U.S. patent application number 16/460226 was filed with the patent office on 2020-05-28 for use of gabaa receptor antagonists for the treatment of excessive sleepiness and disorders associated with excessive sleepiness.
This patent application is currently assigned to EMORY UNIVERSITY. The applicant listed for this patent is EMORY UNIVERSITY. Invention is credited to Andrew Jenkins, Kathy P. Parker, David B. Rye.
Application Number | 20200163974 16/460226 |
Document ID | / |
Family ID | 41065847 |
Filed Date | 2020-05-28 |
United States Patent
Application |
20200163974 |
Kind Code |
A1 |
Parker; Kathy P. ; et
al. |
May 28, 2020 |
USE OF GABAA RECEPTOR ANTAGONISTS FOR THE TREATMENT OF EXCESSIVE
SLEEPINESS AND DISORDERS ASSOCIATED WITH EXCESSIVE SLEEPINESS
Abstract
GABA.sub.A receptor mediated hypersomnia can be treated by
administering a GABA.sub.A receptor antagonist (e.g., flumazenil;
clarithromycin; picrotoxin; bicuculline; cicutoxin; and
oenanthotoxin). In some embodiments, the GABA.sub.A receptor
antagonist is flumazenil or clarithromycin. The GABA.sub.A receptor
mediated hypersomnia includes shift work sleep disorder,
obstructive sleep apnea/hypopnea syndrome, narcolepsy, excessive
sleepiness, hypersomnia (e.g., idiopathic hypersomnia; recurrent
hypersomnia; endozepine related recurrent stupor; and amphetamine
resistant hypersomnia), and excessive sleepiness associated with
shift work sleep disorder, obstructive sleep apnea/hypopnea
syndrome, and hypersomnia (e.g., idiopathic hypersomnia; recurrent
hypersomnia; endozepine related recurrent stupor; and amphetamine
resistant hypersomnia.
Inventors: |
Parker; Kathy P.;
(Rochester, NY) ; Rye; David B.; (Dunwoody,
GA) ; Jenkins; Andrew; (Decatur, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EMORY UNIVERSITY |
ATLANTA |
GA |
US |
|
|
Assignee: |
EMORY UNIVERSITY
ATLANTA
GA
|
Family ID: |
41065847 |
Appl. No.: |
16/460226 |
Filed: |
July 2, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15445131 |
Feb 28, 2017 |
10376524 |
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16460226 |
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12922044 |
Sep 10, 2010 |
9616070 |
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PCT/US2009/037034 |
Mar 12, 2009 |
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15445131 |
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61036047 |
Mar 12, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/006 20130101;
A61K 9/0019 20130101; A61K 31/70 20130101; A61K 31/5517 20130101;
A61P 25/00 20180101; A61K 9/0014 20130101; A61K 9/06 20130101; A61K
9/08 20130101; A61K 9/20 20130101 |
International
Class: |
A61K 31/5517 20060101
A61K031/5517; A61K 9/20 20060101 A61K009/20; A61K 9/08 20060101
A61K009/08; A61K 9/06 20060101 A61K009/06; A61K 9/00 20060101
A61K009/00; A61K 31/70 20060101 A61K031/70 |
Claims
1.-61. (canceled)
62. A method for treating excessive sleepiness in a subject with a
disorder associated with excessive sleepiness, the method
comprising administering an effective amount of a GABA.sub.A
receptor antagonist to the subject with the disorder associated
with excessive sleepiness.
63. The method of claim 62, wherein the disorder is selected from
the group consisting of narcolepsy, obstructive sleep
apnea/hypopnea syndrome, shift work sleep disorder, and
hypersomnia.
64. The method of claim 63, wherein the disorder is narcolepsy.
65. The method of claim 62, wherein the GABA.sub.A receptor
antagonist is selected from the group consisting of flumazenil,
clarithromycin, picrotoxin, bicuculline, cicutoxin, and
oenanthotoxin.
66. The method of claim 66, wherein the GABA.sub.A receptor
antagonist is flumazenil or clarithromycin.
67. The method of claim 66, wherein the GABA.sub.A receptor
antagonist is clarithromycin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn. 119
to U.S. Provisional Application Ser. No. 61/036,047, filed Mar. 12,
2008, incorporated by reference in its entirety herein.
TECHNICAL FIELD
[0002] The present disclosure relates to the treatment of excessive
sleepiness and promotion of wakefulness in a subject. In
particular, a method of treating hypersomnia (e.g., GABA.sub.A
receptor mediated hypersomnia) using a GABA.sub.A receptor
anagonist such as flumazenil (formulated, for example, for I.V.,
transdermal, transmucosal, sublingual, or subdermal administration)
is disclosed.
BACKGROUND
[0003] There are two main categories of hypersomnia: primary
hypersomnia (sometimes called idiopathic hypersomnia) and recurrent
hypersomnia (sometimes called idiopathic recurrent hypersomnia).
Both are characterized by similar signs and symptoms and differ
only in the frequency and regularity with which the symptoms
occur.
[0004] Primary hypersomnia is characterized by excessive daytime
sleepiness over a long period of time. The symptoms are present
all, or nearly all, of the time. Recurring hypersomnia involves
periods of excessive daytime sleepiness that can last from one to
many days, and recur over the course of a year or more. The primary
difference between this and primary hypersomnia is that persons
experiencing recurring hypersomnia will have prolonged periods
where they do not exhibit any signs of hypersomnia, whereas persons
experiencing primary hypersomnia are affected by it nearly all the
time. Idiopathic hypersomnia is much like narcolepsy, except there
is no cataplexy, no sleep paralysis, and no rapid eye movement when
the victim first falls asleep.
[0005] Various treatments including prescription drugs have been
used to treat hypersomnia without significant success, and no
substantial body of evidence supports the effectiveness of any of
these treatments. Stimulants are not generally recommended to treat
hypersomnia as they treat the symptoms but not the base problem.
There is a need for more effective treatments of hypersomnia,
especially using administration routes that allow for better drug
delivery and patient compliance.
SUMMARY
[0006] The inventors have discovered that many patients that suffer
from excessive sleepiness or disorders associated with excessive
sleepiness have one or more endogenous substances present,
typically in excess, in their CSF that act as positive allosteric
modulators of the GABA.sub.A receptor, potentiating the effect of
GABA on the receptor. Treatment of such patients with a GABA.sub.A
receptor antagonist thus can provide a method to treat the
disorders, in particular the symptoms of excessive sleepiness
associated with the disorders.
[0007] Accordingly, provided herein are methods of treating
GABA.sub.A receptor mediated hypersomnia in a subject, the methods
comprising administering to the subject an effective amount of a
GABA.sub.A receptor antagonist. In addition, provided herein is a
method of treating excessive sleepiness associated with GABA.sub.A
receptor mediated hypersomnia in a subject, comprising
administering to the subject an effective amount of a GABA.sub.A
receptor antagonist. In some embodiments, the GABA.sub.A receptor
mediated hypersomnia is selected from one or more of: shift work
sleep disorder; narcolepsy; obstructive sleep apnea/hypopnea
syndrome; REM behavior disorder; frontal nocturnal dystonia;
restless legs syndrome; nocturnal movement disorder; Kleine-Levin
syndrome; Parkinson's disease; excessive sleepiness; hypersomnia;
idiopathic hypersomnia; recurrent hypersomnia; endozepine related
recurrent stupor; and amphetamine resistant hypersomnia. In some
embodiments, the GABA.sub.A receptor mediated hypersomnia is a
result of the production of endogenous somnogenic compounds in a
subject, e.g. excessive amounts of somnogenic compounds. In some
embodiments, the GABA.sub.A receptor antagonist can be a negative
allosteric modulator. In some embodiments, the GABA.sub.A receptor
antagonist is selected from the group consisting of: flumazenil;
clarithromycin; picrotoxin; bicuculline; cicutoxin; and
oenanthotoxin. In some embodiments, the method includes
administering an I.V., transdermal, transmucosal, sublingual, or
subdermal formulation of the GABA.sub.A receptor antagonist to the
subject.
[0008] Also provided herein is a method of treating excessive
sleepiness in a subject. The method comprises the steps of
determining whether the subject has an endogenously produced
somnogenic compound in a CSF sample of the subject, e.g., an
excessive amount of the somnogenic compound; and administering to
the subject an effective amount of a GABA.sub.A receptor
antagonist, e.g., flumazenil. The step of determining whether the
subject has an endogenously produced somnogenic compound, including
an excessive amount of the somnogenic compound, includes the steps
of: a) measuring the potentiation of GABA.sub.A receptors contacted
with the CSF sample of the subject in a whole cell patch clamp
assay, wherein the cells express benzodiazepine sensitive
receptors; b) measuring the potentiation of GABA.sub.A receptors
contacted with the CSF sample of the subject in a whole cell patch
clamp assay, wherein the cells express benzodiazepine insensitive
receptors; and c) comparing the response of step a) to the response
of step b), wherein a persistence of potentiation in step b) to
within .+-.25% of the step a) response is indicative of an
endogenously produced somnogenic compound in the CSF sample of the
subject. In some embodiments, the somnogenic compound is a
non-classical benzodiazepine. In some embodiments, the somnogenic
compound binds to a site on the GABA.sub.A receptor, e.g., an
allosteric site. In some embodiments, the site on the GABA.sub.A
receptor is other than the benzodiazepine binding site.
[0009] A method of treating excessive sleepiness of a subject
endogenously producing a somnogenic compound, e.g., an excessive
amount of a somnogenic compound, is also provided herein, the
method comprising administering to the subject an effective amount
of a GABA.sub.A receptor antagonist, e.g., flumazenil. Further
described herein is a method of determining whether a subject will
benefit from treatment with a GABA.sub.A receptor antagonist, e.g.,
flumazenil, wherein the benefit is a reduction in excessive
sleepiness, the method comprising determining whether the subject
has an endogenously produced somnogenic compound, e.g., an excess
of the somnogenic compound, in a CSF sample of the subject, wherein
the presence of the endogenously produced somnogenic compound is
indicative that the subject will benefit from treatment with
GABA.sub.A receptor antagonist, e.g., flumazenil. In the method,
the determining step comprises: a) measuring the potentiation of
GABA.sub.A receptors contacted with the CSF sample of the subject
in a whole cell patch clamp assay, wherein the cells express
benzodiazepine sensitive receptors; b) measuring the potentiation
of GABA.sub.A receptors contacted with the CSF sample of the
subject in a whole cell patch clamp assay, wherein the cells
express benzodiazepine insensitive receptors; and c) comparing the
response of step a) to the response of step b), wherein a
persistence of potentiation to within .+-.25% of the step a)
response is indicative that the subject will benefit from treatment
with flumazenil.
[0010] In some embodiments of the methods described herein, the
GABA.sub.A receptor antagonist is a negative allosteric modulator.
In some embodiments, the GABA.sub.A receptor antagonist is selected
from the group consisting of: flumazenil; clarithromycin;
picrotoxin; bicuculline; cicutoxin; and oenanthotoxin. In some
embodiments, the GABA.sub.A receptor antagonist is flumazenil. In
some embodiments, the GABA.sub.A receptor antagonist is
clarithromycin.
[0011] Further provided herein are methods of treating disorders
associated with excessive sleepiness (e.g., GABA.sub.A receptor
mediated hypersomnia) and symptoms of excessive sleepiness in a
subject. In some embodiments, the method includes administering an
I.V., transdermal, transmucosal, sublingual, or subdermal
formulation of a GABA.sub.A receptor antagonist, e.g., selected
from flumazenil; clarithromycin; picrotoxin; bicuculline;
cicutoxin; and oenanthotoxin to the subject.
[0012] A disorder associated with excessive sleepiness can be
selected from one or more of: shift work sleep disorder;
narcolepsy; obstructive sleep apnea/hypopnea syndrome; hypersomnia;
REM behavior disorder; frontal nocturnal dystonia; restless legs
syndrome; nocturnal movement disorder; Kleine-Levin syndrome; and
Parkinson's disease. In some embodiments, the disorder is
hypersomnia, for example GABA.sub.A receptor mediated hypersomnia
(e.g., idiopathic hypersomnia; recurrent hypersomnia; endozepine
related recurrent stupor; and amphetamine resistant
hypersomnia).
[0013] A method of treating a disorder associated with excessive
sleepiness in a subject is provided, the method comprising
administering to the subject an effective amount of a transmucosal,
transdermal, or I.V. formulation of a GABA.sub.A receptor
antagonist, e.g., flumazenil. In some embodiments, treating a
disorder associated with excessive sleepiness can include
administering an effective amount of a GABA.sub.A receptor
antagonist, e.g., flumazenil, using a subdermal pump.
[0014] In some embodiments, a transmucosal formulation of a
GABA.sub.A receptor antagonist, e.g., flumazenil, is administered.
The transdermal formulation can be administered supralingually,
sublingually, or buccally.
[0015] In some embodiments, the subject is administered about 2 mg
flumazenil per Body Mass Index unit of the subject over a 24 hour
period. Administration may be self-administered by the patient as
needed, or in the case of an I.V. or subdermal route of
administration, the flumazenil can be administered automatically.
In some embodiments, the effective amount of flumazenil is about 6
mg per dose six times per day.
[0016] Independent of the formulation and route of administration,
any of the methods may further comprise administering a wakefulness
promoting agent (e.g., modafinil and armodafinil). In some
embodiments, the wakefulness promoting agent is modafinil. In some
embodiments, the method comprises administering a time-release
formulation of a GABA.sub.A receptor antagonist, such as a
time-release transdermal formulation.
[0017] Further provided herein is a method of treating a GABA.sub.A
receptor mediated hypersomnia in a subject, the method comprising:
a) administering to the subject a sublingual formulation of a
GABA.sub.A receptor antagonist, e.g., flumazenil; and b)
administering to the subject a wakefulness promoting agent. In some
embodiments, the method comprises: a) administering flumazenil in
an amount of about 2 mg of flumazenil per Body Mass Index unit of
the subject per 24 hour period; and b) administering to the subject
a wakefulness promoting agent. Also provided is a method of
treating a GABA.sub.A receptor mediated hypersomnia in a subject,
the method comprising: a) administering to the subject a GABA.sub.A
receptor antagonist, e.g., flumazenil, using a subdermal pump; and
b) administering to the subject a wakefulness promoting agent. In
some embodiments, a method of treating a GABA.sub.A receptor
mediated hypersomnia in a subject is provided, the method
comprising: a) administering to the subject an I.V. formulation of
flumazenil in an amount of about 0.2 mg to about 2 mg; and b)
administering to the subject a wakefulness promoting agent. In some
embodiments, the methods described above further comprise
administration of a transdermal formulation of a GABA.sub.A
receptor antagonist, e.g., flumazenil.
[0018] A method of treating a disorder associated with excessive
sleepiness in a subject is provided, the method comprising
administering a GABA.sub.A receptor antagonist, e.g., flumazenil,
in an amount effective to decrease the subject's CSF-induced
enhancement of whole cell patch clamp assayed GABA.sub.AR responses
in the presence of GABA such that the responses in the presence of
GABA are within .+-.25% of a control sample. In some embodiments, a
method of treating a disorder associated with excessive sleepiness
in a subject is provided, the method comprising administering a
GABA.sub.A receptor antagonist, e.g., flumazenil, in an amount
effective to modulate the response of a CSF sample of the subject
as measured in a GABA whole cell patch clamp assay to within
.+-.25% of the response of a control sample. In some embodiments,
the modulation is a decrease in the response of the CSF sample of
the subject in the presence of a GABA.sub.A receptor antagonist,
e.g., flumazenil.
[0019] A method of testing a subject for the presence of a positive
allosteric modulator of GABA.sub.A receptor function in a CSF or
blood sample is also provided, the method comprising measuring the
response of GABA.sub.A Receptors contacted with the CSF or blood
and with GABA in a whole cell patch clamp assay, and comparing the
response to a control sample, wherein a greater than 50% increase
in the response relative to the control is indicative of the
presence of a positive allosteric modulator of GABA.sub.A receptor
function.
[0020] Also provided herein are methods of treating shift work
sleep disorder, obstructive sleep apnea/hypopnea syndrome, and
narcolepsy in a subject, the methods comprising administering to
the subject an effective amount of a GABA.sub.A receptor
antagonist, e.g., flumazenil. A method of treating excessive
sleepiness associated with shift work sleep disorder, obstructive
sleep apnea/hypopnea syndrome, hypersomnia (e.g., idiopathic
hypersomnia; recurrent hypersomnia; endozepine related recurrent
stupor; and amphetamine resistant hypersomnia), or narcolepsy in a
subject is also provided, the method comprising administering to
the subject an effective amount of a GABA.sub.A receptor
antagonist, e.g., flumazenil. In some embodiments, the a GABA.sub.A
receptor antagonist is an I.V. formulation, a transdermal
formulation, or a transmucosal formulation.
[0021] A method of altering a somnolent state of a subject is
further provided herein, the method comprising administering to the
subject an effective amount of a GABA.sub.A receptor antagonist,
e.g., flumazenil. The somnolent state is selected from one or more
of: narcolepsy, obstructive sleep apnea/hypopnea syndrome, shift
work sleep disorder, and hypersomnia (e.g., idiopathic hypersomnia;
recurrent hypersomnia; endozepine related recurrent stupor; and
amphetamine resistant hypersomnia). In some embodiments, the a
GABA.sub.A receptor antagonist is an I.V. formulation, a
transdermal formulation, or a transmucosal formulation.
[0022] Also provided herein are methods for enhancing alertness or
increasing regularity of sleep rhythms in a subject; promoting
wakefulness in a subject; improving cognitive dysfunction in a
subject; and restoring a normal sleep pattern and improving the
quality of psychosocial life and relationships in a subject, each
method comprising administering to the subject an effective amount
of a GABA.sub.A receptor antagonist, e.g., flumazenil. In some
embodiments, the a GABA.sub.A receptor antagonist is an I.V.
formulation, a transdermal formulation, or a transmucosal
formulation.
[0023] A method of characterizing the phenotypic spectrum of
GABA.sub.A receptor mediated hypersomnia is also provided, the
method comprising measuring the potentiation of GABA.sub.A receptor
function of a CSF or plasma sample of at least one subject having a
disorder associated with excessive sleepiness, and correlating the
potentiation with at least one measure of sleep or sleepiness of
the subject, wherein a positive correlation is indicative that the
subject's disorder is within the phenotypic spectrum of a
GABA.sub.A receptor mediated hypersomnia. In some embodiments, the
measure of sleep and sleepiness is a behavioral assessment, an
electroencephalographic assessment, or a subjective assessment. The
method can further comprise quantifying GABA.sub.A receptor
function.
[0024] Further provided herein are uses of a GABA.sub.A receptor
antagonist such as flumazenil for the manufacture of medicaments
for the treatment of the following disorders and conditions:
obstructive sleep apnea/hypopnea syndrome; shift work sleep
disorder; narcolepsy; hypersomnia; and excessive sleepiness
associated with shift work sleep disorder, obstructive sleep
apnea/hypopnea syndrome, hypersomnia, or narcolepsy. In some
embodiments, the hypersomnia is selected from one or more of:
idiopathic hypersomnia; recurrent hypersomnia; endozepine related
recurrent stupor; and amphetamine resistant hypersomnia.
[0025] Also provided herein are uses of a GABA.sub.A receptor
antagonist such as flumazenil for the manufacture of medicaments
for altering a somnolent state of a subject; enhancing alertness or
increasing regularity of sleep rhythms in a subject; promoting
wakefulness in a subject; improving cognitive dysfunction in a
subject; and restoring a normal sleep pattern and improving the
quality of psychosocial life and relationships in a subject. In
some embodiments, the somnolent state is selected from one or more
of: narcolepsy; obstructive sleep apnea/hypopnea syndrome; shift
work sleep disorder; and hypersomnia. In some embodiments, the
hypersomnia is selected from one or more of: idiopathic
hypersomnia; recurrent hypersomnia; endozepine related recurrent
stupor; and amphetamine resistant hypersomnia. In some embodiments,
a GABA.sub.A receptor antagonist such as flumazenil is formulated
for administration by a transdermal, transmucosal, or intravenous
route for the uses described herein.
[0026] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0027] FIG. 1 illustrates whole cell patch clamp recordings of
GABA.sub.AR function with and without flumazenil.
[0028] FIG. 2 shows that human .alpha.1.beta.2.gamma.2s GABA.sub.A
receptor function is enhanced by the plasma of a subject suffering
from hypersomnia. Enhancement is reduced following administration
of 12 mg of a sublingual formulation of flumazenil.
[0029] FIG. 3 is a graph illustrating that potentiation of
GABA.sub.A function is evident in human controls absent sleep
related complaints, non-human (rhesus) primates, and in excess in
many hypersomnic patients.
[0030] FIG. 4 shows the power spectrum analyses results obtained
from processing 27 minutes of non-artifactual data for subject
DS122 after infusion with 2.0 mg flumazenil.
[0031] FIG. 5 shows the power spectrum analyses results obtained
from processing 19 minutes of non-artifactual data for subject DT74
after infusion with 2.0 mg flumazenil.
[0032] FIG. 6 shows a histogram displaying the results of the
psychomotor vigilance task (PVT) performance before administration
of I.V. flumazenil for case 74.
[0033] FIG. 7 shows a histogram displaying the results of the
psychomotor vigilance task (PVT) performance after 2.0 mg dose of
I.V. flumazenil for case 74.
[0034] FIG. 8 shows a histogram displaying the results of the
psychomotor vigilance task (PVT) performance before administration
of I.V. flumazenil for case 102.
[0035] FIG. 9 shows a histogram displaying the results of the
psychomotor vigilance task (PVT) performance after 2.0 mg dose of
I.V. flumazenil for case 102.
[0036] FIG. 10 shows a histogram displaying the results of the
psychomotor vigilance task (PVT) performance before administration
of I.V. flumazenil for case 122.
[0037] FIG. 11 shows a histogram displaying the results of the
psychomotor vigilance task (PVT) performance after 2.0 mg dose of
I.V. flumazenil for case 122.
[0038] FIG. 12 shows a histogram displaying the results of the
psychomotor vigilance task (PVT) performance before administration
of I.V. flumazenil for case 124.
[0039] FIG. 13 shows a histogram displaying the results of the
psychomotor vigilance task (PVT) performance after 1.2 mg dose of
I.V. flumazenil for case 124.
[0040] FIG. 14 shows a graph displaying the results of the
psychomotor vigilance task (PVT) performance before and after
treatment with I.V. flumazenil for case 74.
[0041] FIG. 15 shows a graph displaying the results of the
psychomotor vigilance task (PVT) performance before and after
treatment with I.V. flumazenil for case 102.
[0042] FIG. 16 shows a graph displaying the results of the
psychomotor vigilance task (PVT) performance before and after
treatment with I.V. flumazenil for case 122.
[0043] FIG. 17 shows a graph displaying the results of the
psychomotor vigilance task (PVT) performance before and after
treatment with I.V. flumazenil for case 124.
[0044] FIG. 18a is an illustration of the rest-activity cycle of
patient AS99 before treatment with flumazenil.
[0045] FIG. 18b is an illustration of the rest-activity cycle of
patient AS99 after treatment with flumazenil.
[0046] FIG. 19a illustrates a whole cell patch clamp recording from
a cell expressing human .alpha.1.beta.2.gamma.2s receptors. Bars
above the traces indicate duration of GABA and CSF application.
[0047] FIG. 19b illustrates a whole cell patch clamp recording from
a cell expressing the benzodiazepine insensitive subunit
.alpha.1(H102R). Bars above the traces indicate duration of GABA
and CSF application.
DETAILED DESCRIPTION
[0048] Ionotropic GABA.sub.A receptors (GABA.sub.AR) are the most
recognized therapeutic targets for anesthetics and
sedative/hypnotic drugs. Mutations in the .alpha.1, .gamma.2, and
delta subunits of GABA.sub.A R account for several of the heritable
epilepsies, endogenous positive allosteric neurosteroid modulators
contribute to fluctuations in mood due to developmental changes in
expression of the .alpha.4.delta. GABA.sub.AR, and mutation of the
.beta.3 subunit has been associated with chronic insomnia. The
inventors have found that a naturally occurring endogenous,
positive, allosteric modulator of recombinant .alpha.1, .beta.2,
.gamma.2 short splice variant GABA.sub.AR is present in CSF plasma
in normal humans and non-human primates, and when present in
excess, produces hypersomnia and excessive daytime sleepiness, or
GABA.sub.A receptor mediated hypersomnia (GRH) as described herein.
Accordingly, treatment of such patients with a GABA.sub.A receptor
antagonist thus can provide a method to treat patients having
various disorders associated with excessive sleepiness, and in
particular treat the symptoms of excessive sleepiness associated
with the various disorders.
I. Methods of Treating GABA.sub.A Receptor Mediated Hypersomnia and
Disorders Associated with Excessive Sleepiness
[0049] Provided herein are methods of treating GABA.sub.A receptor
mediated hypersomnia in a subject, the methods comprising
administering to the subject an effective amount of a GABA.sub.A
receptor antagonist. In addition, provided herein is a method of
treating excessive sleepiness associated with GABA.sub.A receptor
mediated hypersomnia in a subject, comprising administering to the
subject an effective amount of a GABA.sub.A receptor antagonist. In
some embodiments of the methods described herein, the GABA.sub.A
receptor antagonist is a negative allosteric modulator. In some
embodiments, the GABA.sub.A receptor antagonist is selected from
the group consisting of: flumazenil; clarithromycin; picrotoxin;
bicuculline; cicutoxin; and oenanthotoxin. In some embodiments, the
GABA.sub.A receptor antagonist is flumazenil. In some embodiments,
the GABA.sub.A receptor antagonist is clarithromycin. In some
embodiments, the method includes administering a I.V., transdermal,
transmucosal, sublingual, or subdermal formulation of flumazenil to
the subject. The administration of flumazenil can be combined with
administration of other agents, including wakefulness promoting
agents and transdermal formulations of flumazenil.
[0050] GABA.sub.A receptor mediated hypersomnia or disorders
associated with excessive sleepiness are selected from one or more
of: shift work sleep disorder; narcolepsy; obstructive sleep
apnea/hypopnea syndrome; REM behavior disorder; frontal nocturnal
dystonia; restless legs syndrome; nocturnal movement disorder;
Kleine-Levin syndrome; Parkinson's disease; excessive sleepiness;
hypersomnia; idiopathic hypersomnia; recurrent hypersomnia;
endozepine related recurrent stupor; and amphetamine resistant
hypersomnia. In some embodiments, the GABA.sub.A receptor mediated
hypersomnia is selected from idiopathic hypersomnia; recurrent
hypersomnia; endozepine related recurrent stupor; and amphetamine
resistant hypersomnia. In some embodiments, the hypersomnia is
idiopathic hypersomnia. In some embodiments, the hypersomnia is
endozepine related recurrent stupor. In some embodiments, the
hypersomnia is amphetamine resistant hypersomnia.
[0051] Such disorders can be characterized by many objective and
subjective tests known in the art. For example, the Epworth
Sleepiness Scale; the Stanford Sleepiness Scale; the Pittsburgh
Sleep Quality Index; an Activity-Rest and Symptom Diary;
Actigraphy; Psychomotor Vigilance Task; Polysomnography; Functional
Magnetic Resonance Imaging; Profile of Mood States; Functional
Outcomes of Sleep Questionnaire; Medical Outcomes Study Short-Form
36; and Neurophysical Testing, such as the Cambridge Neurophysical
Test Automated Battery (CANTAB) (e.g., physcomotor speed,
attention, working memory, and executive function).
[0052] In addition, GABA.sub.A receptor mediated hypersomnia can be
characterized by demonstration of enhanced GABA.sub.A Receptor
function of a subject's CSF or plasma as compared to a control,
e.g., see Example 1 and Example 14.
II. Methods of Promoting Wakefulness and Enhancing Alertness in
Sleepiness Associated Disorders
[0053] Further provided herein are methods of treating GABA.sub.A
mediated hypersomnia disorders, including shift work sleep
disorder, obstructive sleep apnea/hypopnea syndrome, narcolepsy,
and excessive sleepiness associated with shift work sleep disorder,
obstructive sleep apnea/hypopnea syndrome, hypersomnia, and
narcolepsy. In some embodiments, the GABA.sub.A mediated
hypersomnia is idiopathic hypersomnia; recurrent hypersomnia;
endozepine related recurrent stupor; or amphetamine resistant
hypersomnia. The method comprises administering to the subject an
effective amount of a GABA.sub.A receptor antagonist, such as
flumazenil. In some embodiments, the GABA.sub.A receptor antagonist
is an I.V. formulation, a transdermal formulation, or a
transmucosal formulation.
[0054] A method of altering a somnolent state of a subject is
further provided herein, the method comprising administering to the
subject an effective amount of GABA.sub.A receptor antagonist,
e.g., flumazenil. The somnolent state is selected from one or more
of: narcolepsy, obstructive sleep apnea/hypopnea syndrome, shift
work sleep disorder, and hypersomnia (e.g., idiopathic hypersomnia;
recurrent hypersomnia; endozepine related recurrent stupor; and
amphetamine resistant hypersomnia). In some embodiments, the
GABA.sub.A receptor antagonist is an I.V. formulation, a
transdermal formulation, or a transmucosal formulation.
[0055] Also provided herein are methods for enhancing alertness or
increasing regularity of sleep rhythms in a subject; promoting
wakefulness in a subject; improving cognitive dysfunction in a
subject; and restoring a normal sleep pattern and improving the
quality of psychosocial life and relationships in a subject, each
method comprising administering to the subject an effective amount
of GABA.sub.A receptor antagonist, e.g., flumazenil. In some
embodiments, the GABA.sub.A receptor antagonist is an 1. V.
formulation, a transdermal formulation, or a transmucosal
formulation.
[0056] As used herein, the term "promoting wakefulness" refers to a
decrease in sleepiness, tendency to fall asleep, or other symptoms
of undesired or reduced alertness or consciousness compared with
sleepiness, tendency to fall asleep, or other symptoms of undesired
or reduced alertness or consciousness expected or observed without
treatment. Promoting wakefulness refers to a decrease in any stage
of sleep, including light sleep, deeper sleep characterized by the
presence of high amplitude, low wave brain activity termed "slow
wave sleep", and rapid eye movement (REM) sleep.
[0057] A determination of whether the treatment is useful in
performing the methods described herein can be made, for example,
by direct observation of behavioral or physiological properties of
mammalian sleep, by self-reporting, or by various well-known
methods, including electrophysiological methods. Such methods
include, for example, examining electroencephalograph (EEG)
activity amplitude and frequency patterns, examining electromyogram
activity, and examining the amount of time during a measurement
time period, in which a mammal is awake or exhibits a behavioral or
physiological property characteristic of wakefulness.
[0058] The effectiveness of the treatments can also be
characterized by the objective and subjective tests described
herein, including the Epworth Sleepiness Scale; the Stanford
Sleepiness Scale; the Pittsburgh Sleep Quality Index; an
Activity-Rest and Symptom Diary; Actigraphy; Psychomotor Vigilance
Task; Polysomnography; Functional Magnetic Resonance Imaging;
Profile of Mood States; Functional Outcomes of Sleep Questionnaire;
Medical Outcomes Study Short-Form 36; and Neurophysical Testing,
such as the Cambridge Neurophysical Test Automated Battery (CANTAB)
(e.g., physcomotor speed, attention, working memory, and executive
function).
III. Formulation and Administration of A GABA.sub.A Receptor
Antagonist
[0059] A GABA.sub.A receptor antagonist can be selected from
flumazenil; clarithromycin; picrotoxin; bicuculline; cicutoxin; and
oenanthotoxin and can be formulated for I.V., transdermal,
transmucosal, sublingual, oral, and subdermal administration for
use with the methods described herein. A transmucosal formulation
can include sublingual, supralingual, and buccal administration.
For transmucosal administration, the antagonist may be combined
with one or more inactive ingredients for the preparation of a
tablet, packed powder, edible film strip, soft gel capsule, hard
gel capsule, lozenge, or troches. For example, in some embodiments,
the antagonists such as flumazenil may be combined with at least
one excipient such as fillers, binders, humectants, disintegrating
agents, solution retarders, absorption accelerators, wetting agents
absorbents, or lubricating agents. According to some embodiments,
the antagonist may be combined with one or more of a polyol (e.g.,
lactose, sucrose, mannitol, or mixtures thereof), an alcohol (e.g.,
ethanol), and a gum (e.g., acacia and guar), and then formed into a
lozenge by conventional methods.
[0060] In some embodiments, the formulation is a hard, compressed,
rapidly dissolving tablet adapted for direct sublingual dosing. The
tablet includes particles made of the antagonist and a protective
material. In some embodiments, these particles are provided in an
amount of between about 0.01 and about 75% by weight based on the
weight of the tablet (e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,
45%, 50%, 60%, 70%, and 75%). In some embodiments, the tablet may
also include a matrix made from a nondirect compression filler, a
wicking agent, and a hydrophobic lubricant. In some embodiments,
the tablet is adapted to dissolve spontaneously in the mouth of a
patient in less than about 60 seconds (and, in some cases, in less
than about 30 seconds).
[0061] In some embodiments, the formulation can be a compressed
rapidly dissolving tablet comprising effervescent agents. These
effervescent agents allow enhanced adsorption of the antagonist
across the mucosal membranes (e.g., tongue, cheek, and gums) in the
oral cavity. An example of effervescent pharmaceutical compositions
suitable for use in conjunction with the methods described herein
are the compositions described in U.S. Pat. No. 6,200,604.
[0062] In some embodiments, the antagonist can be administered
transmucosally using an edible film. Such films can include a
carrier comprising water-soluble polymers in combination with
certain ingredients and provides a therapeutic effect. In some
embodiments, the film is coated and dried utilizing existing
coating technology and exhibits instant wettability followed by
rapid dissolution/disintegration upon administration in the oral
cavity. In some embodiments, an edible film can contain as the
essential components a water-soluble polymer or a combination of
water-soluble polymers, one or more plasticizers or surfactants,
one or more polyalcohols, and flumazenil. Non-limiting examples of
edible films can be found in U.S. Pat. Nos. 5,948,430; 6,177,096;
6,284,264; 6,592,887; and 6,709671.
[0063] Further examples of additional pharmaceutical compositions
suitable for transmucosal administration include those described in
U.S. Pat. Nos. 5,178,878; 5,223,264; and 6,024,981.
[0064] In some embodiments, the antagonist is combined with
inactive ingredients. Such ingredients may be necessary, for
example, to add bulk to the pharmaceutical preparation, to bind the
preparation, to add color or flavor to the preparation, and to
prevent degradation or growth of contaminants.
[0065] In some embodiments, administration of the antagonist may be
performed using an implantable device, for example, an implantable,
self-regulating mechanochemical subdermal pump. In some
embodiments, the device may administer the antagonist on a set
dosage program. In some embodiments, the device may administer the
antagonist on demand as determined by the subject. In some
embodiments, the device may administer the antagonist on a constant
release profile. In some embodiments, the device may administer the
antagonist automatically. These devices are known in the art for
the treatment of other disorders, for example, diabetes.
Non-limiting examples of various embodiments of this mode of
administration are detailed in U.S. Pat. Nos. 5,062,841; 5,324,518;
and 6,852,104.
[0066] In some embodiments, a transmucosal administration of an
antagonist may be combined with transdermal administration of the
same or another antagonist. Without being bound by theory, such a
delivery mechanism may be useful for nocturnal application to
assist the subject with morning wakefulness.
[0067] Transdermal administration of the antagonist can be
accomplished by mixing the antagonist with suitable pharmaceutical
carriers, preservatives, optional penetration enhancers, and
optional gelling agents to form ointments, emulsions, lotions,
solutions, creams, gels, patches or the like, wherein a fixed
amount of the preparation is applied onto a certain area of
skin.
[0068] By the term "suitable pharmaceutical carrier" is meant a
non-toxic pharmaceutically acceptable vehicle including, for
example, polyethylene glycol, propylene glycol, isopropanol,
ethanol, oleic acid, N-methylpyrrolidone, sesame oil, olive oil,
wood alcohol ointments, vaseline, and paraffin or a mixture
thereof.
[0069] Suitable penetration enhancers include, for example,
saturated and unsaturated fatty acids and their esters, alcohols,
monoglycerides, diethanolamines, N,N-dimethylamines such as
linolenic acid, linolenyl alcohol, oleic acid, oleyl alcohol,
stearic acid, stearyl alcohol, palmitic acid, palmityl alcohol,
myristic acid, myristyl alcohol, 1-dodecanol, 2-dodecanol, lauric
acid, decanol, capric acid, octanol, caprylic acid,
1-dodecylazacycloheptan-2-one sold under the trademark AZONE
(Nelson Research and Development; Irvine, Calif.), ethyl caprylate,
isopropyl myristate, hexamethylene lauramide, hexamethylene
palmitate, capryl alcohol, decyl methyl sulfoxide, dimethyl
sulfoxide, salicylic acid and its derivatives,
N,N-diethyl-m-toluamide, crotamiton, 1-substituted
azacycloalkan-2-ones, polyethylene glycol monolaurate and any other
compounds compatible with medetomidine and its optically active
enantiomers and the packages and having transdermal permeation
enhancing activity.
[0070] Suitable gelling agents include, for example, hydroxy methyl
cellulose, hydroxypropyl cellulose sold under the trademark KLUCEL
HF (Hercules Inc.; Wilmington, Del.), tragacanth, sodium alginate,
gelatin, methylcellulose, sodium carboxymethylcellulose, and
polyvinyl alcohols. Suitable preservatives include, for example,
parabens, benzoic acid, and chlorocresol.
[0071] Antioxidants can be included in the formulations described
herein. Suitable antioxidants include, for example, ascorbyl
palmirate, butylated hydroxyanisole, butylated hydroxytoluene,
potassium sorbate, sodium bisulfate, sorbic acid, propyl gallate,
and sodium metabisulfite.
[0072] In some embodiments, the antagonist is administered by a
transdermal patch. Adhesives for making transdermal patches for use
in the methods described herein include polyisobutylene, silicone
based adhesives, and acrylic polymers. The adhesive polymers can be
mixed with other excipients such as waxes and oils (e.g., mineral
oil). A protective liner can be placed in contact with the adhesive
layer to protect against drug release from the patch prior to
application. Liners for use with the transdermal patches described
herein include, for example, polyethylene terephthalate film,
polyester membrane, and polycarbonate film.
[0073] The backing membrane of the transdermal patch for use with
the methods described herein constitutes the top face surface of
the transdermal patch. It may be made of a single layer or film of
polymer, or be a laminate of one or more polymer layers and metal
foil. Examples of polymers suitable for use in making backing films
include, for example, polyester films, ethyl vinyl acetate,
polypropylene, polyethylene, and polyvinyl-chloride.
[0074] In some embodiments, the administration rate of the drug is
0.1-1000 .mu.g/h through a skin area of about 2-90 cm.sup.2 (e.g.,
10-30 cm.sup.2). The amount of drug delivered into the skin can be
controlled by a number of factors including skin patch size, degree
of drug loading, the use of rate controlling membranes, permeation
enhancers, and the like.
[0075] In some embodiments, the transmucosal and/or the transdermal
formulation may be a time-release or slow-release formulation. In
some embodiments, the transdermal formulation may be a time-release
or slow-release formulation. The transmucosal or transdermal
formulation described herein may also be formulated so as to
provide slow or controlled release of the antagonist using, for
example, hydropropylmethyl cellulose in varying proportions to
provide the desired release profile, other polymer matrices, gels,
permeable membranes, osmotic systems, multilayer coatings,
microparticles, liposomes and/or microspheres. In general, a
controlled-release preparation is a pharmaceutical composition
capable of releasing the active ingredient at the required rate to
maintain constant pharmacological activity for a desirable period
of time. Such dosage forms provide a supply of a drug to the body
during a predetermined period of time and thus maintain drug levels
in the therapeutic range for longer periods of time than
conventional non-controlled formulations.
[0076] U.S. Pat. No. 5,591,767 describes a liquid reservoir
transdermal patch for the controlled administration of ketorolac, a
non-steroidal anti-inflammatory agent with potent analgesic
properties. U.S. Pat. No. 5,120,548 discloses a controlled-release
drug delivery device comprised of swellable polymers. U.S. Pat. No.
5,073,543 describes controlled-release formulations containing a
trophic factor entrapped by a ganglioside-liposome vehicle. U.S.
Pat. No. 5,639,476 discloses a stable solid controlled-release
formulation having a coating derived from an aqueous dispersion of
a hydrophobic acrylic polymer. Biodegradable microparticles are
known for use in controlled-release formulations. U.S. Pat. No.
5,354,566 discloses a controlled-release powder that contains the
active ingredient. U.S. Pat. No. 5,733,566 describes the use of
polymeric microparticles that release antiparasitic
compositions.
[0077] The controlled-release of the active ingredient may be
stimulated by various inducers, for example, pH, temperature,
enzymes, water, or other physiological conditions or compounds.
Various mechanisms of drug release exist. For example, in one
embodiment, the controlled-release component may swell and form
porous openings large enough to release the antagonist after
administration to a patient. The term "controlled-release
component" means a compound or compounds, such as polymers, polymer
matrices, gels, permeable membranes, liposomes and/or microspheres
that facilitate the controlled-release of the active ingredient in
the pharmaceutical composition. In another embodiment, the
controlled-release component is biodegradable, induced by exposure
to the aqueous environment, pH, temperature, or enzymes in the
body.
[0078] The specific dose of an antagonist required to obtain
therapeutic benefit in the methods of treatment described herein
will, usually be determined by the particular circumstances of the
individual patient including the size, weight, age, and sex of the
subject, the nature and stage of the disorder being treated, the
aggressiveness of the disorder, and the route of administration of
the compound.
[0079] For transmucosal administration (e.g., sublingual
administration), for example, a daily dosage of flumazenil, for
example, can range from about 0.5 mg to about 10 mg per Body Mass
Index (BMI) unit (e.g., about 0.5 mg to about 5 mg; about 1 mg to
about 3 mg; about 1.5 mg to about 4 mg; about 2 mg to about 6 mg;
about 1.25 mg to about 8 mg; and about 4 mg to about 10 mg). In
some embodiments, a daily dosage of flumazenil can range from about
1 mg per BMI to about 5 mg per BMI. In some embodiments, a daily
dosage of flumazenil can be about 1.5 mg per BMI. In some
embodiments, a daily dosage of flumazenil can be about 2 mg per BMI
unit. In some embodiments, a daily dosage of flumazenil can be
about 3 mg per BMI unit. For example, a subject with a BMI of 20
could be administered a daily dosage of about 40 mg of flumazenil,
in other words, a daily dosage of 2 mg per BMI unit. Higher or
lower doses are also contemplated, as it may be necessary to use
dosages outside these ranges in some cases.
[0080] The transmucosal formulation can be administered in one
single dosage or the daily dosage may be divided, such as being
divided equally into two to six times per day daily dosing. In some
embodiments, the transmucosal formulation is administered at least
twice daily. In some embodiments, the transmucosal formulation is
administered at least three times daily. In some embodiments, the
transmucosal formulation is administered about every one to six
hours (e.g., about every one hour; about every two hours; about
every three hours; about every three and a half hours; about every
four hours; about every five hours; and about every six hours). In
some embodiments, the transmucosal formulation is administered by
the subject as needed, e.g., patient controlled titration to a
desired end effect (e.g., wakefulness or reduced sleepiness).
[0081] A transmucosal formulation may be formulated in a unit
dosage form, each dosage containing from about 0.5 to about 20 mg
of the antagonist, e.g., flumazenil, per unit dosage (e.g., about
0.5 mg to about 15 mg; about 1 mg to about 10 mg; about 1.5 mg to
about 8 mg; about 2 mg to about 7 mg; about 3 mg to about 6 mg;
about 4 mg to about 8 mg; about 5 mg to about 10 mg; about 6 mg to
about 12 mg; and about 8 mg to about 20 mg). In some embodiments,
each dosage can contain about 5 to about 10 mg of the antagonist
per unit dosage. In some embodiments, each dosage contains about 6
mg of the antagonist. The term "unit dosage form" refers to
physically discrete units suitable as a unitary dosage for human
subjects and other mammals, each unit containing a predetermined
quantity of active material calculated to produce the desired
therapeutic effect, in association with a suitable pharmaceutical
excipient.
[0082] For transdermal administration, for example, a daily dosage
of flumazenil can range from about 0.5 mg to about 10 mg (e.g.,
about 0.5 mg to about 5 mg; about 1 mg to about 3 mg; about 1.5 mg
to about 4 mg; about 2 mg to about 6 mg; about 1.25 mg to about 8
mg; and about 4 mg to about 10 mg). In some embodiments, a daily
dosage of transdermal flumazenil can range from about 1 mg to about
5 mg. In some embodiments, a daily dosage of transdermal flumazenil
can be about 1.5 mg. In some embodiments, a daily dosage of
transdermal flumazenil can be about 2 mg. In some embodiments, a
daily dosage of transdermal flumazenil can be about 3 mg. Higher or
lower doses are also contemplated as it may be necessary to use
dosages outside these ranges in some cases.
[0083] The transdermal formulation can be administered in one
single dosage or the daily dosage may be divided, such as being
divided equally into two to six times per day daily dosing. In some
embodiments the transdermal formulation is formulated to a
concentration of about 0.5 mg to about 10 mg per mL (e.g., about
0.5 mg to about 8 mg per mL; about 1 mg to about 6 mg per mL; about
1.5 mg to about 5 mg per mL; about 3 mg to about 7 mg per mL; about
4 mg to about 10 mg per mL; and about 4 mg to about 8 mg per mL).
In some embodiments, the transdermal formulation is formulated to a
concentration of about 4 mg per mL. In some embodiments, the
transdermal formulation is administered once daily (e.g., before
bed). In some embodiments, the transdermal formulation is
administered at least twice daily. In some embodiments, the
transdermal formulation is administered about every eight to about
twenty-four hours (e.g., about every eight hours; about every ten
hours; about every twelve hours; about every sixteen hours; about
every twenty hours; about every twenty-two hours; and about every
twenty-four hours).
[0084] A transdermal formulation may be formulated in a unit dosage
form, each dosage containing from about 0.5 to about 10 mg of
flumazenil per unit dosage (e.g., about 0.5 mg to about 8 mg; about
1 mg to about 5 mg; about 1.5 mg to about 4 mg; about 2 mg to about
6 mg; about 3 mg to about 7 mg; about 4 mg to about 8 mg; and about
5 mg to about 10 mg). In some embodiments, each dosage can contain
about 1 to about 4 mg of flumazenil per unit dosage. In some
embodiments, each dosage contains about 2 mg of flumazenil. The
term "unit dosage form" refers to physically discrete units
suitable as a unitary dosage for human subjects and other mammals,
each unit containing a predetermined quantity of active material
calculated to produce the desired therapeutic effect, in
association with a suitable pharmaceutical excipient.
[0085] The components used to formulate the pharmaceutical
compositions described above are of high purity and are
substantially free of potentially harmful contaminants (e.g., at
least National Food grade, generally at least analytical grade, and
more typically at least pharmaceutical grade). Particularly for
human consumption, the composition is preferably manufactured or
formulated under Good Manufacturing Practice standards as defined
in the applicable regulations of the U.S. Food and Drug
Administration. For example, suitable formulations may be sterile
and/or substantially isotonic and/or in full compliance with all
Good Manufacturing Practice regulations of the U.S. Food and Drug
Administration.
[0086] The antagonist can be administered in combination with other
agents. In one embodiment, the antagonist is administered with a
wakefulness promoting agent (e.g., modafinil and armodafinil). In
some embodiments, the wakefulness promoting agent is modafinil. In
some embodiments, the subject may be resistant to one or more
wakefulness promoting agents prior to administration of the
antagonist. The wakefulness promoting agent can be administered in
an amount less than about 600 mg per day (e.g., less than about 100
mg per day; less than about 200 mg per day; less than about 300 mg
per day; less than about 400 mg per day; less than about 500 mg per
day; and less than about 600 mg per day). The specific dose of a
wakefulness promoting agent required to obtain therapeutic benefit
in the methods of treatment described herein will usually be
determined by the particular circumstances of the individual
subject including the size, weight, age, and sex of the subject,
the nature and stage of the disorder being treated, the
aggressiveness of the disorder, and the route of administration of
the compound. In some embodiments, the wakefulness promoting agent
can be administered twice daily. In some embodiments, the
wakefulness promoting agent can be administered in an amount of 5
mg per BMI unit. In some embodiments, the wakefulness promoting
agent can be administered in an amount of 100 mg per dose. In some
embodiments, the subject exhibits resistance to a wakefulness
promoting agent prior to administration of the antagonist. In some
embodiments, administration of the antagonist can reverse or
decrease a subjects resistance to a wakefulness promoting
agent.
[0087] In some embodiments, treatment of a disorder associated with
excessive sleepiness can include the following: [0088] a)
transmucosal, e.g., sublingual, administration of an antagonist,
e.g., flumazenil; and [0089] b) administration of a wakefulness
promoting agent. In some embodiments, the treatment can further
include: [0090] c) transdermal administration of an antagonist,
e.g., flumazenil.
[0091] For example, in some embodiments, a sublingual formulation
of flumazenil is administered about every 2 to 4 hours during the
waking hours of the day (e.g., every about 3 to 3.5 hours). In some
embodiments, a wakefulness promoting agent is administered from one
to three times during the waking hours of the day (e.g., about
every 4 hours). In some embodiments, the wakefulness promoting
agent is modafinil. In some embodiments, a transdermal or
time-release formulation of flumazenil is administered once daily
(e.g., before bed).
IV. Assay for GABA.sub.A Receptor Mediated Hypersomnia
[0092] The GABA.sub.A receptors are one of several classes of
chemically gated ion channels that incorporate the features of both
"receptors" and "ion channels" into one membrane protein. These
chemically gated channels (ligand gated ion channel: LGIC) can
detect extracellular chemical signals such as neurotransmitters
released from neighboring cells and in response will open an ion
channel to allow specific ions to enter or leave the cell. When
this results in a net movement of positive charge into the cell,
the cell becomes more electrically positive and thus more
excitable. Conversely, when this results in a net flow of negative
ions into the cell, the neuron becomes more electrically negative
and thus more inhibited. In this way, LGICs act as
chemical-to-voltage converters and are fundamental to cell-to-cell
communication and neuronal activity. Drugs and chemicals that
enhance or block these functions have profound effect on brain
circuits and ultimately human behavior. For example, most general
anesthetics render patients unconscious by enhancing the function
of inhibitory LGICs, the most common of which is the GABA.sub.A
receptor.
[0093] The most common inhibitory neurotransmitter in the human
nervous system is .gamma.-aminobutyric acid or GABA. It is released
by neurons at synapses, the specialized junctions between 2 neurons
that permits rapid cell-to-cell communication. After leaving the
presynaptic neuron and crossing the synaptic gap, the molecules of
GABA arrive at the postsynaptic membrane where they can interact
with a LGIC, the Type-A GABA receptor (GABA.sub.AR). After GABA
binds to the receptor, the LGIC changes shape and allows the flow
of negatively charged chloride ions into the neuron, which results
in the neuron becoming inhibited and unable to pass a message onto
another neuron, until GABA unbinds and the inhibition passes.
[0094] If GABA.sub.AR function is blocked, then the brain circuits
in which they are imbedded experience less inhibition. This can
cause the circuits to become hyper-excitable, exhibiting much more
excitation than normal. This will result in convulsions and
seizures if the block is not removed. This can occur in the
presence of a GABA.sub.AR channel blocker toxin or a GABA
antagonist. This can also occur in some patients who have inherited
forms of epilepsy. In these patients, a GABA.sub.AR gene has
mutated to make a dysfunctional GABA.sub.AR that does not function
as well as it should.
[0095] GABA.sub.ARS are enhanced by many chemicals and drugs.
General anesthetics, as already noted, enhance inhibition by making
the channels stay open for longer periods of time, increasing the
duration of inhibition. This is also true for many neurosteroids
(e.g., progesterone metabolites) and for ethanol. GABA.sub.ARS are
also a critical binding site for benzodiazepines, such as valium.
These important anxiolytic and sedative drugs cause the receptors
to bind GABA more tightly, also enhancing inhibition by the
receptor.
[0096] It is important to note that all of these compounds do not
activate the channel. They are all "allosteric modulators". They
bind to sites separate from the GABA binding sites and simply
enhance or amplify the effect of GABA. In the absence of GABA,
physiologic and/or therapeutic concentrations of these different
compounds have no effect on the channel. GABA must be present for
them to have an effect. Similarly, the benzodiazepine antagonist
flumazenil is not a GABA.sub.AR blocker. It occupies the
benzodiazepine binding site, thus blocking drugs like valium from
acting on the channel. Although it is bound to the receptor,
flumazenil does not have an effect on the channel. Its functional
effect can only be observed when both GABA and a benzodiazepine are
present.
[0097] Developed in the late 1970s, the single cell
electrophysiology method known as patch clamp is a standard for
measuring the function of ion channels in research laboratories.
The techniques takes advantage of the high electrical resistance
between a cell surface and specially constructed microelectrodes,
and capacitative feedback electronics which combine to give ultra
low noise (<100 fA) recordings of ions flowing through single
ion channels.
[0098] Provided herein is a method of diagnosing and treating a
patient suffering from hypersomnia associated with the endogenous
production of GABA.sub.A receptor modulators, e.g., excessive
production of such modulators. There are many reports of
hypersomnia disorders in subjects who do not respond well to
conventional stimulant (e.g., amphetamine) therapies. These
subjects may be suffering from a form of hypersomnia referred to as
amphetamine resistant hypersomnia, from an increased production of
endozepines (e.g., hemin and protoporphyrin IX), or from an
increased production of another substance that binds to the
GABA.sub.A receptor. Without being bound by theory, the subject may
be producing endogenous benzodiazepines (i.e. "endozepines") or
other somnogenic compound(s) that interact directly or indirectly
with the benzodiazepine binding site on the GABA.sub.AR, enhancing
receptor function as classic benzodiazepines such as valium.
[0099] A method of diagnosing a patient suffering from GABA.sub.A
mediated hypersomnia associated with increased production of
endozepines or other somnogenic substance(s) can be performed by
measuring the effect of a subjects' cerebral spinal fluid (CSF) or
blood or plasma on recombinant GABA.sub.AR function under whole
cell patch clamp conditions (see, e.g., FIG. 1 and Example 1 or
FIGS. 19A and 19B and Example 14). In some embodiments, the effect
of the CSF or blood or plasma can be compared to the effect
observed when the CSF or blood or plasma is co-applied with a
GABA.sub.A receptor antagonist such as flumazenil. In some
embodiments, application of the antagonist such as flumazenil can
modulate the response of a CSF or blood sample of a subject as
measured in a GABA whole cell patch clamp efficacy assay to within
25% of a control sample response. In some embodiments, the
modulation is a decrease in the response of the CSF sample of the
subject in the presence of the antagonist such as flumazenil. In
some embodiments, the effect of the CSF or blood or plasma in an
assay expressing benzodiazepine sensitive receptors can be compared
to the effect observed of the CSF or blood or plasma in an assay
expressing benzodiazepine insensitive receptors. In some
embodiments, the substance in the CSF or blood or plasma sample of
a subject potentiates the response of GABA as measured in a GABA
whole cell patch clamp efficacy assay. In some embodiments, the
potentiation of the GABA response in the benzodiazepine sensitive
receptors and the potentiation of the GABA response in the
benzodiazepine insensitive receptors are within .+-.25% of each
other. In some embodiments, the persistence of potentiation within
.+-.25% of the GABA responses in benzodiazepine sensitive and
insensitive receptor assays is indicative that the subject would
benefit from treatment with a GABA.sub.A receptor antagonist. In
some embodiments, the GABA.sub.A receptor antagonist is
flumazenil.
[0100] Further, a method of diagnosing a patient suffering from
GABA.sub.A mediated hypersomnia associated with increased
production of endozepines or other somnogenic substances can be
performed by measuring the effect of a subjects' cerebral spinal
fluid (CSF) or blood or plasma on recombinant GABA.sub.AR function
under whole cell patch clamp conditions.
V. Kits
[0101] Also provided herein are kits for treating disorders
associated with excessive sleepiness. A kit can include an I.V.,
transdermal, oral, or transmucosal (e.g., sublingual, supralingual,
and buccal) formulation of a GABA.sub.A receptor antagonist. In
some embodiments, the GABA.sub.A receptor antagonist is flumazenil.
In some embodiments, the kit can further includes one or more of a
wakefulness promoting agent (e.g., modafinil) and a transdermal
formulation of a GABA.sub.A receptor antagonist. In some
embodiments, a kit can include one or more delivery systems and
directions for use of the kit (e.g., instructions for treating a
subject). In some embodiments, a kit can include a sublingual
formulation of flumazenil and a transdermal formulation of
flumazenil. In another embodiment, a kit can include a sublingual
formulation of flumazenil and a wakefulness promoting agent. In
some embodiments, the kit can include a sublingual formulation of
flumazenil and a label that indicates that the contents are to be
administered to a subject resistant to amphetamines. In another
embodiment, the kit can include a sublingual formulation of a
GABA.sub.A receptor antagonist such as flumazenil and a label that
indicates that the contents are to be administered to a subject
positive for increased production of endozepines or other
somnogenic compounds, as described herein. In a further embodiment,
a kit can include a sublingual formulation of flumazenil and a
label that indicates that the contents are to be administered with
a wakefulness promoting agent and/or a transdermal formulation of
flumazenil.
[0102] Also provided herein are kits for performing a diagnostic
assay. In some embodiments, the diagnostic assay can be used to
diagnose subjects suffering from a GABA.sub.A receptor mediated
hypersomnia and/or to determine subjects that would benefit from
treatment with a GABA.sub.A receptor antagonist. In some
embodiments, a kit for use as a diagnostic assay is provided with
the components for carrying out a patch clamp assay as described
herein. In some embodiments, the kit can include a GABA.sub.A
receptor antagonist and cells which transiently or stably express
human .alpha.1.beta.2.gamma.2s GABA.sub.A receptors. In some
embodiments, the kit can include cells which transiently and stably
express human .alpha.1.beta.2.gamma.2s GABA.sub.A receptors and
cells which transiently and stably express a benzodiazepine
insensitive subunit (e.g., a1(H102R). In some embodiments, the kit
further comprises one or more of an extracellular solution that can
function as a control sample, e.g., a control CSF sample; an
intracellular solution; an extracellular medium, a motor-driven
solution exchange device; and instructions for use of the kit.
VI. Definitions
[0103] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this disclosure belongs. All
patents, applications, published applications, and other
publications are incorporated by reference in their entirety. In
the event that there is a plurality of definitions for a term
herein, those in this section prevail unless stated otherwise.
[0104] As used herein, the singular forms "a," "an," and "the"
include plural referents unless the context clearly dictates
otherwise.
[0105] A "subject" can include both mammals and non-mammals.
Mammals include, for example, humans; nonhuman primates, e.g. apes
and monkeys; cattle; horses; sheep; rats; mice; pigs; and goats.
Non mammals include, for example, fish and birds.
[0106] The expression "effective amount", when used to describe an
amount of compound in a method, refers to the amount of a compound
that achieves the desired pharmacological effect or other effect,
for example an amount that results in reduced sleepiness.
[0107] The terms "treating" and "treatment" mean causing a
therapeutically beneficial effect, such as ameliorating existing
symptoms, preventing additional symptoms, ameliorating or
preventing the underlying metabolic causes of symptoms, postponing
or preventing the further development of a disorder and/or reducing
the severity of symptoms that will or are expected to develop.
EXAMPLES
Example 1
Endozepine Modulation of GABA.sub.AR Function
[0108] HEK293 cells transiently expressing human .alpha.1, .beta.2,
and .gamma.2s subunits were superfused at 1 mL/min with an
extracellular solution (ACSF) containing 145 mm NaCl, 3 mm KCl, 1.5
mm CaCl.sub.2, 1 mm MgCl.sub.2, 6 mm d-glucose, and 10 mm
HEPES-NaOH adjusted to pH 7.4. Whole cell patch clamp recordings
from cells voltage clamped at -60 mV were made using the Multiclamp
700B amplifier (Molecular Devices, Sunnyvale, Calif.). The
resistance of the patch pipette was 4-6 M when filled with
intracellular solution (145 mm N-methyl-d-glucamine hydrochloride,
5 mm dipotassium ATP, 1.1 mm EGTA, 2 mm MgCl.sub.2, 5 mm HEPES-KOH,
and 0.1 mm CaCl.sub.2 adjusted to pH 7.2). In addition to the
continuous bath perfusion with extracellular medium, solutions
including AS99-CSF (as described below), GABA and/or flumazenil
were applied rapidly to the cell by local perfusion using a
motor-driven solution exchange device (Rapid Solution Changer
RSC-160; Molecular Kinetics, Indianapolis, Ind.). Solutions were
exchanged within approximately 50 ms. Laminar flow out of the rapid
solution changer head was achieved by driving all solutions at
identical flow rates (1.0 mL/min) via a multichannel infusion pump
(KD Scientific, Holliston, Mass.). The solution changer was driven
by protocols in the acquisition program of pCLAMP version 9.2
(Molecular Devices, Sunnyvale, Calif.). AS-CSF was isolated from
AS99, a patient experiencing hypersomnia. The patient also
exhibited apparent resistance to amphetamine treatment. All other
compounds were obtained from the Sigma-Aldrich Co.
[0109] Results indicated that AS-CSF had no intrinsic GABA
efficacy, but it enhanced the amplitude of response to EC.sub.20
concentrations of GABA (see FIG. 1a). In a second experiment, 4
.mu.M flumazenil was co-applied with AS-CSF. The flumazenil
immediately reversed the enhancing effect of the AS-CSF (see FIG.
1b).
[0110] Positive modulation of GABA.sub.A receptor function by 100%
or more is normal for concentrations of general anesthetic drugs
that would anesthetize a human. The results indicate that AS-CSF
contains a positive allosteric modulator of GABA.sub.A Receptor
that would have potent sedative effects in a human. The reversal of
this effect by flumazenil suggests that the positive modulator
likely acts directly or indirectly at the benzodiazepine binding
site on the GABA.sub.A receptor.
[0111] Accordingly, patients experiencing disorders associated with
excessive sleepiness (e.g., idiopathic or amphetamine resistant
hypersomnia) who test positive for a positive allosteric modulator
of GABA.sub.A receptor function may likely benefit from
administration of flumazenil.
Example 2
Formulation of Flumazenil as Tablet for Sublingual Dosing
TABLE-US-00001 [0112] Ingredient: Amount added: Flumazenil 0.3
grams Tablet triturate base (20%/80% powder) 4.7 grams Tablet
triturate exipient (flavorless) 2 milliliters Flavor, PCCA
Bittershop 4 drops Stevia concentrate (250 mg/mL) 2 drops
[0113] Procedure: The ingredients were combined and mixed to form a
thick paste. After the thick paste was formed, a flavor was added.
The flavor added was selected from the following: [0114] a) 2 drops
lemon, 1 drop marshmallow, 4 milligrams yellow color [0115] b) 2
drops creme de mint, 4 mg green color [0116] c) 2 drops tangerine,
1 drop marshmallow, 4 mg orange. The formulation provided 50
tablets.
Example 3
Formulation of Flunzazenil as Tablet Triturate for Sublingual
Dosing
TABLE-US-00002 [0117] Ingredient: Amount added: Flumazenil 0.6
grams Tablet triturate base (20%/80% powder) 9.4 grams Tablet
triturate exipent (flavorless) 4 milliliters Flavor, PCCA
Bittershop 8 drops Stevia concentrate solution (250 mg/mL) 4
drops
[0118] Procedure: The ingredients were combined and mixed to form a
thick paste. See Example 4 for tablet triturate base (20%/80%
powder) formulation and Example 5 for stevia concentrate solution
formulation. After the thick paste was formed, a flavor was added.
The flavor added was selected from the following (quantities given
are per 50 tablets): [0119] a) 2 drops lemon, 1 drop marshmallow, 4
milligrams yellow color [0120] b) 2 drops creme de mint, 4 mg green
color [0121] c) 2 drops tangerine, 1 drop marshmallow, 4 mg orange
[0122] d) 5 drops cherry, 2 drops vanilla, 4 mg red color. The
formulation provided 100 tablets.
Example 4
Formulation of Tablet Triturate Base 20%/80% Powder
TABLE-US-00003 [0123] Ingredient: Amount added: Sucrose powdered
(confectioners) 20 grams Lactose monohydrate (hydrous) 80 grams
[0124] Procedure: The sucrose and lactose monohydrate were sieved
through 120 or smaller mesh. After adding the active ingredient
(e.g., flumazenil), the mixture was wetted with an excipient of 40%
distilled water and 60% alcohol. The formulation provided 100 grams
of table triturate base 20%/80% powder.
Example 5
Formulation of Stevia Concentrate Solution (250 mg/mL)
TABLE-US-00004 [0125] Ingredient: Amount added: Stevia powder
extract 25 grams Sodium benzoate 0.6 grams Water preserved liquid
100 milliliters
[0126] Procedure: The stevia powder and sodium benzoate were
dissolved in the water preserved. See Example 6 for water preserved
liquid formulation. The mixture was warmed to aid in dissolution.
The formulation prepared 100 mL of stevia concentrate solution.
Example 6
Formulation of Water Preserved (Paraben) Liquid
TABLE-US-00005 [0127] Ingredient: Amount added: Water preserved
concentrate liquid 10 milliliters Water distilled liquid 3780
mL
[0128] Procedure: The liquids were mixed to prepare the water
preserved (paraben) liquid. See Example 7 for water preserved
concentrate liquid formulation.
Example 7
Formulation of Water Preserved Concentrate Liquid
TABLE-US-00006 [0129] Ingredient: Amount added: Methylparaben 19
grams Propylparaben NF 9.6 grams Propylene glycol USP 100 mL
[0130] Procedure: The ingredients were mixed together and stirred
until the methylparaben and propylparaben NF were completely
dissolved.
Example 8
Formulation of Flumazenil as Cream for Transdermal Dosing
TABLE-US-00007 [0131] Ingredient: Amount added: Flumazenil 0.04
grams Prophlene glycol USP 0.1 milligrams Food color, pink (powder)
0.03 milligrams Versabase cream 10 grams
[0132] Procedure: The ingredients were combined and mixed. The
formulation provided 10 milliliters of cream.
Example 9
Formulation of Flumazenil as Cream for Transdermal Dosing
TABLE-US-00008 [0133] Ingredient: Amount added: Flumazenil 0.25
grams Prophlene glycol USP 0.25 milliliters Food color, red
(powder) 0.0075 milligrams Versabase cream 25 grams
[0134] Procedure: The ingredients were combined and mixed. The
formulation provided 25 milliliters of cream.
Example 10
Characterization of the Spectrum of GABA.sub.A Receptor Mediated
Hypersomnia
[0135] An organized, multidimensional approach to characterizing
the phenotypic spectrum of GRH will be employed to determine who is
affected, and how it manifests with specific attention to overlap
with ICSD-2 defined sleep and circadian rhythm disorders. This will
involve recruiting and extensively characterizing and correlating
biological activity at the GABA.sub.A receptor with behavior in 70
individuals suffering from sleepiness or hypersomnia. Ten, age and
sex-matched controls deemed `affected` or `unaffected` by
sleepiness will also be studied. Initial identification,
recruitment, and biological sample procurement will take place in
the outpatient clinic and diagnostic sleep laboratory which share
dedicated space. After satisfying inclusion/exclusion criteria and
upon providing consent, additional behavioral, wake/sleep, and
rest-activity cycle assessments will be conducted along with
quantification endogenous GABA.sub.A receptor bioactivity. Subjects
will then be admitted to a clinical setting for 24 hours and their
clinical response to single-blind intravenous delivery of saline,
0.5, and 2.0 mg flumazenil will be determined. Other known causes
of hypersomnia, such as hypocretin deficient narcolepsy, exogenous
BZD use, iatrogenic effects of common medications known to
positively or negatively modulate GABA.sub.AR (e.g., steroids,
methylxanthines and many antibiotics), and metabolic disorders
(e.g., urea cycle disorders) will be excluded. Finally, to offer
some further sense of the commonality and phenotypic spectrum
associated with plasma potentiation of GABA.sub.AR function, this
activity will be quantified in a population-based sample of
subjects.
Inclusion/Exclusion Criteria
[0136] Patients complaining of daytime sleepiness/hypersomnia with
an Epworth Sleepiness Scale or >15 and who exhibit either
objective sleepiness (MSL<8 minutes), REM-sleep propensity
during their diagnostic evaluation, or treatment resistant
sleepiness will be recruited. Patients with DSM-IV Axis I disorders
such as depression, bipolar disease, serious medical co-morbidities
such as stroke, congestive heart failure, active cancer, severe
obstructive pulmonary disease, asthma, or uncontrolled type I or II
diabetes will be excluded. Any patient with a history of CNS
trauma, infection, or neurodegenerative condition will be excluded.
Patients with, treated or untreated sleep disordered breathing
(AHI>10) will also be excluded. Subjects with chronic health
conditions otherwise well-controlled with medication (e.g.,
hypertension, hypothyroidism, arthritis) will be allowed to
participate. Potential controls and subjects will be excluded if
they are ingesting psychoactive medications including
sedative-hypnotics, anxiolytics, mood-stabilizers presumed to act
via GABAcrgic mechanisms, neuroleptics, and anti-depressants. In
addition, given the known ability of steroids, methylxanthines, and
many antibiotics to allosterically modulate GABA.sub.AR, potential
subjects taking gluco- or mineralo-corticoids, theophylline, or
certain antibiotics will be excluded (at least while they are
ingesting these agents). Three mls each of plasma and urine will be
sent to MedTox Laboratories (Burlington, N.C.) to be analyzed for
classic BZDs and their metabolites by gas chromatography (GC) and
high performance liquid chromatography (HPLC). The specific agents
and respective reporting limits (i.e., thresholds for detection)
will include: Desalkylflurazepan (flurazepam metabolite) 10 ng/ml;
Nordiazepam 50 ng/ml; oxazepam 50 ng/ml, lorazepam 10 ng/ml,
diazepam 50 ng/ml, hydroxyflurazepam 10 ng/ml, temazepam 50 ng/ml,
chordiazepoxide, 50 ng/ml, midazolam 10 ng/ml, flurazepam 10 ng/ml,
alpha-hydroxyalprazolam 50 ng/ml, alprozolam, 13 ng/ml,
hydroxytriazolam 10 ng/ml, triazolam 10 ng/ml and estazolam 10
ng/ml. Additional, individual samples will be sent to NMS Labs
(Willow Grove, Pa.) for GC quantification of zolpidem (4-5 ng/ml),
HPLC quantification for zaleplon (3 ng/ml), and HPLC tandem mass
spectrometry (LC-MS/MS) quantification of eszopiclone.
[0137] In order to more carefully delineate a provisional diagnosis
of GRH and to provide additional potentially important biochemical
data relevant to the spectrum of hypersomnolence disorders such as
narcolepsy with cataplexy, CSF for hypocretin (HCRT-1) will be
assayed using a commercially available RIA (Orexin A RIA kit,
Phoenix Pharmaceuticals, Belmont, Calif.). This assay has an
intra-assay variability of <5%. Other recognized metabolic
causes of hypersomnolence will also be screened. For example,
disorders of the urea cycle and the catabolic enzymes for GABA
(e.g., GABA-transaminase and succinic semialdehyde dehydrogenase)
have been associated with lassitude and hypersomnia, albeit,
incompletely characterized by MSLT or ICSD-2. These must be ruled
out as a potential contributors to hypersomnia by assessing
arterial ammonia and urine and plasma organic and amino acids. The
latter analyses will be performed in a CLIA certified laboratory
employing ion exchange chromatography.
Flumazenil Infusion
[0138] The subject will be instructed in the proper use and care of
the Actiwatch and completion of the sleep/wake diary within one
month following the lumbar puncture. The subject will complete all
study inventories (see below), and within two weeks, will undergo
48-hours of ambulatory polysomnography (see below). Five
home/clinic visits will be made during that period to hook-up the
subject, check the integrity of the electrodes, and to disconnect
the subject from the equipment. Within two weeks, subjects will be
scheduled for a 24-hour admission to the ACTSI and after a
full-night of recorded sleep receive saline (control), 0.5 mg, and
2.0 mg flumazenil at roughly 2.5 hour intervals while undergoing
continuous EEG monitoring and hourly monitoring of vital signs. All
subjects will complete a baseline Stanford Sleepiness Scale (SSS)
and Psychomotor Vigilance Task (PVT) (see below) which will be
repeated at 10, 30, 60, 90, 120, and 150 minutes following each
injection.
Polysomnographic Recording
[0139] Diagnostic nocturnal polysomnography (NPSG) and subsequent
daytime testing will b recorded with the Embla digital PSG system
(Medcare Corporation, Buffalo, N.Y.) with a sampling rate of 512
Hz, as this allows for Fast Fourier Transform of EEG signals. The
system employs a Windows XP platform and uses proprietary software
(Somnologica Science). Spectral analyses, or the Welch method of
FFT smoothing, that provide an average of several FFT's will be
useful to more fully characterize the signature of fingerprint of
endogenous GABAAR like activity given the known effects of GABA on
corticothalamic excitability as manifest in the EEG.
Multiple Sleep Latency Testing
[0140] Daytime sleepiness will be objectively assessed with the
MSLT, which is a clinical and research tool that uses standard
guidelines for testing and scoring. Sleep latencies and number of
REM onsets will be determined according to standard criteria. The
MSLT displays excellent interrater and intrarater reliabilities for
sleep latency (coefficients of 0.81-0.88) and REM onset scores
(kappa coefficients of 0.78-0.88). The stability of the MSL on
repeat testing in known narcoleptics is high (r=0.81, p<0.01)
with test-retest reliability improving vis a vis diagnostic
certainty with the additional ICSD-2 requirement of two or more
sleep onset REM-sleep periods (Kappa=0.95; variance=0.08; Z=2.33;
p<0.05).
Blood Collection
[0141] Thirty mL of venous blood will be drawn for: 1)
lymphoblastoid cell line generation to establish a permanent source
of DNA and cells for future investigations; 2) clear plasma
aliquoted and frozen for future analytic studies; and 3) buffy coat
and purified DNA banked for future genetic studies. The DNA will be
purified from 200 .mu.l of buffy coat using a Qiagen kit protocol
(Qiagen, Valencia, Calif.). The cell lines, buffy coat, plasma and
purified DNA will all be labeled with barcode compatible labels and
banked at -80 .degree. C. within CRIN dedicated resources.
De-identified DNA from all participants will be assigned a 6-digit
reference number. Aliquots with this reference number will be
forwarded to a laboratory for testing.
Collection of Lumbar CSF
[0142] All patients and family members (afflicted, unafflicted)
will provide Informed Consent for collection of cerebrospinal fluid
(CSF). Lumbar punctures (LP) will be performed under sterile
conditions using standard procedures, subcutaneous administration
of 4% lidocaine, and collection of 15-20 ml CSF with a 22 gauge
spinal needle inserted at L3/L4 or L4/L5. One ml fractions will be
labeled with the participants 6-digit reference number and frozen
immediately upon dry ice and then stored at -80 degrees Centigrade
for future analyses. LPs will be performed between 0830 and 0930
after completion of the first MSLT nap. This will obviate the need
to control for subsequent daytime activity levels and extent of
food intake which hypothetically could affect endogenous activity
at the GABA.sub.AR.
Questionnaire Assessments
[0143] Administered questionnaires serve as both screening
instruments and as predictors in the regression models described
below. Subjective sleepiness as a trait variable will be assessed
using the Epworth Sleepiness Scale (ESS) and overall quality of
sleep will be assessed using the Pittsburgh Sleep Quality Index
(PSQI). State and trait anxiety will be assessed with the
State-Trait Anxiety Inventory (STAI) and mood will be assessed with
the Beck Depression Inventory (BDI). These are all standardized
scales with population based norms. Data on functional impairments
related to sleepiness using the Functional Outcomes of Sleep
Questionnaire (FOSQ) will also be collected. The FOSQ is a
self-report measure designed to assess the impact of excessive
sleepiness on multiple activities of daily living.
Actigraphy
[0144] The Actiwatch wrist-worn monitor, manufactured by
Respironics (Murrysville, Pa.), will be used to assess
characteristic sleep durations in patients for two weeks prior the
infusion protocol. Patients will also be provided a sleep log to
keep during the two weeks to generate data on timing of sleep and
napping.
Ambulatory Polysomnography
[0145] Ambulatory PSG over a 48 hour period will be conducted using
the same equipment cited above which can be adapted for this use to
document the degree of `hypersomnia` suggested by actigraphy. Only
EEG, EOG, submental EMG lead, ECG, and pulse oxymetry will be
conducted. No limb leads will be used for patient safety reasons.
Sleep stages, episodes of desaturation, and ECG will be
analyzed.
Psychomotor Vigilance Task (PVT)
[0146] The Psychomotor Vigilance Task (PVT) provides a sensitive
marker of minute-to-minute fluctuations in alertness during the
flumazenil infusion protocol. The PVT is a 10-minute, simple,
portable reaction time test (finger button press response to light)
designed to evaluate the ability to sustain attention and respond
in a timely manner to salient signals. Data to be generated
include: 1) frequency of lapses, which refer to the number of times
the subjects fail to respond to the signal or fail to respond in a
timely manner; 2) the median reaction time (RT) over the 10-minute
interval. Additionally, as a measure of state sleepiness, the
Stanford Sleepiness Scale (SSS) will be administered immediately
prior to each trial. The PVT/SSS will be administered at 10, 30,
60, 90, 120, and 150 minutes following each infusion of saline or
flumazenil.
Statistical Analysis/Power Calculations
[0147] The relationship between the extent of GABA.sub.A
potentiation and behavioral outcomes will be examined using
regression models. Separate models will be run for each type of
specimen source (e.g., CSF and plasma derived markers of
potentiation). A simple bivariatc relationship between the two
measures of GABA potentiation using correlational models will be
examined, relying on non-parametric alternatives (Spearman) should
the measure present with a non-normal distribution. The extent of
GABA potentiation in the Baseline condition among patients will be
predicted using predictors such as standard demographics (e.g.,
gender, age), psychometrics (e.g., STAI, BDI), recent sleep history
(e.g., cumulative sleep over the preceding 2 weeks as measured with
actigraphy, daytime naps on sleep log), and laboratory-based
measurements of nocturnal sleep (e.g., FFT derived relative delta
power or beta power) or daytime alertness (e.g., MSLT sleep
latency, PVT-derived median reaction time). Because multiple
measurements in each domain and the sheer number of domains
increase the likelihood of Type I error, such error will be
minimized by first carefully examining the intercorrelations among
measures within each domain. Substantial collinearity is expected
to be among many of these. For example, trait anxiety (STAI) and
depressed mood (BDI) are likely to be highly intercorrelated, as
are Baseline PVT median reaction times and MSLT-defined sleep
latency. The specific approach to deriving variables to employ in
the regression might include a selection of a single variable from
each domain chosen on the basis of a more normally distributed
range of scores across subjects. Alternatively, the data reduction
techniques can be relied on such as principal components analyses
(PCA) to determine a single measure in each domain that best
captures variance within that domain. Thus, a single score (or
composite score, if PCA was used) from each domain will be entered
in the regression predicting potentiation. Based on the data
presented in FIG. 3, large effects will be displayed. GABA
potentiation differences between controls and patients will be
substantial (d=3.095). Assuming effects of this size are maintained
in the work proposed here, and assuming a 2-tailed alpha of 0.01,
an N of 60 cases would yield 99% power to reject the null of
hypothesis of the contribution for any single domain to GABA
potentiation. It is fully recognized that, in multivariate models
encompassing each of the five domains listed above, actual power
might be somewhat reduced because of the contribution of multiple
variables to the prediction. Nonetheless, given the substantial
effects observed in FIG. 3, sufficient power to understand how
different variables may predict potentiation when considered
simultaneously should be retained. Regression models will also be
used to determine what factors may predict change in GABA
potentiation under flumazenil infusion. Each patient's Baseline
potentiation level (measured under saline infusion) will be forced
and it is determined whether either low or high dosage of
flumazenil predicts change subsequent to infusion. Domain variables
selected for entry into these models are limited only to those
shown to relevant to the prediction of Baseline potentiation, thus
saving degrees of freedom whenever possible. This modeling allows
the determining of the extent that other variables (demographic,
recent sleep history, etc) may have to moderate or mediate the
GABA-mediated response to flumazenil. The behavioral response to
flumazenil (performed separately by dose) will also be examined,
defined as the mean of the median RTs for the 4 PVT measurements
closest to point of infusion. Each patient's Baseline median RT
(mean of 4 Baseline/saline measurements) (see FIGS. 6, 8, 10, and
12) and Baseline GABA potentiation levels will be forced initially
in these regressions, followed by entry of significant predictors
of Baseline potentiation found in the analyses described above.
[0148] Further, the plasma-measured GABA potentiation will be
examined as the dependent variable among 227 individuals, all of
whom will have received two nights of PSG and an intervening day of
MSLT. The hypersomnolence demonstrated by the index cases will
represent a more extreme form of a continuous trait present in
segment of the population generally. To that end, the initial
review of the data indicated that 58 had mean MSLT-defined sleep
latencies of less than 5 minutes that could not be accounted for by
known sleep disorders. If the MSLTs across all 227 cases show a
bimodal distribution, the analyses would be limited to only those
cases at the extremes (e.g., mean latencies<5 minutes versus
mean latencies>15 minutes) and employing an ANCOVA approach.
However, the distribution of mean sleep latencies is more
continuous and, as is often the case of studies using MSLT, sharply
skewed to the right. In this case, log transforms are performed on
these mean values before proceeding. The overall approach will be
similar to those described above, though they are somewhat more
limited by the range of variables collected.
Example 11
Electroencephalography (EEG) Power Spectrum Analysis
[0149] Quantitative analysis of delta (0.4-3.99 Hz), theta
(4.00-7.99 Hz), alpha (8.00-12.99 Hz) and beta power (13.00-16.00
Hz) was obtained from EEG spectral analyses of the C4-M1 electrode.
Manual and automated artifact removal methods were utilized prior
to EEG spectral analyses to prevent erroneous results. Spectral
analyses were conducted utilizing the computational software
program MATLAB v 7.1. The Welch method of FFT smoothing was
employed to obtain power spectrum values from an average of several
FFT's. The FFT contained a minimum of 512 data samples with a 50%
overlap moving window of the subsequent 512 data samples. EEG data
collected at 200 Hz provided an FFT window comprised of
approximately 2.56 seconds of data. Parameters for Welch spectral
analyses were user adjustable within the MATLAB program such that
user defined frequency bands for specific frequency resolutions
were obtained. Power values for the defined frequency bands were
represented by mV.sup.2/Hz (microvolts squared divided by
hertz).
[0150] The EEG power spectrum analyses for two subjects (DS122 and
DT74) were obtained (see FIGS. 4 and 5, respectively). Table 1
provides the corresponding sampling frequency and FFT window size
for each patient data set. There was a spectral change
approximately five minutes after intravenous infusion of 2.0 mg
flumazenil that manifested as diminution of delta frequencies and
emergence of higher EEG frequencies emblematic of improved
vigilance/arousal.
TABLE-US-00009 TABLE 1 Patient Sampling Frequency FFT size DS 500
Hz 1024 DT 200 Hz 512
[0151] Table 2 displays mean relative band power results obtained
from EEG power spectrum (i.e., delta, theta, alpha, beta) analyses
of subject ED102 and DS122 for each clinical treatment (i.e.,
saline, 0.5 mg flumazenil, and 2.0 mg flumazenil). Ten minute data
segments were selected 30 minutes following each clinical treatment
and were analyzed via a three second processing window to obtain
the relative power spectrum results provided in Table 2.
TABLE-US-00010 TABLE 2 Delta Theta Alpha Beta Gamma Subject
Treatment Power Power Power Power Power ED102 Saline 0.5314 0.1824
0.1090 0.1207 0.0565 ED102 0.5 mg 0.5199 0.1755 0.1017 0.1387
0.0642 flumazenil ED102 2.0 mg 0.4820 0.1479 0.0866 0.1803 0.1032
flumazenil D5122 Saline 0.4122 0.2777 0.1966 0.1011 0.0124 D5122
0.5 mg 0.3515 0.2995 0.2183 0.1165 0.0142 flumazenil D5122 2.0 mg
0.3128 0.3776 0.1798 0.1141 0.0157 flumazenil
Example 12
Psychomotor Vigilance Task (PVT)
[0152] The dose and temporal reversibility of the sleepiness of
patients to intravenous flumazenil were determined employing the
PVT/SSS paradigm as described in Example 10. Five hypersomnic
patients demonstrated dose-dependent improvements in vigilance and
subjective alertness with intravenous delivery of flumazenil as
shown in Table 3.
TABLE-US-00011 TABLE 3 Baseline/Saline % Stanford GABA Reaction
Sleepiness FLU 0.325- 0.5 mg FLU 1.2 - 2 mg potenti- time (RT)
Scale RTs in RTs in Case ation in ms lapses (SSS) ms lapses SSS ms
lapses SSS 74 200 +/- 432.3 +/- 31.0 6 236.8 +/- 0.7 4 207 +/- 0.7
3 21.7 84.8 47.7 4.97 99 160 +/- 285.5 +/- N/A 6 225.5 +/- N/A 3
255.5 +/- N/A 1 9.2 13 6.3 2.3 102 189 +/- 1962 +/- 16.4 6 1642 +/-
3.1 3 363.2 +/- 3.8 4 24.3 1478 1036 38.2 122 149 +/- 369.5 +/-
17.2 5 297.8 +/- 0.6 1 269.3 +/- 0.6 1 20.4 78.9 16.2 6.8 124 58.5
+/- 327.8 +/- 5.8 6 259.8 +/- 1.3 2 250 +/- 1.0 2 3.5 22.96 3.8
1.2
[0153] Administration of flumazenil (FLU) was associated with
dramatic and substantial improvement in reaction time performance
on the PVT and subjective alertness on the SSS. Relative to
baseline, median RTs decreases at low (t=2.56, p=0.063) dose, and
number of lapses decreased both at low (t=3.03, p=0.056) and high
(t=3.51, p=0.039) dose. When compared to the worst Baseline measure
for each case, SSS showed significant improvement for both low
(t=8.55, p=0.001) and high (t=7.06, p=0.002) dose. Raw histograms
for cases 74, 102, 122, and 124 displaying baseline PVT performance
and PVT performance after 2.0 mg are shown in FIGS. 6-17.
Example 13
Clinical Study of GABA.sub.A Receptor Mediated Hypersomnia
(GRH)
[0154] Patient AS99 with a diagnosis of "narcolepsy" and restless
legs syndrome (RLS) complained of "craving" sleep, and of long,
unrefreshing sleep periods. Polysomnography revealed periodic leg
movements (31 per hour), but was otherwise normal (TST=444 min). A
mean sleep nap latency of 2.6 minutes absent intrusion of REM sleep
confirmed pathological sleepiness and a diagnosis of idiopathic
hypersomnia. Patient AS99's examination was normal with a BMI of
22.3, and urine drug screens (repeated.times.3), serum ammonia
(n=2), thyroid functions (n=2), complete blood counts (n=5),
vitamin B12, and comprehensive metabolic screens (n=2) were normal.
Ferritin (23 ng/ml) and % transferrin saturation (13%) were low
with otherwise normal serum iron. The RLS was successfully treated
with iron supplementation and pramipexole;
[0155] however, hypersomnia persisted despite maximum doses of
dextroamphetamine (60 mg) in combination with modafinil (800 mg).
Actigraphy confirmed resolution of RLS/PLMs, yet revealed erratic
rest-activity cycles with sleep periods varying from 5 to 10 hours
per night. Patient AS99's condition progressed and weight decreased
(BMI=20), and patient AS99 developed anxiety and hypertension
requiring treatment with metoprolol attributed to supratherapeutic
doses of psychostimulants. Affective and factitious disorders were
ruled out by two independent psychiatric assessments. Weaned off
all medications, CSF was obtained and hypocretin determined to be
high-normal (401 pg/ml) thus ruling out a diagnosis of narcolepsy.
Electrophysiological analysis for bioactivity in CSF and plasma
revealed the presence of a positive allosteric modulator of the
GABA.sub.A receptor reversible with the competitive BZD antagonist
flumazenil. The dose and temporal reversibility of sleepiness to
intravenous flumazenil were then determined employing the PVT/SSS
paradigm. The rest-activity cycles of patient AS99 improved with
chronic sublingual flumazenil administration (see FIGS. 18a and
18b). The sleep, mood, sleepiness, and quality of life improved
dramatically and are sustainable with sublingual flumazenil in
patient AS99 (see Table 4) as shown through the Pittsburgh Sleep
Quality Index, Beck Depression Inventory, Epworth Sleepiness Scale,
Functional Outcomes of Sleep, and SF-36 Health Survey.
TABLE-US-00012 TABLE 4 February 22.sup.nd March 31.sup.st April
28.sup.th Variable (pre-treatment) (1 month post) (2 months post)
Pittsburgh Sleep 4 2 1 Quality Index Beck Depression 7 2 2
Inventory Epworth Sleepiness 18 3 3 Scale Functional Outcomes of
Sleep General Productivity 6 23 24 Social Activity 12 24 24
Activity 3.6 19.6 21.3 Vigilance 8 22 24 Total 29.5 88.6 93.3 Total
Mean 7.4 22.1 23.3 SF-36 Health Survey Physical 39.5 54.4 66.2
Mental 49.7 64.0 56.2
[0156] Patient AS99 continued use of sublingual flumazenil for 9
months with positive results. When prescribed clarithromycin,
patient AS99 suddenly developed 4 nights of insomnia that reversed
promptly upon discontinuation. Clarithromycin is an antibiotic with
a high incidence of hypomania/insomnia associated with its use, and
it functions as a negative allosteric modulator at GABA.sub.A
receptors.
Example 14
Identification of Substance Causing Potentiation at GABA.sub.A
Receptors
[0157] Several studies were performed in order to identify the
substance accounting for potentiation at GABA.sub.A receptors. It
was determined that adenosine is not the substance, as several
concentrations (1 mM, 100 .mu.M, and 10 .mu.M) of adenosine in
artificial CSF exhibited no activity at GABA.sub.A receptors.
[0158] In addition, it was shown that the substance accounting for
potentiation at GABA.sub.A receptors is not a neurosteroid.
Cerebrospinal fluid from four hypersomnic cases were tested in
duplicate by quantitative HPLC for endogenous neuroactive GABAergic
steroids (i.e., neurosteroids). The controls revealed no
differences in the levels of pregnenolone, DHEA,
3.alpha.,5.alpha.-THP, 3.alpha.,5.beta.-androstandiol,
3.alpha.,5.alpha.-androsterone, and 3.alpha.,5.beta.-androsterone.
Controls and subjects exhibited undetectable quantities of
3.alpha.,5.beta.-THP, 3.alpha.,5.alpha.-THDOC,
3.alpha.,5.beta.-THDOC, and 3.alpha.,5.alpha.-androstandiol.
[0159] Further, it was shown that the substance accounting for
potentiation at GABA.sub.A receptors has a molecular weight less
than 3,000. Pooled CSFs from confirmed GRH subjects versus controls
were fractionated with filters having approximately 3,000 molecular
weight cut-off. Bioactivity at GABA.sub.A receptors in both samples
was completely retained within the smaller molecular weight
fractions.
[0160] It was also found that the substance accounting for
potentiation at GABA.sub.A receptors may act at a non-traditional
benzodiazepine site (see FIGS. 19a and 19b). In a whole cell patch
clamp current recording from a cell expressing human
.alpha.1.beta.2.gamma.2s receptors, the response to 10 .mu.M GABA
is potentiated by the co-application of a 50% CSF, indicating the
presence of a positive allosteric modulator (see FIG. 19a). A
recording from a different cell expressing the benzodiazepine
insensitive subunit .alpha.1(H102R) shows that the enhancement
persists (see FIG. 19b). Not to be bound by theory, this data
indicates that the somnogenic compound is not a classical
benzodiazepine, or does not act conventionally at the classical
high-affinity benzodiazepine binding site on the GABA.sub.A
receptor.
Example 15
Patch Clamp Analysis of CSF Bioactivity
[0161] Patch clamp analyses of CSFs from non-human primates, drawn
from animals under different conditions, was also used to identify
the somnogenic GABAergic substance. In this experiment, CSF was
drawn from 4 monkeys (Canjala, Santiaga, Penelope, and Cricket) at
3 different time points. 1) early morning, 2) late afternoon and 3)
very late evening, having been kept awake throughout (when they
would normally be asleep). A whole cell patch clamp current was
recorded (as described in Example 1), and the response to 10 .mu.M
GABA co-administered with primate CSF was determined. The
bioactivity of the CSF is expressed as a percent increase, or
potentiation, of the control current by the CSF (see Table 5). The
first two columns show the normal diurnal variation of this
somnogenic compound. It appears that this substance waxes and wanes
in animals as it does in humans during the normal day night cycle.
More interestingly, in 2 of the 4 animals, the bioactivity
increased still further after the animals were "wake-extended".
These promising results indicate that under sleep deprived
conditions, humans may also benefit from flumzenil or other
GABAergic therapy to relieve the symptoms of fatigue they
experience from the accumulation of this somnogenic compound.
TABLE-US-00013 TABLE 5 Morning Evening Wake Enhanced Canjala 79.9
.+-. 2.7 76.6 .+-. 4.6 73.4 .+-. 4.7 Santiaga 49.0 .+-. 2.0 58.7
.+-. 0.8 64.9 .+-. 1.7 Penelope 54.6 .+-. 3.5 77.1 .+-. 0.5 98.4
.+-. 23.1 Cricket 51.3 .+-. 0.7 58.1 .+-. 4.2 63.9 .+-. 6.9
[0162] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *