal muscle
contraction, disorders of smooth muscle contraction, disorders of
muscle contraction associated with a gland, and disorders of
cardiac muscle contraction such as congestive heart failure; these
and other disorders to be prevented or reduced in severity
according to a method of the invention include those in which the
myocytes are innervated as well as those in which the myocytes are
not innervated. As non-limiting examples, the methods of the
invention can be useful for preventing or reducing the severity of
a disorder of muscle contraction such as back or other muscle
spasm; muscle contraction associated with cystitis; muscle
contraction associated with non-bacterial prostatitis; muscle
contraction associated with teeth grinding; muscle contraction
associated with tension type headache; and muscle contraction
associated with congestive heart failure.
[0032] The methods of the invention can be useful, for example, for
preventing or reducing the severity of a muscle spasm such as a
back spasm. Muscle spasms are well known in the art. As used
herein, the term "spasm" means a sudden, involuntary contraction of
a muscle or a group of muscles, accompanied by pain and
interference with function. A spasm can produce, for example,
involuntary movement or distortion. In one embodiment, a method of
the invention prevents or reduces the severity of a back spasm.
[0033] In one embodiment, a method of the invention is useful for
preventing or reducing the severity of muscle contraction
associated with cystitis. As used herein, the term "cystitis" means
inflammation of the urinary bladder. The term cystitis encompasses,
yet is not limited to, allergic cystitis, bacterial cystitis, acute
catarrhal cystitis, cystic cystitis, diphtheritic (croupous)
cystitis, eosinophilic cystitis, exfoliative cystitis, cystitis
follicularis, cystitis glandularis, incrusted cystitis, chronic
interstitial (panmural, submucous) cystitis, mechanical cystitis,
cystitis papillomatosa and cystitis senilis feminarum. See, for
example, Anderson, supra, 1994. Cystitis can be accompanied by one
or more of the following clinical symptoms: frequent urination,
burning on urination, suprapubic discomfort, lassitude, cloudy or
blood-tinged urine and sometimes low-grade fever (Bennett and Plum
(Eds.), Cecil Textbook of Medicine Sixth Edition, W.B. Saunders
Company, Philadelphia 1996). One skilled in the art understands
that the muscle contraction associated with any of these or other
forms of mild, severe, acute or chronic cystitis can be treated
according to a method of the invention.
[0034] As disclosed herein, a method of the invention also can be
useful for preventing or reducing the severity of muscle
contraction associated with non-bacterial prostatitis. Symptoms of
prostatic inflammation are experienced by about 50% of men in adult
life; of these, about 95% result from factors other than bacterial
infection. As used herein, the term "non-bacterial prostatitis" is
synonymous with "abacterial prostatitis" and means inflammation of
the prostate not resulting from bacterial infection. Non-bacterial
prostatitis encompasses, yet is not limited to, chronic
non-bacterial prostatitis, allergic or eosinophilic prostatitis and
non-specific granulomatous prostatitis. It is understood that the
term non-bacterial prostatitis includes, without limitation,
prostatitis of unknown etiology characterized by abnormal expressed
prostatic secretions (EPS) and normal bacterial cultures. In some
cases, non-bacterial prostatitis can be effectively treated with
antibiotics or stress management (Bennett and Plum, supra, 1996).
It is understood that muscle contraction associated with these or
other forms of mild, severe, acute or chronic non-bacterial
prostatitis can be treated according to a method of the
invention.
[0035] In another embodiment, a method of the invention is useful
for preventing or reducing the severity of muscle contraction
associated with tension type headache (TTH), which is a common form
of headache affecting as many as 90% of adult Americans. As used
herein, the term "tension type headache" means a headache caused,
at least in part, by muscle contraction, which may be triggered,
for example, by stress or exertion. The term "tension type
headache" encompasses episodic and chronic headache and includes
but is not limited to common tension headaches. Tension type
headaches generally involve the posterior of the head and neck,
although they may also appear at the top or front of the skull and
are further generally characterized by symmetry and a non-disabling
severity. Although not all may be present, diagnostic features of
tension type headache include bilateral pain; mild to moderate
severity; pressing-like character with a stable profile;
accentuation as the day progresses; possible high frequency such as
daily or continuously; and relative rarity of migrainous features
such as nausea, photosensitivity, phonosensitivity and aggravation
by physical activity such as head movement.
[0036] Tension type headaches result from tightening of muscles of
the face, neck and scalp due, for example, to stress, overwork,
eyestrain or poor posture. Such headaches can last for days or
weeks and can cause pain of varying intensity. Tension type
headaches occurring over an extended period of time such as several
weeks or months are denoted chronic tension headaches and are
encompassed by the term tension type headache as used herein.
[0037] Tension type headaches can be distinguished from migraines
by the absence of vascular features and symptoms such as nausea,
vomiting and sensitivity to light and the absence of an aura
(Spira, Austr. Family Phys. 27: 597-599 (1998). The term tension
type headache, which refers to headaches without a significant
vascular component, is used in contradistinction to
tension-vascular headaches, cluster headaches, migrainous headaches
and other headaches with a major vascular component. However, the
methods of the invention also can be useful for preventing or
reducing the severity of sensory hypersensitivity associated with
other headaches including, but not limited to, cervicogenic
headache, post-traumatic headache, cluster headache and
temporomandibular joint disorder (TMJ).
[0038] The methods of the invention also can be useful for
preventing or reducing the severity of sensory hypersensitivity
associated with migraine, a headache that plagues more than 10% of
the population and that may be associated with a vascular
component. In one embodiment, the methods of the invention prevent
or reduce the severity of an ocular hypersensitivity associated
with migraine, for example, photophobia. The methods of the
invention are useful for preventing or reducing the severity of
sensory hypersensitivity associated with any of a variety of forms
of migraine including, but not limited to, migraine without aura
("MO"), migraine with aura ("MA"), and migrainous disorder. Sensory
hypersensitivity to be prevented or reduced in severity according
to a method of the invention further can be associated with, for
example, abdominal migraine, acute confusional migraine, basilar
(basilar artery) migraine, hemiplegic or familial hemiplegic
migraine, fulgurating migraine, ocular (ophthalmic) migraine,
opthalmoplegic migraine or retinal migraine. In addition, the
methods of the invention can be useful for preventing or reducing
the severity of sensory hypersensitivity associated with a migraine
equivalent, in which there is a migraine aura without headache.
Migraine auras are the abnormal visual, motor, psychic, paresthesic
or other neurologic abnormalities that accompany a migraine. See
Elrington, J. Neurol. Neurosurg. Psychiatry 72 Supple. II:ii10-ii15
(2002); Anderson, supra, 1994; Bennett and Plum, supra, 1996.
[0039] The methods of the invention can be useful for preventing or
reducing the severity of one or more of a variety of types of
sensory hypersensitivity associated with migraine. Such sensory
hypersensitivity includes, but is not limited to, nausea; vomiting;
diarrhea; photophobia (light intolerance); and phonophobia (noise
intolerance). Such sensory hypersensitivity also includes visual
abnormalities such as bright flashing lights (scintillation or
fortification scotomata) or a monocular (retinal) visual
abnormality or hemianoptic loss of vision; paresthesia (abnormal
touch sensation) such as unilateral paresthesia; aphasia (loss of
speech or comprehension); hemiparesis (muscular weakness or
incomplete paralysis on one side of the body); hemisensory defect;
or vertigo, ataxia (loss of muscular coordination) or diplopia. It
is understood that the methods of the invention can be useful for
preventing or reducing the severity of one of these or other types
of sensory hypersensitivity occurring prior to, during, or
subsequent to migraine headache, or occurring in the absence of
headache as part of a migraine equivalent.
[0040] The methods of the invention also can be useful for
preventing or reducing the severity of one or more of a variety of
types of sensory hypersensitivity associated with other disorders
such as fibromyalgia, also known as fibrositis. Fibromyalgia is a
disorder involving chronic, widespread musculoskeletal pain and
tenderness at multiple sites in the absence of signs of connective
tissue or other musculoskeletal disease. In particular,
fibromyalgia is defined by pain or tenderness at 11 of 18 or more
sites as defined by the American College of Rheumatology.
Fibromyalgia frequently is associated with disturbed sleep, chronic
fatigue, headaches and irritable bowel syndrome (Bennett and Plum,
supra, 1996).
[0041] A variety of types of sensory hypersensitivity can be
associated with fibromyalgia and can be prevented or reduced in
severity according to a method of the invention, including, without
limitation, hypersensitivity to light, noise, touch or odors, cold
or heat intolerance, nausea or allergic-like symptoms such as
rhinitis, itching, or rash in the absence of a true allergy. One
skilled in the art understands that the methods of the invention
can be useful for preventing or reducing the severity of any of
these or other types of sensory hypersensitivity associated with
fibromyalgia.
[0042] The methods of the invention further can be useful for
preventing or reducing the severity of a stress-associated
behavioral disorder, which is any behavioral disorder which is
induced or exacerbated by stress. As non-limiting examples, a
stress-associated behavioral disorder can be a compulsive or
repetitive detrimental behavior which is induced or exacerbated by
stress such as, without limitation, over-eating or obesity,
obsessive compulsive disorder (OCD), tics, Tourette syndrome (TS),
alcohol use, drug use, gambling, self-inflicted injurious behavior
such as scratching or hair-pulling, or sexual impotency or arousal.
In one embodiment, the stress-associated behavioral disorder is a
disorder other than drug use. In another embodiment, the
stress-associated behavioral disorder is a disorder other than drug
or alcohol use.
[0043] The methods of the invention further can be useful for
preventing or reducing the severity of a stress-associated
psychiatric disorder, which is any psychiatric disorder which is
induced or exacerbated by stress. As a non-limiting example, the
methods of the invention can be used to prevent or reduce the
severity of a psychiatric disorder such as schizophrenia.
[0044] Also provided herein is a method of preventing or reducing
the severity of an ocular condition in a subject by systemically
administering to the subject an effective amount of brimonidine or
a pharmaceutically acceptable salt, ester, amide, sterioisomer or
racemic mixture thereof. As disclosed herein, brimonidine can act
as a neuroprotective agent, preventing, for example, retinal damage
in a number of ocular conditions affecting the neurosensory retina.
Ocular conditions which can be prevented or reduced in severity
using brimonidine according to a method of the invention include,
without limitation, diabetic retinopathy; macular edema such as
macular edema associated with diabetes mellitus or other
conditions; retinal degeneration such as age-related macular
degeneration or retinitis pigmentosa; inflammatory disorders of the
retina; vascular occlusive conditions of the retina such as retinal
vein occlusions or branch or central retinal artery occlusions;
retinopathy of prematurity; retinopathy associated with blood
disorders such as sickle cell anemia; damage following retinal
detachment; damage or insult due to vitrectomy surgery or retinal
surgery; and other retinal damage including therapeutic damage such
as that resulting from laser treatment of the retina, for example,
pan-retinal photocoagulation for diabetic retinopathy or
photodynamic therapy of the retina, for example, for age-related
macular degeneration as well as other ocular conditions such as
ocular itch. Ocular conditions that can be prevented or reduced in
severity according to a method of the invention further include,
without limitation, genetic and acquired optic neuropathies such as
optic neuropathies characterized primarily by loss of central
vision, for example, Leber's hereditary optic neuropathy (LHON),
autosomal dominant optic atrophy (Kjer disease) and other optic
neuropathies such as those involving mitochondrial defects,
aberrant dynamin-related proteins or inappropriate apoptosis. See,
for example, Carelli et al., Neurochem. Intl. 40:573-584 (2002);
and Olichon et al., J. Biol. Chem. 278:7743-7746 (2003).
[0045] The methods of the invention can be useful for preventing or
reducing the severity of a stress-associated condition without
concomitant sedation. Sedation, as used herein, is a term that
means a reduction in motor activity. The phrase "without
concomitant sedation," as used herein, means that relatively little
reduction in motor activity accompanies the reduction in severity
of one or more symptoms of a stress-associated condition at one or
more doses of drug. A drug generally acts "without concomitant
sedation" if, upon peripheral administration, the dose required to
produce a 20% reduction in motor activity is at least 3-fold
greater than the dose required to produce a significant reduction
in one or more symptoms of the stress-associated condition. As
shown in FIG. 6, brimonidine but not tizanidine or clonidine could
be administered at doses that produced a reduction in the
sensitization score (solid line, left axis) with less than a 20%
increase in sedation (broken line, right axis). As non-limiting
examples, the dose required to produce a 20% reduction in motor
activity can be at least 4-fold greater than, 5-fold greater than,
6-fold greater than, 7-fold greater than, 8-fold greater than,
9-fold greater than, 10-fold greater than, 25-fold greater than,
50-fold greater than, 100-fold greater than, 200-fold greater than,
500-fold greater than, 1000-fold greater than, 2000-fold greater
than, or 5000-fold greater than the dose required to produce a
significant reduction in one or more symptoms of a
stress-associated condition. Methods of determining the extent of a
reduction in severity of symptoms of a stress-associated condition
and the extent of sedation are well known in the art.
[0046] The term "brimonidine," as used herein, means a compound
having the formula
##STR00001##
or a pharmaceutically acceptable derivative thereof such as a salt,
ester, amide, sterioisomer, racemic mixture, polymorph, hydrate or
solvate. Such a pharmaceutically acceptable derivative can have
substantially the activity of
5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline D-tartrate (1:1) in
reducing tactile hypersensitivity without concomitant sedation in
sulprostone-treated mice. The term brimonidine encompasses, without
limitation, Alphagan.TM. and UK14304. Brimonidine, and
pharmaceutically acceptable salts, esters, amides, sterioisomers
and racemic mixtures thereof, is commercially available, for
example, as Alphagan.TM. (Allergan). In addition, brimonidine and
pharmaceutically acceptable salts, esters, amides, sterioisomers
and racemic mixtures thereof can be prepared by routine methods as
described below in Example I. See, also, U.S. Pat. No.
6,323,204.
[0047] Thus, it is understood that the methods of the invention
encompass the use of pharmaceutically acceptable salts, esters and
amides derived from the formula representing brimonidine. Suitable
pharmaceutically acceptable salts of brimonidine include, without
limitation, acid addition salts, which can be formed, for example,
by mixing a solution of brimonidine with a solution of an
appropriate acid such as hydrochloric acid, sulfuric acid, fumaric
acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric
acid, tartaric acid, carbonic acid or phosphoric acid.
Pharmaceutically acceptable salts further include, yet are not
limited to, acid phosphate, acetate, benzenesulfonate, benzoate,
bicarbonate, bisulfate, bitartrate, borate, bromide, calcium
edetate, camsylate, carbonate, chloride, clavulanate, citrate,
dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,
gluceptate, gluconate, glutamate, glycollylarsanilate,
hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,
hydroiodide, hydroxynaphthoate, iodide, isothionate, lactate,
lactobionate, laurate, malate, maleate, mandelate, mesylate,
methylbromide, methylnitrate, methylsulfate, mucate, napsylate,
nitrate, N-methylglucamine ammonium, oleate, oxalate, pamoate
(embonate), palmitate, pantothenate, phosphate/diphosphate,
polygalacturonate, saccharate, salicylate, stearate, sulfate,
subacetate, succinate, tannate, tartrate, teoclate, p-toluene
sulphonate salts, tosylate, triethiodide and valerate. In one
embodiment, a method of the invention is practiced with brimonidine
tartrate.
[0048] It further is understood that functional groups of
brimonidine can be modified, for example, to enhance the
pharmacological utility of the compound. Such modifications, which
are well within the knowledge of the skilled chemist and include,
without limitation, esters, amides, ethers, N-oxides, and pro-drugs
of brimonidine, are encompassed within the term "brimonidine" as
used herein. Examples of modifications that can enhance activity
include, for example, esterification such as the formation of
C.sub.1 to C.sub.6 alkyl esters, preferably C.sub.1 to C.sub.4
alkyl esters, wherein the alkyl group is a straight or branched
chain. Other acceptable esters include, for example, C.sub.5 to
C.sub.7 cycloalkyl esters and arylalkyl esters such as benzyl
esters. Such esters can be prepared from the compounds described
herein using conventional methods well known in the art of organic
chemistry.
[0049] Other pharmaceutically acceptable modifications include the
formation of amides. Useful amide modifications include, for
example, those derived from ammonia; primary C.sub.1 to C.sub.6
dialkyl amines, where the alkyl groups are straight or branched
chain; and arylamines having various substitutions. In the case of
secondary amines, the amine also can be in the form of a 5 or 6
membered ring. Methods for preparing these and other amides are
well known in the art.
[0050] It is further understood that chemically distinct
enantiomers and tautomers of brimonidine are encompassed within the
term "brimonidine" and can be useful in the methods of the
invention. Furthermore, in crystalline form, a compound may exist
as polymorphs; in the presence of a solvent, a compound may form a
solvate, for example, with water or a common organic solvent. Such
polymorphs, hydrates and other solvates of brimonidine also are
encompassed within the term "brimonidine" and can be useful in the
methods of the invention disclosed herein.
[0051] It is understood that pharmaceutical compositions containing
brimonidine can be useful in the methods of the invention. Such a
pharmaceutical composition includes brimonidine and optionally
includes an excipient such as a pharmaceutically acceptable carrier
or a diluent, which is any carrier or diluent that has
substantially no long term or permanent detrimental effect when
administered to a subject. An excipient generally is mixed with
active compound, or permitted to dilute or enclose the active
compound. A carrier can be a solid, semi-solid, or liquid agent
that acts as an excipient or vehicle for the active compound.
Examples of solid carriers include, without limitation,
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharin, polyalkylene glycols, talcum,
cellulose, glucose, sucrose and magnesium carbonate. Suppository
formulations can include, for example, propylene glycol as a
carrier. Examples of pharmaceutically acceptable carriers and
diluents include, without limitation, water, such as distilled or
deionized water; saline; aqueous dextrose, glycerol, ethanol and
the like. It is understood that the active ingredients can be
soluble or can be delivered as a suspension in the desired carrier
or diluent.
[0052] A pharmaceutical composition also can optionally include one
or more agents such as, without limitation, emulsifying agents,
wetting agents, sweetening or flavoring agents, tonicity adjusters,
preservatives, buffers or anti-oxidants. Tonicity adjustors useful
in a pharmaceutical composition include, but are not limited to,
salts such as sodium acetate, sodium chloride, potassium chloride,
mannitol or glycerin and other pharmaceutically acceptable tonicity
adjustors. Preservatives useful in pharmaceutical compositions
include, without limitation, benzalkonium chloride, chlorobutanol,
thimerosal, phenylmercuric acetate, and phenylmercuric nitrate.
Various buffers and means for adjusting pH can be used to prepare a
pharmaceutical composition, including, but not limited to, acetate
buffers, citrate buffers, phosphate buffers and borate buffers.
Similarly, anti-oxidants useful in pharmaceutical compositions are
well known in the art and include, for example, sodium
metabisulfite, sodium thiosulfate, acetylcysteine, butylated
hydroxyanisole and butylated hydroxytoluene. It is understood that
these and other substances known in the art of pharmacology can be
included in a pharmaceutical composition useful in the methods of
the invention. See, for example, Remington's Pharmaceutical
Sciences Mack Publishing Company, Easton, Pa. 16.sup.th Edition
1980. Furthermore, a composition containing brimonidine may be
administered in conjunction with one or more other therapeutic
substances, in the same or different pharmaceutical composition and
by the same or different routes of administration.
[0053] Brimonidine, or a pharmaceutically acceptable salt, ester,
amide, sterioisomer or racemic mixture thereof, is administered in
an effective amount. Such an effective amount generally is the
minimum dose necessary to achieve the desired prevention or
reduction in severity of one or more symptoms of a
stress-associated condition, for example, that amount roughly
necessary to reduce the discomfort caused by the stress-associated
condition to tolerable levels. Such a dose generally is in the
range of 0.1-1000 mg/day and can be, for example, in the range of
0.1-500 mg/day, 0.5-500 mg/day, 0.5-100 mg/day, 0.5-50 mg/day,
0.5-20 mg/day, 0.5-10 mg/day or 0.5-5 mg/day, with the actual
amount to be administered determined by a physician taking into
account the relevant circumstances including the severity and type
of stress-associated condition, the age and weight of the patient,
the patient's general physical condition, and the pharmaceutical
formulation and route of administration. Suppositories and extended
release formulations also can be useful in the methods of the
invention, including, for example, dermal patches, formulations for
deposit on or under the skin and formulations for intramuscular
injection.
[0054] A pharmaceutical composition useful in the methods of the
invention can be administered to a subject by a variety of means
depending, for example, on the type of condition to be treated, the
pharmaceutical formulation, and the history, risk factors and
symptoms of the subject. Routes of administration suitable for the
methods of the invention include both systemic and local
administration. As non-limiting examples, a pharmaceutical
composition useful for preventing or reducing the severity of a
stress-associated condition can be administered orally;
parenterally; by subcutaneous pump; by dermal patch; by
intravenous, intra-articular, subcutaneous or intramuscular
injection; by topical drops, creams, gels or ointments; as an
implanted or injected extended release formulation; by subcutaneous
minipump or other implanted device; by intrathecal pump or
injection; or by epidural injection. Depending on the mode of
administration, brimonidine can be incorporated in any
pharmaceutically acceptable dosage form such as, without
limitation, a tablet, pill, capsule, suppository, powder, liquid,
suspension, emulsion, aerosol or the like, and can optionally be
packaged in unit dosage form suitable for single administration of
precise dosages, or sustained release dosage forms for continuous
controlled administration.
[0055] A method of the invention can be practiced by peripheral
administration of brimonidine, or a pharmaceutically acceptable
salt, ester, amide, sterioisomer or racemic mixture thereof. As
used herein, the term "peripheral administration" or "administered
peripherally" means introducing brimonidine, or a pharmaceutically
acceptable salt, ester, amide, sterioisomer or racemic mixture
thereof, into a subject outside of the central nervous system.
Peripheral administration encompasses any route of administration
other than direct administration to the spine or brain.
[0056] Peripheral administration can be local or systemic. Local
administration results in significantly more of a pharmaceutical
composition being delivered to and about the site of local
administration than to regions distal to the site of
administration. Systemic administration results in delivery of a
pharmaceutical composition essentially throughout at least the
entire peripheral system of the subject.
[0057] Routes of peripheral administration useful in the methods of
the invention encompass, without limitation, oral administration,
topical administration, intravenous or other injection, and
implanted minipumps or other extended release devices or
formulations. A pharmaceutical composition useful in the invention
can be peripherally administered, for example, orally in any
acceptable form such as in a tablet, liquid, capsule, powder, or
the like; by intravenous, intraperitoneal, intramuscular,
subcutaneous or parenteral injection; by transdermal diffusion or
electrophoresis; topically in any acceptable form such as in drops,
creams, gels or ointments; and by minipump or other implanted
extended release device or formulation.
[0058] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE I
Preparation of Brimonidine
[0059] This example describes preparation of brimonidine
(5-bromo-6-(2-imidazolin-2-ylamino) quinoxaline).
Preparation 6-Amino-5-bromoquinoxaline hydrobromide
[0060] 6-Aminoquinoxaline (2.08 g, 14.4 mmol) was dissolved in 11.5
ml glacial acetic acid. The solution was cooled in water while a
solution of bromine (0.74 ml, 2.3 g, 14.4 mmol) in 1.5 ml glacial
acetic acid was added slowly over 15 minutes. After stirring for an
additional 30 minutes, the orange red solid formed was filtered off
and washed thoroughly with dry ether. The solid was dried in vacuo
overnight to yield 4.44 g crude product (a yield of 100%). The
compound, 6-amino-5-bromoquinoxaline hydrobromide, had no definite
melting point. A phase change from fine powder to red crystals was
observed at about 220.degree. C. Decomposition was observed at
about 245.degree. C. The material was used directly for preparation
of 6-amino-5-bromoquinoxaline as follows.
6-Amino-5-Bromoquinoxaline
[0061] Crude 6-amino-5-bromoquinoxaline from above was dissolved in
water, and saturated sodium bisulfite solution was added until the
resulting solution tested negative with starch-iodide paper. The
solution was then basified with 2N sodium hydroxide and extracted
thoroughly--with ethyl acetate. The organic extract was dried over
magnesium sulfate and concentrated under reduced pressure to give
the free base. The crude product was recrystallized from boiling
benzene to give yellow crystals, m.p. 155-6.degree. C. Using
various analytical procedures, the yellow crystals were determined
to be 6-amino-5-bromoquinoxaline. The yield was 82%.
6-Bromo-6-isothiocyanatoquinoxaline
[0062] The crude hydrobromide product described above (4.27 g, 14.0
mmol) was dissolved in 60 ml of water; thiophosgene (Aldrich, 1.28
ml, 16.8 mmol) was added in small portions with vigorous stirring.
After 2 hours, the red color of the solution was discharged. The
solid formed was filtered off and washed thoroughly with water.
After drying in vacuo at 25.degree. C., 3.38 g of brick red
crystals were obtained, m.p. 157-8.degree. C., representing a yield
of 90%. A portion of this material was further purified by column
chromatography to give white crystals, m.p. 157-8.degree. C. Using
various analytical procedures, these crystals were determined to be
5-bromo-6-isothiocyanatoquinoxaline.
5-Bromo-6(-N-(2-aminoethyl)thioureido)quinoxaline
[0063] A solution of the isothiocyanate (3.25 g, 12.2 mmol) in 145
ml benzene was added to a solution of ethylenediamine (Aldrich,
5.43 g, 90.0 mmol) in 18 ml benzene at 25.degree. C. over 2 hours.
After stirring for a further 30 minutes, the supernatant was poured
off. The oil which remained was washed by swirling with dry ether
three times and used directly for the next step.
[0064] A portion of this product was further purified by column
chromatography (SiO.sub.2, CHCl.sub.3) for characterization. A
white solid was recovered which decomposed at 175.degree. C. with
gas evolution (puffing). This white solid was determined to be
5-bromo-6(-N-2-(aminoethyl)thioureido) quinoxaline.
5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline
[0065] The crude product from above was dissolved in 100 ml dry
methanol and the brown solution was refluxed for 19 hours until
hydrogen sulfide gas was no longer evolved. The mixture was cooled
to room temperature and concentrated to about 50 ml. The yellow
solid was filtered off and dried in vacuo; the solid weighed 2.52 g
(a yield of 70%) and had a melting point of 242-4.degree. C.
[0066] As the crude product was insoluble in most common organic
solvents, initial purification was achieved by an acid-base
extraction procedure. Crude product (23 g) was dissolved in 100 ml
0.5N hydrochloric acid. The turbid yellow solution was filtered to
give a clear orange yellow solution which was extracted twice with
ethyl acetate (10 ml each extraction). The aqueous phase was cooled
to 0.degree. C. and basified with 6N sodium hydroxide, keeping the
temperature of the solution below 15.degree. C. at all times. The
yellow solid which precipitated was filtered off and washed
thoroughly with water until the washings were neutral to pH paper.
The solid was dried overnight in vacuo to give 1.97 g yellow solid,
m.p. 249-250.degree. C. The recovery was about 88%.
[0067] Further purification was achieved by recrystallization. The
partially purified product from above was dissolved in
N,N-dimethylformamide (about 17 ml/g) at 100.degree. C. with
vigorous stirring. The solution was filtered hot and set aside to
cool overnight. The bright yellow crystals were collected by
filtration, m.p. 252-253.degree. C. Recovery was from 65-77%. Using
various analytical procedures, the bright yellow solid was
determined to be 5-bromo-6-(2-imidazolin-2-ylamino)
quinoxaline.
EXAMPLE II
Mouse Models with Different Mechanisms of Sensory Sensitization
[0068] This example demonstrates that the increased sympathetic
tone of .alpha.-2A and .alpha.-2C knockout mice enhances induction
of tactile hypersensitivity by .alpha.-1 receptor activation.
A. Sulprostone-Induced Tactile Hypersensitivity is Driven by the
Sympathetic Nervous System While Phenylephrine-Induced Tactile
Hypersensitivity is Independent of Sympathetic Nervous System
Input
[0069] To dissect the contribution of the sympathetic nervous
system to sensory sensitization, mouse models having different
mechanisms of sensory sensitization were developed. Tactile
hypersensitivity was measured in mice following intrathecal or
intraperitoneal injection of an inducing agent by scoring the
response to light stroking of the mouse flank with a paintbrush. To
mimic increased sympathetic tone, phenylephrine, an .alpha.-1
adrenergic receptor agonist, was injected. As shown in FIGS. 1a and
1b, intrathecal (i.t.) or intraperitoneal (i.p.) dosing of
phenylephrine caused tactile hypersensitivity, with significant
responses observed starting at doses of 3 ng i.t. and 3 ng/kg i.p.
Induction of tactile hypersensitivity was .alpha.-1 receptor
dependent, as evidenced by the ability of the .alpha.-1 receptor
antagonist 5-methyl urapidil (5-MU) to block the hypersensitive
response when injected intraperitoneally.
[0070] The activity of a synthetic EP.sub.1/EP.sub.3
receptor-selective prostaglandin agonist, sulprostone, also was
assayed. As shown in FIG. 1c, increasing doses of intrathecal
sulprostone elicited dose-dependent tactile hypersensitivity; doses
of 100 and 200 ng caused a significant hypersensitive response.
Coadministration of a specific EP.sub.1 receptor antagonist
completely blocked the sulprostone-induced tactile
hypersensitivity, demonstrating that sulprostone mediates tactile
hypersensitivity through activation of the EP.sub.1 receptor.
[0071] In a third mouse model, chemical sensitization was induced
by injection of increasing intrathecal doses of NMDA, which may
activate NMDA channels on post-synaptic dorsal horn neurons (Woolf
et al., Science 288:1765-1769 (2000)). Intrathecal NMDA resulted in
a dose-dependent tactile hypersensitivity with a maximal effect at
a 100 ng dose. The hypersensitivity was blocked with the NMDA
antagonist, memantine, as shown in FIG. 1d.
[0072] To assess whether the three stimuli sensitize sensory
pathways by different mechanisms, a set of pharmacological agents
was assayed for the ability to prevent or ameliorate tactile
hypersensitivity. As shown in Table 1, each receptor antagonist
(5-MU, the EP.sub.1 receptor antagonist or memantine) blocked only
tactile hypersensitivity resulting from the corresponding receptor
agonist (phenylephrine, sulprostone or NMDA, respectively).
Gabapentin, which is used clinically to alleviate neuropathic pain
by reducing spinal sensitization, also was assayed for the ability
to block tactile hypersensitivity. Gabapentin inhibited tactile
hypersensitivity elicited by sulprostone and NMDA, but not by
phenylephrine, further demonstrating differences between the
sensory pathways involved by different stimuli.
TABLE-US-00001 TABLE 1 Receptor antagonists and clinically used
analgesics inhibit chemically-induced mechanical hypersensitivity
Vehi- EP.sub.1 Gaba- cle 5-MU antagonist Memantine pentin
Phenylephrine 14.3 .+-. 5.0 .+-. 9.8 .+-. 11.0 .+-. 13.0 (100 ng/kg
0.7** 1.0 0.7** 0.7** (.+-.0.6)** I.P.) Sulprostone 13.2 .+-. 12.0
.+-. 4.0 .+-. 14.3 .+-. 3.2 .+-. (200 ng IT) 0.8** 1.0** 1.2 0.8**
0.5 NMDA 14.2 .+-. 13.3 .+-. 11.4 .+-. 4.2 .+-. 3.7 .+-. (100 ng
IT) 1.0** 0.8** 1.53* 0.9 0.8 *indicates p < 0.01 **indicates p
< 0.001
[0073] .alpha.-2 knockout mice were provided by Dr. Brian Kobilka
(Stanford University; Link et al., Mol. Pharmacol. 48:48-55 (1995);
Altman et al., Mol. Pharmacol. 56:154-161 (1999)). The .alpha.-2
knockout mice have a C57BL/6 background and were bred from
homozygous knockout mice breeding pairs. Age and sex matched
C57BL/6 wildtype mice were used as controls.
[0074] Sulprostone (Cayman Chemical; Ann Arbor, Mich.) and NMDA
(Sigma; St Louis, Mo.) were dissolved in dimethyl sulfoxide (DMSO).
The EP.sub.1 receptor antagonist
##STR00002##
synthesized essentially as described in U.S. Pat. No. 5,843,942,
and gabapentin (Victor Medical; Irvine, Calif.) were dissolved in
50% DMSO, 50% saline. Memantine (1-amino-3,5-dimethyladamantane
hydrochloride), an analog of the well known anti-viral agent
amantadine (1-adamantanamine hydrochloride), was synthesized
essentially as described in U.S. Pat. No. 5,061,703 (see, also,
Schneider et al., Dtsch Med. Wochenschr. 109:987 (1984)).
5-methylurapidil, brimonidine, phenylephrine, clonidine and
guanethidine were obtained from Sigma and dissolved in saline.
Prazosin (Sigma) and tizanidine (Biomol; Plymouth Meeting, Pa.)
were dissolved in distilled water.
[0075] Spinal drug injections were performed as follows. Mice
(20-30 g) were injected intrathecally as described in Hylden and
Wilcox, Eur. J. Pharmacol. 67:313-316 (1980). Briefly, a sterile
30-gauge 1/2 inch needle attached to a microsyringe was inserted
between the L5 and L6 vertebrae. The mouse was held firmly by the
pelvic girdle in one hand, while the syringe was held in the other
hand at an angle of approximately 20.degree. above the vertebral
column. The needle was inserted into the tissue to one side of the
L6 spinous process, into the groove between the spinous and
transverse processes. The needle angle was decreased to about
10.degree., and the needle slowly advanced forward into the
intervertebral space until a pop was felt and there was a visible
serpentine tail movement. Compounds were slowly injected in the
subarachnoid space in a volume of 5 .mu.l. Each compound was tested
at multiple doses. The minimal efficacious dose was used for all
subsequent experiments.
[0076] Sensitivity to light touch was quantified by scoring the
response of mice to light stroking of their flanks with a small
paintbrush, which is not normally painful. The mice were rated on
the following scale once every 5 minutes between 15 and 50 minutes
post injection: a score of "2" was given to animals showing
aggressive escape responses along with squeaking and biting at the
brush; a score of "1" was given to animals exhibiting mild
squeaking with attempts to escape; and a score of "0" was given if
the animal showed no response to the light stroking of the
paintbrush. The scores were summed to generate a cumulative score
of 0 to 16 as described in Minami et al., Pain 57:217-223 (1994).
Statistical calculations of significance for in vivo studies were
done using a two-tailed Students t-test.
[0077] Guanethidine sympathectomies were performed essentially as
follows. Animals were injected intraperitoneally with 50 mg/kg
guanethidine (Malmberg and Basbaum, Pain 76:215-222 (1998)) before
being assessed for baseline tactile sensitivity 24 hours later.
Animals that exhibited normal tactile sensitivity were assayed for
sensitivity to chemical induction of tactile hypersensitivity. Mice
recovered from the sympathectomy six to eight days later as
demonstrated by a return to pre-sympathectomy responsiveness.
B. Increased Sympathetic Tone of .alpha.-2A and .alpha.-2C Knockout
Mice Enhances their Sensitivity to Induction of Tactile
Hypersensitivity by .alpha.-1 Receptor Activation
[0078] To assess whether sympathetic tone can influence
susceptibility to sensory sensitization, the sensitivity of
.alpha.-2A and .alpha.-2C knockout mice to chemical induction of
tactile hypersensitivity was compared to the sensitivity of
wildtype mice. The .alpha.-2A and .alpha.-2C knockout mice did not
exhibit baseline tactile hypersensitivity when compared to wildtype
controls. First, the concentration of phenylephrine that elicits
tactile hypersensitivity was compared in the knockout and wildtype
mice. As shown in FIG. 2, there was a dramatic leftward shift in
the phenylephrine dose response in both the .alpha.-2A and
.alpha.-2C knockout mice. These results demonstrate that the
ability of phenylephrine to cause tactile hypersensitivity was
enhanced in both .alpha.-2 knockout mouse lines, with a greater
enhancement in the .alpha.-2C knockout mice. In particular,
compared with a strongly tactile hypersensitivity-inducing dose of
30 ng/kg phenylephrine in the wildtype line, 0.1 and 0.3 ng/kg
phenylephrine resulted in maximal hypersensitivity in the
.alpha.-2C and .alpha.-2A knockout mice, respectively. As further
evidenced in FIG. 2, the gradual biphasic dose-response in the
wildtype mice became a steeper dose-response in both lines of
knockout mice.
[0079] Systemic administration of guanethidine results in a
functional sympathectomy by depleting noradrenaline from
sympathetic terminals. In order to test if shifts in the
phenylephrine dose response curves were due to increased
sympathetic tone in the .alpha.-2 knockout mice, .alpha.-2A
knockout mice were chemically sympathectomized by guanethidine
treatment (50 mg/kg i.p.) and assayed for phenylephrine-induced
sensitivity 24-30 hours later. In guanethidine-treated .alpha.-2A
mice, the increased sensitivity to phenylephrine was partly ablated
so that the dose response was similar to the biphasic dose response
observed in wildtype mice (see FIG. 2). These results confirm that
increased sympathetic tone enhances sensory sensitization in
.alpha.-2A knockout mice.
C. The Sympathetic Nervous System Enhances Sulprostone-Induced
Tactile Hypersensitivity
[0080] Sulprostone was injected intrathecally at increasing
concentrations into wildtype and .alpha.-2 knockout mice in order
to determine whether the knockout mice were more sensitive to
sensitization of primary afferents. As shown in FIG. 3, the dose
response of sulprostone was identical in the wildtype and
.alpha.-2C knockout mice, but was shifted to the left in the
.alpha.-2A knockout mice. In particular, a 30 ng dose was maximally
effective in the .alpha.-2A knockout mice compared to a partially
hypersensitivity-inducing dose of 100 ng and a maximal dose of 200
ng in the wild-type and .alpha.-2C knockout mice. A guanethidine
(50 mg/kg i.p.) chemical sympathectomy decreased the sensitivity of
the .alpha.-2A knockout mice to sulprostone. As shown in FIG. 3,
the dose response of sulprostone-induced tactile hypersensitivity
was shifted approximately 10-fold to the right in the .alpha.-2A
knockout mice treated with guanethidine. These results demonstrate
that the sympathetic nervous system enhances sulprostone
sensitization.
D. The Sympathetic Nervous System Does not Contribute to
NMDA-Induced Tactle Hypersensitivity
[0081] To assess whether .alpha.-2 knockout mice are more sensitive
to dorsal horn sensitization by NMDA, wildtype and .alpha.-2
knockout mice were injected with varying concentrations of NMDA. As
shown in FIG. 4, .alpha.-2A and .alpha.-2C knockout mice are not
more sensitive to NMDA than wildtype mice. These results indicate
that the sympathetic nervous system does not appear to contribute
to NMDA-induced tactile hypersensitivity.
[0082] In sum, these results demonstrate that .alpha.-2 knockout
mice exhibit elevated levels of sympathetic nerve activity and
further indicate that these mice exhibit enhanced sensitization
which is specific to the site and mode of stimulation.
EXAMPLE III
Comparison of Activity of .alpha.-2 Agonists Brimonidine and
Clonidine
[0083] This example demonstrates that .alpha.-adrenergic agonists
differ in their ability to alleviate sensory hypersensitivity that
is enhanced by the sympathetic nervous system.
A. Brimonidine, But not Clonidine, Alleviates
Sympathetically-Enhanced Tactile Hypersensitivity
[0084] Spinally administered .alpha.-2 adrenergic agonists
alleviate neuropathic pain through a spinal .alpha.-2A receptor. To
determine if the increased sympathetic activity in .alpha.-2
knockout mice alters the analgesic activity of the .alpha.-2
agonists, several agonists were assayed for activity. The .alpha.-2
agonists brimonidine and clonidine were first tested in the NMDA
model in which sensitization is not influenced by the basal
sympathetic tone of the knockout mice. Intrathecal
co-administration of NMDA with either clonidine or brimonidine
resulted in complete inhibition of tactile hypersensitivity in the
wildtype and .alpha.-2C (FIGS. 5a and c, respectively) knockout
mice. As expected, neither clonidine nor brimonidine inhibited
NMDA-induced tactile hypersensitivity in the .alpha.-2A knockout
mice (FIG. 5c), consistent with previous studies showing that a
spinal .alpha.-2A adrenergic receptor subtype mediates analgesic
actions of .alpha.-2 adrenergic agonists (Lakhlani et al., Proc.
Natl. Acad. Sci. USA 94:9950-9955 (1997); Stone et al., J.
Neurosci. 17:7157-1765 (1997); Hunter et al., Br. J. Pharmacol.
122:1339-1344 (1997)). The same pattern of analgesic activity of
brimonidine also was observed in the sulprostone-induced tactile
hypersensitivity model, which is sensitive to sympathetic tone (see
FIGS. 5b and d). In contrast, the results obtained with clonidine
were strikingly different: clonidine was analgesic in wildtype
mice, but not in .alpha.-2A or .alpha.-2C knockout mice (compare
FIGS. 5b and d). These results demonstrate that .alpha.-2
pan-agonists can have differential activity in
sympathetically-enhanced conditions, with brimonidine exhibiting
activity while clonidine is inactive.
B. Brimonidine, But Not Clonidine or Tizanidine, Alleviates
Sulprostone-Induced Hypersensitivity in the Absence of Sedation
[0085] Sedation limits the utility of many pharmaceuticals,
including .alpha.-2 agonists. The .alpha.-2 agonists were therefore
compared to test whether there was a difference in the dose that
resulted in alleviation of sensory hypersensitivity relative to the
dose that resulted in sedation.
[0086] For three .alpha.-2 agonists (tizanidine, clonidine and
brimonidine), sedative effects and the ability to block tactile
hypersensitivity were compared at various doses in models of
locomoter activity and sulprostone-induced tactile
hypersensitivity, respectively. The tactile hypersensitivity of 5-6
mice per group was scored every five minutes between 15 and 50
minutes following intraperitoneal dosing. Vehicle treated animals
typically had a score of about 4. In addition, the locomoter
activity of 5-6 mice per group was measured in a five minute period
30 minutes following intraperitoneal dosing. The locomoter activity
relative to vehicle-treated animals was expressed as a percentage;
percentage sedation was calculated as 100% minus the percent
locomoter activity. As shown in FIG. 6, of the three
.alpha.-adrenergic agonists assayed, only brimonidine produced an
analgesic effect that was separable from sedation. These results
demonstrate that brimonidine is distinct from other .alpha.-2
pan-agonists such as clonidine and tizanidine in the ability to
alleviate sympathetically-enhanced disorders such as
sulprostone-induced tactile hypersensitivity without concomitant
sedation.
C. Variable .alpha.-2 Versus .alpha.-1 Functional Selectivity of
.alpha.-Adrenergic Pan-Agonists
[0087] The .alpha.-adrenergic receptor pharmacological profiles of
brimonidine and clonidine were analyzed in assays using cell lines
stably expressing .alpha.-2A, .alpha.-2C, .alpha.-1A and .alpha.-1B
receptors.
[0088] Consistent with previous studies, the order of potency for
inhibiting forskolin-induced cAMP accumulation in PC12 cells stably
expressing either .alpha.-2A or .alpha.-2C receptor (FIGS. 7a, b;
Table 2) was dexmedetomidine, which was greater than or equal to
brimonidine, which was greater than clonidine, which was greater
than tizanidine, which was greater than or equal to phenylephrine
(Jasper et al., Biochem. Pharmacol. 55:1035-1043 (1998); Pihlavisto
et al., Eur. J. Pharmacol. 385:247-253 (1999)). Brimonidine,
clonidine and tizanidine were approximately 10-fold more potent at
the .alpha.-2A receptor than the .alpha.-2C receptor.
[0089] The same compounds were functionally tested for the ability
to stimulate .alpha.-1-mediated increases in intracellular calcium
in HEK293 cells stably expressing the .alpha.-1A and .alpha.-1B
receptor (FIGS. 7c, d; Table 2). The order of potency at the
.alpha.-1A and .alpha.-1B receptors was phenylephrine, which was
greater than clonidine, which was greater than tizanidine, which
was equal to dexmedetomidine, which was greater than brimonidine.
The .alpha.-2 agonists, clonidine, tizanidine and dexmedetomidine,
were partial agonists while brimonidine exhibited weak activity at
the .alpha.-1A receptor and no activity at the .alpha.-1B receptor.
Thus, although clonidine and tizanidine have previously been
characterized as ".alpha.-2 selective" agonists in binding assays,
these compounds display a less than 10-fold selectivity between
.alpha.-2 and .alpha.-1 receptor activation in functional assays.
In contrast, dexmedetomidine was approximately 300-fold selective
in functional assays, and brimonidine, the most highly selective
compound in functional assays, exhibited greater than 1000-fold
selectivity for .alpha.-2 receptors relative to .alpha.-1 receptors
(see Table 2). These results demonstrate that brimonidine is a
highly selective .alpha.-2 versus .alpha.-1 agonist and that the
differential .alpha.-2/.alpha.-1 selectivity of brimonidine
contrasts with the selectivity of other pan-agonists such as
clonidine.
[0090] The difference in .alpha.-2/.alpha.-1 selectivity between
clonidine and brimonidine indicates that the .alpha.-1 agonist
activity of clonidine can augment the increased sympathetic tone of
the .alpha.-2C knockout mice and mask the analgesic activity of
clonidine in the sulprostone model. These results are supported by
the ability of co-administration of the .alpha.-1 antagonist
prazosin with clonidine to restore the analgesic activity of
clonidine in .alpha.-2C knockout mice (FIG. 7e). Prazosin had no
analgesic activity by itself in wildtype or .alpha.-2C knockout
mice.
[0091] In sum, these results indicate that the loss of clonidine,
but not brimonidine, analgesic activity in the .alpha.-2C knockout
mice can be a result of clonidine's .alpha.-1 agonist activity and
that the .alpha.-1 agonist activity of many ".alpha.-2 agonists"
can limit their ability to treat stress-associated and other
sympathetically-enhanced disorders.
[0092] Stable cell lines expressing an adrenergic receptor were
established as follows. The bovine .alpha.-1A, hamster .alpha.-1B,
human .alpha.-2A and human .alpha.-2C receptor cDNAs were blunt-end
subcloned into the NheI-EcoRI sites in the retroviral vector pCL
BABE Puro. The retroviral constructs were verified by double
stranded DNA sequencing. High titer pseudotyped retroviral
particles were produced by co-transfecting HEK293GP, a HEK293 cell
line stably expressing Gag-Pol of the Maloney leukemia virus, with
the appropriate retroviral vector and pMD.G, an expression vector
for the vesicular stomatitis virus envelope protein, VSV-G. Sixteen
hours after transfection, the media (DMEM, 10% FCS) was changed;
the high titer (.about.1.times.10.sup.6 pfu/mL) media was then
harvested forty-eight hours later. The supernatant was filtered
through a 0.4 uM filter.
[0093] The human .alpha.-2A and .alpha.-2C receptor supernatants
were added, in varying amounts, to naive PC12 cells, which were
then incubated for 48 hours. The transduced cell populations were
replated at a lower density and grown in media containing 100 ug/ml
puromycin. Non-transduced cells were killed within three days, and
single foci grew within two months. The foci were picked, expanded,
and assayed for receptor density by brimonidine radioligand
binding. Functional .alpha.-2 receptor activity was confirmed by
inhibition of forskolin-induced cAMP accumulation.
[0094] The bovine .alpha.-1A and hamster .alpha.-1B receptor
supernatants were added, in varying amounts, to naive HEK293 cells,
which were then incubated for 48 hours. The transduced cell
populations were replated at a lower density and grown in media
containing 0.25 ug/ml puromycin. Significant cell death was evident
within three days, with single foci appearing within two weeks.
After the foci were picked and expanded, expanded subclones were
functionally assayed for .alpha.-1 receptor expression by measuring
phenylephrine-induced intracellular Ca.sup.+2 accumulation.
Receptor density was measured in a prazosin radioligand binding
assay.
[0095] Intracellular Ca.sup.+2 responses were measured as follows
in HEK293 cells stably expressing either the bovine .alpha.-1A or
hamster .alpha.-1B adrenergic receptor. Between 40,000 to 50,000
cells were plated per well in 96-well poly-D-lysine coated plates
in 0.2 ml DMEM containing 10% heat-inactivated fetal calf serum, 1%
antibiotic-antimycotic and 0.25 .mu.g/ml puromycin one day prior to
use. Cells were washed twice with HBSS supplemented with 10 mM
HEPES, 2.0 mM CaCl.sub.2 and 2.5 mM probenicid, and subsequently
incubated at 37.degree. C. for 60 minutes with 4 .mu.M Fluo-4
(Molecular Probes; Eugene, Oreg.). The extracellular dye was washed
from the plates twice prior to placing the plates in the
fluorometric imaging plate reader (FLIPR; Molecular Devices;
Sunnyvale, Calif.). Ligands were diluted in HBSS and aliquoted into
a 96-well microplate. Drugs were tested over the concentration
range of 0.64 nM to 10,000 nM. Data for Ca.sup.+2 responses were
obtained in arbitrary fluorescence units.
TABLE-US-00002 TABLE 2 Functional .alpha.-2 versus .alpha.-1
selectivity of .alpha.-adrenergic agonists human .alpha..sub.2A
human .alpha..sub.2C bovine .alpha..sub.1A hamster .alpha..sub.1B
Compound EC.sub.50 % E EC.sub.50 % E EC.sub.50 % E EC.sub.50 % E
.alpha..sub.1A/.alpha..sub.2A Brimonidine 0.86 .+-. 0.1 91 8 .+-. 3
93 1132 .+-. 281 15 943 .+-. 247 12 1316 Dexmedetomidine 0.8 .+-.
.01 93 0.48 .+-. .2 90 376 .+-. 97 59 364 .+-. 72 62 289 Clonidine
10 .+-. 1 94 56 .+-. 28 84 89 .+-. 16 62 83 .+-. 10 63 8.9
Tizanidine 86 .+-. 35 93 1231 .+-. 376 85 264 .+-. 37 63 322 .+-.
31 61 3.1 Phenylephrine 306 .+-. 19 94 340 .+-. 131 87 9 .+-. 1 110
10 .+-. 1 110 .03 The percent efficacy (% E) was determined by
comparing the maximum effect of each agonist to the maximum effect
of a standard full agonist (phenylephrine for .alpha.-1 receptors
and brimonidine for .alpha.-2 receptors). The values represent the
mean and SEM from 3-15 independent experiments. The
fold-selectivity of the agonists for .alpha.-2 receptors relative
to .alpha.-1 receptors was calculated from the ratio of their mean
EC.sub.50s for activating the .alpha.-1A and .alpha.-2A
receptors.
[0096] Intracellular cAMP measurement was performed as follows.
PC12 cells stably expressing the human .alpha.-2A or human
.alpha.-2C adrenergic receptors were plated in 96-well
poly-D-lysine coated plates at a density of 30,000 cells per well
in 100 .mu.l DMEM supplemented with 10% horse serum, 5% heat
inactivated fetal bovine serum, 1% antibiotic-antimycotic and 100
.mu.g/ml puromycin. Cells were grown overnight at 37.degree. C. and
5% C0.sub.2. Cells were dosed by adding an equal volume of media
containing IBMX (to a final concentration of 1 mM), forskolin (to a
final concentration of 10 .mu.M) and the appropriate drug dilution
(to a final concentration of between 10.sup.-5 M and 10.sup.-12 M).
After a 10 minute incubation, the media was aspirated and the cells
lysed with 200 .mu.l lysis buffer (Amersham Biosciences;
Piscataway, N.J.). Plates were stored at -20.degree. C. for up to
24 hours prior to assay. Intracellular cAMP was determined using
the Biotrak cAMP enzyme immunoassay system (Amersham Biosciences)
according to the manufacturer's instructions. Plates were read on a
plate reader at 450 nm.
[0097] Dose response curves for in vitro assays were generated
using KaleidaGraph (Synergy Software; Reading, Pa.) by least
squares fits to the equation, response=maximum response+((minimum
response-maximum response)/(1+(concentration of ligand/EC.sub.50)).
The percent efficacy was determined by comparing the maximum effect
of the compound to the effect of a standard full agonist, which was
phenylephrine for .alpha.-1 receptors and brimonidine for .alpha.-2
receptors.
[0098] All journal article, reference and patent citations provided
above, in parentheses or otherwise, whether previously stated or
not, are incorporated herein by reference in their entirety.
[0099] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
claims.
* * * * *