U.S. patent application number 17/249123 was filed with the patent office on 2021-07-08 for treatments for migraine and related disorders.
This patent application is currently assigned to The University of Chicago. The applicant listed for this patent is The University of Chicago. Invention is credited to Yelena GRINBERG, Marcia KRAIG, Richard KRAIG, Heidi MITCHELL, Aya PUSIC.
Application Number | 20210205414 17/249123 |
Document ID | / |
Family ID | 1000005447966 |
Filed Date | 2021-07-08 |
United States Patent
Application |
20210205414 |
Kind Code |
A1 |
KRAIG; Richard ; et
al. |
July 8, 2021 |
TREATMENTS FOR MIGRAINE AND RELATED DISORDERS
Abstract
Embodiments are directed to compositions and methods of treating
migraine and related neurological disorders. In certain aspects,
methods and compositions are for reducing cortical spreading
depression and/or suppressing the neurochemical basis for chronic
and acute migraine events, and provide methods and pharmaceutical
compositions related to both acute and preventive therapies for
migraine events and related headaches.
Inventors: |
KRAIG; Richard; (Chicago,
IL) ; PUSIC; Aya; (Chicago, IL) ; MITCHELL;
Heidi; (Dubuque, IA) ; GRINBERG; Yelena;
(Riverside, CA) ; KRAIG; Marcia; (Chicago,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Chicago |
Chicago |
IL |
US |
|
|
Assignee: |
The University of Chicago
Chicago
IL
|
Family ID: |
1000005447966 |
Appl. No.: |
17/249123 |
Filed: |
February 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16551032 |
Aug 26, 2019 |
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17249123 |
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15791802 |
Oct 24, 2017 |
10391150 |
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16551032 |
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15171327 |
Jun 2, 2016 |
9827294 |
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15791802 |
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14234276 |
May 27, 2014 |
9399053 |
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PCT/US2012/047683 |
Jul 20, 2012 |
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15171327 |
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61510673 |
Jul 22, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/217 20130101;
A61K 9/0014 20130101; A61K 45/06 20130101; A61K 9/0043 20130101;
A61K 38/28 20130101; A61K 38/30 20130101; A61K 31/00 20130101; A61K
38/18 20130101; A61K 38/2073 20130101 |
International
Class: |
A61K 38/21 20060101
A61K038/21; A61K 38/20 20060101 A61K038/20; A61K 38/28 20060101
A61K038/28; A61K 38/30 20060101 A61K038/30; A61K 31/00 20060101
A61K031/00; A61K 45/06 20060101 A61K045/06; A61K 38/18 20060101
A61K038/18; A61K 9/00 20060101 A61K009/00 |
Goverment Interests
STATEMENT OF FEDERAL FUNDING
[0002] This invention was made with government support under grant
number NS019108 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1.-46. (canceled)
47. A method for treating a patient subject to seizures comprising
administering to the patient an effective amount of a composition
comprising interleukin-11 (IL-11), wherein treating excludes
administering a cytokine to the patient.
48. The method of claim 47, wherein the composition is administered
intranasally.
49. The method of claim 47, wherein the composition is administered
multiple times.
50. The method of claim 47, wherein the composition is administered
on multiple days.
51. The method of claim 47, wherein the composition is administered
on multiple consecutive days.
52. The method of claim 47, wherein the patient is suffering from a
seizure when the composition is administered.
53. The method of claim 47, wherein the patient is not suffering
from a seizure when the composition is administered.
54. The method of claim 47, wherein the patient is administered the
composition once a day.
55. The method of claim 47, wherein the composition is administered
to the patient's brain cells.
56. The method of claim 47, wherein the composition comprises
between about 0.1 ng and about 2.0 g of IL-11.
57. A method for reducing spreading depression (SD) susceptibility
in a patient in need thereof comprising administering to the
patient an effective amount of a composition comprising IL-11,
wherein reducing SD susceptibility excludes administering a
cytokine to the patient.
58. The method of claim 57, wherein the composition is administered
intranasally.
59. The method of claim 57, wherein the composition is administered
multiple times.
60. The method of claim 57, wherein the composition is administered
on multiple days.
61. The method of claim 57, wherein the patient needs protection
from demyelination.
62. The method of claim 57, wherein the patient has a demyelinating
disease.
63. The method of claim 57, wherein the patient is administered the
composition once a day.
64. The method of claim 57, wherein the composition is administered
to the patient's brain cells.
65. The method of claim 57, wherein the composition comprises
between about 0.1 ng and about 2.0 g of IL-11.
66. A method for reducing spreading depression (SD) susceptibility
in a patient having a demyelinating disease comprising intranasally
administering multiple times on multiple days to the patient a
composition comprising interleukin-11 (IL-11), wherein reducing
spreading depression excludes administering a cytokine to the
patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/551,032 filed Aug. 26, 2019, which is a
continuation of U.S. patent application Ser. No. 15/791,802 filed
Oct. 24, 2017, now U.S. Pat. No. 10,391,150, which is a
continuation of U.S. patent application Ser. No. 15/171,327 filed
Jun. 2, 2016, now U.S. Pat. No. 9,827,294, which is a continuation
of U.S. patent application Ser. No. 14/234,276 filed Jan. 22, 2014,
now U.S. Pat. No. 9,399,053, which is a national phase application
under 35 U.S.C. .sctn. 371 of International Application No.
PCT/US2012/047683 filed Jul. 20, 2012, which claims priority to
U.S. Application No. 61/510,673 filed on Jul. 22, 2011. The entire
contents of each of the above-referenced disclosures are
specifically incorporated herein by reference without
disclaimer.
FIELD OF THE INVENTION
[0003] The present invention relates generally to medicine and
neurology. In particular, embodiments are directed to the treatment
of migraine and related neurological disorders.
BACKGROUND OF THE INVENTION
[0004] Migraine headache is a complex, recurrent disorder that is
one of the most common complaints in medicine. In the United
States, more than 30 million people have one or more migraine
headaches per year. Approximately 75% of all persons who experience
migraines are women.
[0005] Migraine was previously considered a vascular phenomenon
that resulted from intracranial vasoconstriction followed by
rebound vasodilation. Currently, however, the neurovascular theory
describes migraine as primarily a neurogenic process with secondary
changes in cerebral perfusion. The neurovascular theory holds that
a complex series of neural and vascular events initiates
migraine.
[0006] The theory of cortical spreading depression (CSD) has been
advanced to explain the neurologic mechanism of migraine with aura.
CSD is a well-defined wave of initial neuronal excitation followed
by neuronal silence and then again excitation that returns to
normal in cortical gray matter areas that spreads from its site of
origin. This transient cellular depolarization is understood to
cause the primary cortical phenomenon or aura phase; in turn, it
activates trigeminal fibers causing the headache phase. Similar
changes are understood to cause pain from migraine with and without
aura. CSD is a wave of electrophysiological hyperactivity followed
by a wave of inhibition, most often noted in the visual cortex. The
scintillating scotoma (visual aura) of migraine in humans may be
related to the neurophysiologic phenomenon termed the spreading
depression of Leao.
[0007] Migraine treatment involves acute (abortive) and preventive
(prophylactic) therapy. Patients with frequent attacks may require
both. Acute treatments are intended to stop or prevent the
progression of a headache or reverse a headache that has started.
Preventive treatment, which is given even in the absence of a
headache, is intended to reduce the frequency and severity of the
migraine attack, make acute attacks more responsive to abortive
therapy, and perhaps also improve the patient's quality of life.
There remains a need for additional therapies for treating migraine
or other neurological disorders.
SUMMARY OF THE INVENTION
[0008] Methods and compositions are provided based on experimental
data showing that they can achieve a desired physiological effect
that may be implemented to treat migraine patients--patients who
have previously suffered from a migraine and who are at risk for
suffering from future migraines. Treatment of a migraine patient
will be understood to reduce or limit the frequency, severity,
and/or duration of migraines. It is also contemplated that methods
and compositions can also be implemented as discussed in
embodiments below to effect prevention of migraines. In certain
aspects, methods and compositions inhibit spreading depression and
migraine events. Such methods and compositions are contemplated in
some embodiments for use on a human subject.
[0009] In some embodiments, methods are provided for treating a
migraine patient comprising administering to the patient
interleukin-11 (IL-11) or a composition comprising IL-11. In other
embodiments there are methods for treating chronic or recurrent
migraines in a patient comprising administering to the patient
IL-11 or a composition comprising IL-11.
[0010] In some embodiments, methods are provided for treating a
migraine patient comprising administering to the patient an
insulin-like growth factor receptor (IGFR) inducer or a composition
comprising an IGFR inducer. In certain embodiments, the IGFR
inducer is IGF-1 or insulin. In specific embodiments, the IGFR
inducer is IGF-1. In other embodiments, the IGFR inducer is
insulin. In further embodiments there are methods for treating
chronic or recurrent migraines in a patient comprising
administering to the patient IGF-1 or a composition comprising
IGF-1. In additional embodiments, there are methods for treating a
migraine patient comprising administering to the patient insulin or
a composition comprising insulin. In other embodiments there are
methods for treating chronic or recurrent migraines in a patient
comprising administering to the patient insulin or a composition
comprising insulin. In particular embodiments, there are methods
for treating a migraine patient comprising administering to the
patient an IGFR inducer or a composition comprising an IGFR
inducer, wherein the IGFR inducer is a polypeptide. In other
embodiments, the IGFR inducer is not insulin. Moreover, it is
specifically contemplated that when insulin is included in some
embodiments for treating migraine or a migraine patient that the
insulin is not ingested or administered subcutaneously.
[0011] Additional aspects include methods for treating a migraine
patient comprising administering to the patient interferon-gamma
(IFN-.gamma.) or a composition comprising IFN-.gamma.. In other
embodiments there are methods for treating chronic or recurrent
migraines in a patient comprising administering to the patient
IFN-.gamma. or a composition comprising IFN-.gamma..
[0012] In further embodiments there are methods for treating a
migraine patient comprising administering to the patient IL-11,
IFN-.gamma., and/or IGF-1. In additional aspects, there are methods
for treating a migraine patient comprising administering
intranasally to brain cells of the patient an effective amount of a
composition comprising IL-11, IFN-.gamma., IGF-1, and/or insulin.
In other embodiments there are methods for treating chronic or
recurrent migraines in a patient comprising administering
intranasally to the patient an effective amount IL-11, IFN-.gamma.,
IGF-1, and/or insulin.
[0013] In certain embodiments, the patient is suffering from
symptoms of a migraine headache when the composition is
administered. A migraine typically includes a unilateral, throbbing
moderate to severe headache. Other symptoms of migraines include,
but are not limited to, nausea, aura, blurred vision, delirium,
nasal stuffiness, diarrhea, tinnitus, polyuria, pallor, sweating,
localized edema of the scalp or face, scalp tenderness, prominence
of a vein or artery in the temple, stiffness and tenderness of the
neck, impairment of concentration and mood, or cold and moist
feeling in appendages.
[0014] In further embodiments, the patient is a chronic or
recurrent migraine patient, which means the patient experiences
headaches more than half the time, for 15 days or more in a month,
for at least three months. In certain embodiments, the patient has
tried one or more acute treatment options such as simple
analgesics, non-steroidal anti-inflammatory drugs (NSAIDS),
triptans, and ergotamines and has not experienced significant pain
relief.
[0015] It is contemplated that the patient is administered an
amount that is considered effective or has been effective in other
patients for achieving a beneficial effect with respect to
migraines or symptoms of a migraine. An effective amount of IL-11,
IFN-.gamma., IGF-1 and/or insulin is administered to or by a
patient.
[0016] The rationale for phasic delivery of the proposed
therapeutic agents for migraine begins to be illustrated in FIG.
1B(3-4). In general, this means that patient exposure to the
therapeutic agents is not constant. Instead, agents are
administered to initiate an adaptive response (3), which is then
allowed to develop (4), in the absence of continued agent exposure,
before the agent is delivered again. Exemplary evidence supporting
the advantage of this approach is provided for IGF-1 using
hippocampal slice cultures (FIGS. 5, 8), where for seven days,
IGF-1 was administered every 12 hours and removed for successive
intervening 12 hours before testing SD susceptibility. This
afforded maximal and continued protection against CSD. Also,
maximal IFN-.gamma.-based protection against CSD was seen when
IFN-.gamma. was pulsed onto slice cultures for only 12 hours once a
week (FIGS. 5, 15, 16). In each case this pattern of agent delivery
was chosen to mimic phasic effects of environmental enrichment (EE)
that consists of exercise-rest-exercise cycles.
[0017] In some methods, the composition is administered to the
patient intranasally. In certain embodiments the composition is
administered to the patient's brain cells or brain tissue. In
additional embodiments, the composition is administered to
microglia in the patient's brain. It is specifically contemplated
that neurons, microglia, oligodendrocytes or astrocytes in the
patient's brain are contacted with a composition comprising IL-11,
IFN-.gamma., IGF-1, and/or insulin.
[0018] The IL-11, IFN-.gamma., IGF-1, or insulin is purified and/or
isolated in embodiments described herein. These polypeptides may be
recombinantly produced or they may be synthetic.
[0019] In some embodiments, the composition is a liquid. In other
embodiments, the composition is a gel or a powder. It is
specifically contemplated that the composition may be a liquid that
is provided to the patient as a mist.
[0020] Methods may involve administering a composition containing
about, at least about, or at most about 0.01, 0.02, 0.03, 0.04,
0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,
2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,
3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,
4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,
6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,
7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,
8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,
9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5,
15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115,
120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,
185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,
250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,
315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375,
380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460,
470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570,
575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675,
680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780,
790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890,
900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000,
1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100,
2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200,
3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300,
4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000,
10000 nanograms (ng), micrograms (mcg), milligrams (mg), or grams
of IL-11, IFN-.gamma., IGF-1, and/or insulin, or any range
derivable therein.
[0021] Alternatively, embodiments may involve providing or
administering to the patient or to cells or tissue of the patient
about, at least about, or at most about 0.01, 0.02, 0.03, 0.04,
0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,
2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,
3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,
4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,
6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,
7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,
8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,
9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5,
15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115,
120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,
185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,
250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,
315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375,
380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460,
470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570,
575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675,
680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780,
790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890,
900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000,
1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100,
2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200,
3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300,
4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000,
10000 nanograms (ng), micrograms (mcg), milligrams (mg), or grams
of IL-11, IFN-.gamma., IGF-1, and/or insulin, or any range
derivable therein, in one dose or collectively in multiple doses.
In some embodiments, the composition comprises between about 0.1 ng
and about 2.0 g of IL-11, IFN-.gamma., IGF-1, and/or insulin. The
above numerical values may also be the dosage that is administered
to the patient based on the patient's weight, expressed as ng/kg,
mcg/kg, or mg/kg, and any range derivable from those values.
[0022] Alternatively, the composition may have a concentration of
IL-11 that is 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,
1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9,
4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,
5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,
6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,
7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,
9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5,
12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0,
17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,
155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215,
220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280,
285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345,
350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420,
425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520,
525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625,
630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730,
740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840,
850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950,
960, 970, 975, 980, 990, 1000 ng/ml, .mu.g/ml, mg/ml, or g/ml, or
any range derivable therein.
[0023] If a liquid, gel, or semi-solid composition, the volume of
the composition that is administered to the patient may be about,
at least about, or at most about 0.01, 0.02, 0.03, 0.04, 0.05,
0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,
2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4,
3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,
4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,
6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,
7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,
8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9,
10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0,
15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 microliters (.mu.l)
or milliliters (ml), or any range derivable therein. In certain
embodiments, the patient is administered up to about 10 ml of the
composition.
[0024] The amount of IL-11, IFN-.gamma., IGF-1, and/or insulin that
is administered or taken by the patient may be based on the
patient's weight (in kilograms). Therefore, in some embodiments,
the patient is administered or takes a dose or multiple doses
amounting to about, at least about, or at most about 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,
3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4,
4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,
5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,
7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3,
8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6,
9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5,
14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0.
19.5, 20.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105,
110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,
175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235,
240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300,
305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365,
370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441,
450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550,
560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660,
670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770,
775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875,
880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980,
990, 1000 .mu.g/kilogram (kg) or mg/kg, or any range derivable
therein.
[0025] The composition may be administered to (or taken by) the
patient 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20 or more times, or any range derivable therein, and they
may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1, 2, 3,
4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12 months, or any range derivable therein. It is
specifically contemplated that the composition may be administered
once daily, twice daily, three times daily, four times daily, five
times daily, or six times daily (or any range derivable therein)
and/or as needed to the patient. Alternatively, the composition may
be administered every 2, 4, 6, 8, 12 or 24 hours (or any range
derivable therein) to or by the patient. In some embodiments, the
patient is administered the composition for a certain period of
time or with a certain number of doses after experiencing symptoms
of a migraine. In particular embodiments, IGF-1 may be administered
once a day; IFN-.gamma. may be administered once every week.
[0026] In particular embodiments, the composition may be
administered to the patient in a phases or cycles. For example, the
composition may be administered to the patient, wherein the
composition is at an amount that the active compound (IL-11,
IFN-.gamma., IGF-1, or insulin) in the composition is no longer
bioavailable or therapeutically effective within a first period of
time of administration, such as after 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours,
preferably 12 hours. The composition may be re-administered to the
patient after a second period of time from the end of the first
period of time, such as after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours,
preferably 12 hours. The compound may be IL-11, IFN-.gamma., IGF-1,
or insulin. For example, IFN-.gamma. or IGF-1 may be administered
and they may be cleared from the body after 12 hours, but the
second dose of IFN-.gamma. or IGF-1 may not be resumed until after
another 12 hours--during the second 12 hours the patient does not
have the administered IFN-.gamma. or IGF-1.
[0027] In some embodiments, methods also include administering to
the patient more than one of the following compounds: IL-11,
IFN-.gamma., IGF-1, or insulin. It is contemplated that the
combination may be administered to the patient concurrently (at the
same time) and in the same composition, concurrently but in
separate compositions, or serially.
[0028] In certain embodiments, IL-11 and IFN-.gamma. are
administered to or taken by the patient. In some embodiments, IL-11
and IGF-1 are administered to or taken by the patient. In
additional embodiments, IL-11 and insulin are administered to or
taken by the patient. In embodiments involving IL-11, IL-11 may be
administered to or by the patient prior to or after the other
compound.
[0029] In other embodiments, IFN-.gamma. and IGF-1 are administered
to or taken by the patient. In some embodiments, IFN-.gamma. and
insulin are administered to or taken by the patient. In embodiments
involving IFN-.gamma., IFN-.gamma. may be administered to or by the
patient prior to or after the other compound.
[0030] In some aspects, IGF-1 and insulin are administered to or
taken by the patient. IGF-1 may be administered to or by the
patient prior to or after the insulin.
[0031] In other embodiments, one or more of IL-11, IFN-.gamma.,
IGF-1, or insulin may be administered to or by a patient who is
also given or taking an anti-migraine drug. In certain aspects the
composition also includes an anti-migraine drug, which may be
either a pain-relieving medication or a preventative medication.
Pain relieving medications include but are not limited to pain
relievers such as NSAIDs or acetaminophen, a combination of
acetaminophen, aspirin, and caffeine, triptans, ergotamine and
caffeine combination drugs, anti-nausea medication, opiates, such
as codeine, and a corticosteroid such as dexamethasone.
Preventative medications include but are not limited to beta
blockers, antidepressants such as tricyclic antidepressants, an
anti-seizure drug, cyproheptadine, or botulinum toxin type A. In
some embodiments, the patient is administered the anti-migraine
drug within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, or 24 hours, or any range derivable
therein, of being administered the composition containing one or
more of IL-11, IFN-.gamma., IGF-1, and/or insulin.
[0032] In some embodiments a polypeptide such as IL-11,
IFN-.gamma., or IGF-1 is not provided directly to the patient or to
cells of the patient, and instead, the patient is provided with an
expression vector that comprises a nucleic acid sequence encoding
the polypeptide under the control of a promoter, wherein the
polypeptide is expressed in a cell containing the vector.
Consequently, embodiments involving polypeptides may be implemented
with an expression vector to achieve a treatment for migraine
patients.
[0033] Other embodiments are discussed throughout this application.
Any embodiment discussed with respect to one aspect applies to
other aspects as well and vice versa. The embodiments in the
Example section are understood to be embodiments of the invention
that are applicable to all aspects of the invention.
[0034] The terms "ameliorating," "inhibiting," or "reducing," or
any variation of these terms, when used in the claims and/or the
specification includes any measurable decrease or complete
inhibition to achieve a desired result.
[0035] Throughout this application, the term "effective amount" is
used to indicate that the compounds are administered at an amount
sufficient to treat a condition in a subject in need thereof. In
some embodiments, the condition is, but is not limited to, acute or
chronic migraine, or other conditions associated with acute or
chronic migraine, or conditions associated with cortical spreading
depression (CSD).
[0036] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one."
[0037] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device or method being employed to determine the value.
[0038] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0039] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0040] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0042] FIGS. 1A-1B. Illustrates an outline of a suggested pattern
of increased brain vitality versus neural activity (FIG. 1A and
FIG. 1B). Irritative (e.g., TNF-.alpha., IL-11, IGF-1, or
IFN-.alpha.) initiative stimuli (1) and adaptive response (2) that,
when delivered phasically reduces SD susceptibility (3-4). With
recurrent activating stimuli (3) and sufficient time (4), brain
increases its resilience to disease. Maladaptive changes within
brain may ensue when irritative stimuli occur without sufficient
time to allow for adaptive nutritive changes (5) or become constant
(6), for example, as can occur with high frequency or chronic
migraine.
[0043] FIGS. 2A-2E. SD-induced increased susceptibility to SD.
(FIG. 2A) Experiments begin with determination of the current
needed to maximize standard CA3 area field potential responses.
Half-maximal intensity is then used to elicit CA3 area evoked field
potentials from dentate gyrus bipolar electrical stimulation. This
documents the normalcy of evoked synaptic responses between
preparations. If a slice's field excitatory post synaptic
potentials are not at least 3 mV, slices are discarded. Next, field
potential trains (i) are noted to verify that preparations are
healthy enough to follow 10 Hz stimulation (e.g., amplitudes of
vertical deviations in fast potential records are similar) and the
current needed to trigger SD (ii) is registered (Pusic and Kraig,
2010). (FIG. 2B) For example, SD threshold is significantly
(p<0.001; n=13 & 17, respectively) reduced 3 days after 6
SDs elicited over an hr compared to control. (FIG. 2C) This effect
is significantly (p<0.002; n=37 & 10, respectively) related
to TNF-.alpha. since it can be mimicked by 3 day exposure (100
ng/mL) to TNF-.alpha. compared to control. (FIG. 2D) Also, SD
threshold is significantly (p<0.001; n=37 & 15,
respectively) increased by removal of TNF-.alpha. signaling via
inclusion of sTNFR1 (100 ng/mL) compared to control. (FIG. 2E)
Finally, minocycline (MinoC; 10 .mu.g/mL), which prevents increased
production of TNF-.alpha. from microglia (Hulse et al., 2008)
significantly (p<0.002; n=9 & 3, respectively) increases SD
threshold compared to control (Mitchell et al., 2010).
[0044] FIGS. 3A-3C. T-cells in slice cultures produce IFN-.gamma.,
which acutely increases SD susceptibility. (FIG. 1A) CD-6 (surface
marker for T-cells; yellow arrow) positive T-cells within the
parenchyma of a mature (>21 days in vitro) hippocampal slice
culture maintained in serum-free media. (Pusic et al., 2011). Cal
bar, 15 .mu.m. (FIG. 1B) When activated, T-cells in slice cultures
are immunopositive for IFN-.gamma. (yellow arrows). Cal bar, 30
.mu.m. (FIG. 1C) Acute (15-60 min) exposure to IFN-.gamma. (500
U/mL) triggers a significant (p<0.001; n=5 for each group)
increase in SD susceptibility.
[0045] FIGS. 4A-4F Oxidative stress (OS) from SD and its reduction
by IGF-1. (FIG. 4A) Normal cytoarchitecture of a hippocampal slice
culture showing pyramidal neuron areas of CA1, dentate gyrus (DG),
and the imaging zone for the inventors' work here (dotted line),
CA3. (FIG. 4B) OS was measured using CellROX, a fixable fluorescent
marker from Invitrogen. Image shows OS 24 hours after 6 SDs. (FIG.
4C) Pre-incubation in IGF-1 (40 ng/mL for 3 days before SD)
triggered a reduction in OS measured a day later. (FIG. 4D) This
reduction versus control SD was significant (p<0.02; n=4 &
5). (FIG. 4E) CellROX can be used to measure relative OS in
specific brain cell types. Image in FIG. 4A shows a pyramidal
neuron from a slice culture labeled with NeuN. (FIG. 4F) Co-labeled
image of CellROX shows associated OS.
[0046] FIGS. 5A-5C Reduced SD susceptibility by small molecules
that mimic enriched environment (EE). Slice cultures were exposed
to agents for 3 days and then SD susceptibility determined. IL-11
(100 ng/mL) provided significant (p=0.001; n=9 and 6, respectively)
(FIG. 5A) as did IFN-.gamma. (500 U/mL; p<0.001; n=9 and 6)
(FIG. 5B) and IGF-1 (40 ng/mL; p<0.001; n=8 and 7) (FIG.
5C).
[0047] FIG. 6. A whole animal recording paradigm was used determine
the threshold for SD in neocortex and hippocampus from anesthetized
rat, with exemplary SDs shown.
[0048] FIGS. 7A-7C Nasal administration of IGF-1, IL-11,
IFN-.gamma., and insulin significantly (p<0.001) reduced SD
susceptibility. Images show experimental setups for nasal
administration and testing. (FIG. 7A) Nasal injection setup showing
heat lamp and thermo-regulator that maintains temperature at
37.degree. C. plus nose cone used to deliver isoflurane anesthesia.
(FIG. 7B) Operator is shown administering nasal IGF-1 solution,
which is performed in a fume hood to adequately vent isoflurane
from nose cone. (FIG. 7C) Image illustrates standard
electrophysiology setup. Anesthetized animal is placed into a
standard electrophysiology holder and anesthesia maintained by a
nose cone delivering 3% isoflurane in oxygen. Arterial oxygen
saturation is monitored with a pulse-oximeter. Animals are warmed
to 37.5.degree. C. with a heating plate. SD is induced by
progressively injecting larger volumes of 0.5 M KCl from an 8 .mu.m
diameter microelectrode. SD is registered with a second
microelectrode used for DC recordings placed more caudally. After
SD is evoked by a given injection pulse, the stimulating electrode
is withdrawn and a volume re-injected under mineral oil. The
diameter of the resultant KCl injection sphere is then measured off
line using a compound microscope and the injection volume
calculated. For example, in the above whole animal experiments it
took 1-8 nL of 0.5 M KCl to evoke SD in control brain regions while
after acute IGF-1 this amount rose to 67-109 nL. A day after IGF-1
treatment the volume needed to evoked SD was 34-48 nL. IFN-.gamma.
treatment required 338 nL of 0.5 M KCl a day after treatment and
IL-11 required 67-200 nL. On the other hand, while still
significantly greater than control, acute insulin treatment only
required 8-34 nL.
[0049] FIGS. 8a-8f IGF-1 reduced spreading depression
susceptibility in hippocampal slice cultures. (FIG. 8a) Exemplary
CA3 area evoked field potential. Experiments begin with
establishment of the current intensity needed to evoke maximal
field potential responses; stimuli of half-maximal intensity were
then used to elicit subsequent field potentials. Only those
cultures with CA3 pyramidal neuron post-synaptic responses of at
least 3 mV were used for experiments. (FIG. 8b) CA3 response to
dentate gyrus bipolar stimulation (10 pulses at 10 Hz, 500 .mu.A)
was used to elicit a spreading depression (SD), as shown in (FIG.
8c). (FIG. 8c) The spreading depression (SD) shown here was induced
by the stimulation/response (arrow) shown in (FIG. 8b). (FIG. 8d)
Average current necessary to induce SD (SD threshold) was
significantly (*p=0.001) higher when slice cultures were exposed to
40 ng/mL IGF-1 acutely (n=6 and 7 for control and experimental
slices, respectively). (FIG. 8e) Similarly, average SD threshold
was also significantly (*p<0.001) increased when slice cultures
were exposed to IGF-1 for 3 days prior to SD (n=8 and 7 for control
and experimental slices, respectively). (FIG. 8f) Finally, average
SD threshold was significantly (*p<0.001) increased when slice
cultures were exposed to IGF-1 for 7 days prior to SD (n=11 and 6
for control and experimental slices, respectively.) Here the
seven-day treatment was phasic--cultures were exposed to IGF-1 for
only 12 hours a day for seven days to better mimic phasic effects
of exercise-rest-exercise seen with EE. Comparisons between groups
made via Student's t-test.
[0050] FIGS. 9a-9f IGF-1 decreased oxidative stress from spreading
depression. (FIG. 9a) NeuN immunohistochemical labeling of a
hippocampal slice culture, for cytoarchitectural reference and to
show the CA3 area of interest (dotted line box) used for
quantification of oxidative stress (OS) via CellROX.TM.
fluorescence intensity. (FIGS. 9b-c) Representative
CellROX.TM.-labeled hippocampal slices exposed to SD (FIG. 9b) and
to 3-day IGF-1 incubation followed by spreading depression (SD)
(FIG. 9c). Dotted line boxes illustrate CA3 areas of interest used
for relative OS quantifications. (FIG. 9d) OS was significantly
(*p=0.008) increased from controls after hippocampal slice cultures
were exposed to SD, and this effect was abrogated when exposed to
IGF-1 acutely (n=21, 12 and 9 for control, `SD` and `SD+IGF-1`
slices, respectively). (FIG. 9e) Similarly, the significantly
(*p=0.007) increased OS induced by SD was abrogated when slices
were exposed to IGF-1 for 3 days prior to SD (n=21, 8 and 6 for
`SD` and `SD+IGF-1` slices, respectively). (FIG. 9f) The
significant increase in OS from SD (*p<0.001), when compared to
controls, was significantly reduced (*p<0.001) in slices exposed
to IGF-1 for 7 days prior to SD induction (n=21, 12 and 3 for `SD`
and `SD+IGF-1` slices, respectively). Again, seven day treatments
consisted of exposing cultures to IGF-1 for 12 hours a day for
seven days. Note: IGF-1 exposure was continued for the additional
24 hour CellROX.TM. incubation. Scale bars=400 .mu.m (a) and 200
.mu.m (b and c). Comparisons between groups were made via ANOVA
plus Holm-Sidak post hoc testing.
[0051] FIGS. 10a-10c The exemplary antioxidant ascorbate reduced,
whereas the oxidizer hydrogen peroxide increased, spreading
depression susceptibility, with the latter effect abrogated by
IGF-1. (FIG. 10a) Average current necessary to induce spreading
depression (SD; i.e., SD threshold) was significantly (*p=0.018)
higher when slice cultures were acutely exposed to ascorbate (AA;
n=8) when compared to controls (n=12). (FIG. 10b) In contrast,
average current necessary to induce SD was significantly lower
(*p<0.001) when slice cultures were exposed to 50 .mu.M hydrogen
peroxide (H.sub.2O.sub.2; n=8 and 11 for control and experimental
slices, respectively). (FIG. 10c) While IGF-1 triggered a
significant protection from SD susceptibility (*p<0.0001) and
this effect continued when co-administered with 50 .mu.M
H.sub.2O.sub.2, the higher dose of 200 .mu.M H.sub.2O.sub.2
abrogated this effect to a non-significant difference from control.
(n=14, 16, 5, 9 for control, IGF-1, IGF-1+50 .mu.M H.sub.2O.sub.2,
and IGF-1+200 .mu.M H.sub.2O.sub.2, respectively). When compared to
IGF-1, SD thresholds of controls and IGF-1+200 .mu.M H.sub.2O.sub.2
were significantly decreased (#p<0.00001). Comparisons between
groups were made via Student's t-test (FIG. 10a, b) or ANOVA plus
Holm-Sidak post hoc testing.
[0052] FIGS. 11a-11g IGF-1 decreased CA3 oxidative stress (OS) and
its related hyperexcitability. (FIG. 11a) Slice culture
excitability in response to OS was further characterized by
classifying evoked potential changes to a single current pulse that
normally triggered a half-maximal field potential response where a
normal field potential (FP; left) was rated "1"; a FP that included
stimulus-related bursting activity (center) was rated "2"; and a
stimulus that resulted in spreading depression (right) was rated is
a "3". Relative evoked excitability was determined as a sum of
responses seen (e.g., responses of "2" and a "3" yielded an overall
excitability score of five). Responses were measured 30 minutes
after exposure to hydrogen peroxide (H.sub.2O.sub.2). (FIG. 11b)
Exposure to H.sub.2O.sub.2 (n=7) triggered a significant
(*p<0.001) increase in evoked excitability compared to control
(n=8) and this increase was abrogated by acute application of IGF-1
(n=4) to a non-significant difference from control. IGF-1 exposure
alone (n=4) had no significant impact on slice CA3 area evoked
excitability (i.e., showed a response of "1"). (FIG. 11c) 3-day
exposure to IGF-1 had a similar impact on slice culture
OS-increased excitability mimicked by application of
H.sub.2O.sub.2. H.sub.2O.sub.2 significantly (*p<0.001)
increased slice excitability (n=15) compared to control (n=18).
Pretreatment with IGF-1 for 3 days (n=9) reduced the
H.sub.2O.sub.2-induced increased excitability to a non-significant
difference from control. (FIGS. 11d-e) Exemplary images of control
(FIG. 11d) slice culture OS compared to increased slice OS induced
by exposure to menadione (FIG. 11e). Calibration bar, 200 Dotted
boxes indicate CA3 areas of interest used for relative OS
quantifications (f and g). (FIG. 11f) Exposure to menadione (M;
n=12) triggered a significant (*p<0.001) increase in OS compared
to control (n=18). Acute treatment with IGF-1 (n=15) reduced OS
from menadione to a non-significant (p=0.15) difference from
control (n=18). Acute IGF-1 exposure alone (n=18) did not reduced
slice culture OS from control. (FIG. 11g) Pretreatment with IGF-1
for 3 days (n=15) also reduced menadione-induced significant
increase in OS (*p<0.001; n=12) to a non-significant (p=0.148)
difference from control (n=15). In addition, IGF-1 pretreatment
alone (n=18) significantly (*p=0.008) reduced slice culture OS
compared to control.
[0053] FIGS. 12a-12e IGF-1 increased spontaneous neuronal spiking
activity. (FIG. 12a) Exemplary recording of unstimulated control
CA3 pyramidal layer spontaneous electrophysiological activity.
(FIG. 12b) Exemplary recording of 3-day IGF-1-exposed CA3 pyramidal
layer spontaneous electrophysiological activity. (FIG. 12c) Acute
IGF-1 treatment (n=9) triggered a significant (*p=0.03) increase in
spontaneous CA3 pyramidal neuron spiking compared to control (n=6).
(FIG. 12d) 3-day IGF-1 treatment also (n=7) triggered a significant
(*p=0.001) increase in spontaneous CA3 pyramidal neuron bursting
compared to control (n=6). (FIG. 12e) Similarly, 7-day IGF-1
treatment also (n=6) triggered a significant (*p<0.001) increase
in spontaneous CA3 pyramidal neuron bursting compared to control
(n=7). Seven day treatments consisted of IGF-1 exposure for only 12
hours daily. Comparisons between groups made via ANOVA plus
Holm-Sidakpost hoc testing.
[0054] FIG. 13 Oxidative stress from SD preferentially rises in
astrocytes and microglia, with the latter effect mitigated by
IGF-1. The inventors have recently shown that spreading depression
(SD), the most likely cause of migraine aura and perhaps migraine
(Lauritzen and Kraig, 2005), occurs with increased oxidative stress
(OS) and that OS, in turn, increases SD susceptibility (Grinberg et
al., 2012). Reactive oxygen and nitrogen species that cause OS have
both autocrine and paracrine signaling capacities that can affect
SD susceptibility by altering excitability (Kishida and Klann,
2007). Accordingly, the inventors looked for the cellular origin of
OS from SD. Here the inventors used hippocampal slice cultures
(HSC) to probe for cell-specific changes in OS from SD. SD was
induced trans-synaptically in rat HSCs using bipolar electrical
stimuli at the dentate gyrus (Pusic et al., 2011). Six SDs were
induced every 7-9 min over an hr, followed by 24 hr incubation in
CellROX.TM., a fixable fluorogenic probe for measuring OS (Grinberg
et al., 2012). HSCs were then fixed in 10% buffered formalin
phosphate. Other fixatives (PLP, 4% paraformaldehyde) prevented
detection of OS change. Tissue was then labeled for neurons
(anti-NeuN), oligodendrocytes (anti-RIP), astrocytes (anti-GFAP),
or for microglia (isolectin GS-IB4). Using confocal microscopy,
followed by MetaMorph analysis of cell-specific fluorescence
intensity, the inventors found that OS from SD significantly
increased in astrocytes (p=0.019) and microglia (p=0.003) but not
in neurons or oligodendrocytes, when compared to sham controls
(n=3-6/group). Since the environmental enrichment mimetic
insulin-like growth factor-1 (IGF-1) mitigates tissue OS from SD
(Grinberg et al., 2012), the inventors next looked for the cell
types responsible for this effect. The inventors applied IGF-1 (100
ng/mL) for three days and observed that the OS from SD seen in
microglia was significantly (p=0.018) decreased by IGF-1, but
astrocytic OS from SD was unchanged. The finding that astrocytes
but not neurons show increased OS from SD provides physiologic
evidence that extends recent work indicating astrocytes have a
higher oxidative metabolism potential (Lovatt et al., 2007).
However, the increased astrocytic OS was surprising given their
expected high antioxidant potential (Belanger et al., 2011).
Furthermore, SD triggers reactive microgliosis (Caggiano and Kraig,
1996), a change that can be expected to occur with increased OS.
The results confirm this, but importantly, show IGF-1 selectively
abrogated microglial OS. Since OS promotes SD (Grinberg et al.,
2012), this work points to microglia and their associated OS as
potential therapeutic targets in novel high-frequency and chronic
migraine therapeutics.
[0055] FIGS. 14A-14C. Reduced myelin basic protein (MBP) from SD
depends on IFN-.gamma./T-cell activation and sphingomylinase. (FIG.
14A) Western blot analyses for MBP confirm that chronically applied
IFN-.gamma. (500 U/mL.times.24 hours) is sufficient to
significantly ("*", p<0.01; n>5 for all groups in figure)
reduce MBP levels. Furthermore, co-administration of TNF-.alpha.
(100 ng/mL) with IFN-.gamma. leads to a further decline in MBP.
(FIG. 14B) Importantly, removal of T cells from hippocampal slice
cultures by exposure to anti-CD4 for 24 hours at 7 days in vitro,
abrogates the decline in MBP that otherwise is seen after SD. Thus,
confirming involvement of T cells. (FIG. 14C) Also, blockade of
neutral sphingomyelinase with GW 4869 prevents the drop in MBP
after SD. Taken together, these results suggest that acute, high
exposure to IFN-.gamma. [recall SD alone triggers an abrupt
elevation in IFN-.gamma. and TNF-.alpha., among other cytokines
(Kunkler et al., 2004)] triggers an abrupt elevation in TNF-.alpha.
that activates sphingomyelinase which is involved in demyelination
from SD.
[0056] FIGS. 15A-15C. Physiological and transient elevation [i.e.,
phasic (see FIG. 1)] of IFN-.gamma. triggers completely opposite
effects. Transient (i.e., 500 U/mL.times.12 hours; all groups
n>5) exposure of hippocampal slice cultures was nutritive when
assessed seven days later. (FIG. 15A) MBP was significantly
(p<0.001) increased above baseline. (FIG. 15B) Importantly, SD
susceptibility was significantly (p<0.001) increased and OS
similarly reduced (FIG. 15C).
[0057] FIGS. 16A-16F. IFN-.gamma., when pulsed onto slice cultures
for 12 hours triggers the release of nutritive exosomes that mimic
the effect of pulsed exposure to IFN-.gamma.. Slice cultures were
exposed to IFN-.gamma. (500 U/mL.times.12 hours) and three days
later exosomes were harvested from their surrounding incubation
media. The latter were then applied to naive slice cultures and
measurements made seven days later. All group sizes were >5; all
significance measurements p<0.001. Electron micrographs show
exosomes at low (FIG. 16A) and high (FIG. 16B) power; cal bars, 200
and 100 nm, respectively. (FIG. 16C) Western blots confirm
exosome-specific protein markers. IFN-.gamma. stimulated exosomes
triggered a significant rise in MBP above baseline levels (FIG.
16D), a significant reduction in SD susceptibility that was greater
than a 200-fold change (FIG. 16E) as well as (FIG. 16F) a
significant reduction in OS.
[0058] FIGS. 17A-17B. Detection of an IFN-.gamma.-induced rise in
slice culture glutathione using Thiol Tracker.TM., a fluorescent
indicator of glutathione. (FIG. 17A) Confocal imaging for
glutathione (long arrow) and a microglia marker (short arrow)
confirmed that pulsed exposure to IFN-.gamma. selectively increases
microglial glutathione. (FIG. 17B) Furthermore, this increase is
significant (p<0.001; n.gtoreq.5/group) and can be mimicked by
exposure to exosomes isolated from slice cultures activated by
pulsed-exposure to IFN-.gamma..
DETAILED DESCRIPTION OF THE INVENTION
[0059] The classic migraine episode is characterized by unilateral
head pain preceded by various visual, sensory, motor symptoms,
collectively known as an aura. Most commonly, the aura consists of
visual manifestations such as scotomas, photophobia, or visual
scintillations (e.g., bright zigzag lines). The typical headache of
migraine is throbbing or pulsatile. (However, more than 50% of
people who suffer from migraines report non-throbbing pain at some
time during the attack.) The headache is initially unilateral and
localized in the frontotemporal and ocular area, but pain can be
felt anywhere around the head or neck. The pain typically builds up
over a period of 1-2 hours, progressing posteriorly and becoming
diffuse. The headache typically lasts from 4-72 hours. Among
females, more than two thirds of patients report attacks lasting
longer than 24 hours. Migraine headaches may be unilateral or
bilateral and may occur with or without an aura. Migraines without
aura are the most common, accounting for more than 80% of all
migraines. Migraine attacks may also include visual manifestations
without headache. The inventors note that migraine is not a
neurodegenerative condition, thus, it is not necessary that a
treatment be neuroprotective. In certain aspects neuroprotective
effects can be explicitly excluded from the scope of the
claims.
[0060] Diagnosis of migraine without aura, according to the
International Headache Society, can be made according to the
following criteria, the "5, 4, 3, 2, 1 criteria": 5 or more
attacks. For migraine with aura, two attacks are sufficient for
diagnosis. 4 hours to 3 days in duration. 2 or more of the
following: Unilateral (affecting half the head); Pulsating;
"Moderate or severe pain intensity"; "Aggravation by or causing
avoidance of routine physical activity". 1 or more of the
following: "Nausea and/or vomiting"; Sensitivity to both light
(photophobia) and sound (phonophobia).
[0061] CSD or Spreading depression (SD) is a paroxysmal
perturbation of brain that is thought to cause migraine aura, and
perhaps migraine (Lauritzen and Kraig, 2005). It is classically
defined as a transient loss in spontaneous and evoked electrical
activity, associated with a large DC potential change in the
interstitial space, which both propagate at a uniquely slow speed
of about 3 mm/min (Bures et al., 1974; Somjen, 2001). SD is
triggered in susceptible gray matter areas of brain where a
sufficient volume is synchronously depolarized (Brazier, 1963).
This triggering effect results from increased excitation, reduced
inhibition, or a combination of these two effects, which results in
a flurry of spontaneous discharges that immediately precede the
loss in activity of SD (Mody et al., 1987; Kruger et al., 1996;
Kunkler and Kraig, 1998). Furthermore, recent evidence showed that
spontaneous and evoked activity is increased long after episodes of
SD.
[0062] Evidence indicates that microglia are activated by increased
synaptic activity (Ziv et al., 2006; Hung et al., 2010) and that
their signaling can also influence synaptic activity (Beattie et
al., 2002; Kaneko et al., 2008; Stellwagen et al., 2005; Stellwagen
and Malenka, 2006). Stellwagen and coworkers show that TNF-.alpha.
enhances neuronal excitation by increasing AMPA receptor cell
surface expression and reducing GABA receptor membrane levels
(Stellwagen et al., 2005; Stellwagen and Malenka, 2006).
Furthermore, Turrigiano and colleagues show that this capacity of
microglia is involved in homeostatic synaptic scaling, an adaptive
response of brain directed toward tuning neural circuit activity to
a functionally optimal state (Steinmetz and Turrigiano, 2010). Thus
by extension, microglia are likely to be involved in SD. Indeed, SD
activates microglia (Caggiano et al., 1996; Hulse et al., 2008).
The inventors show that activated microglia (e.g., that produces
TNF-.alpha. (Hulse et al., 2008); FIG. 2) or oxidative stress
(Grinberg et al., 2012 a, b; FIG. 9) increase spreading depression
susceptibility.
[0063] Microglial motion reflects their activation state. Within
the context of disease, microglia travel directionally toward sites
of irreversible injury (McGlade-McCulloh et al., 1989). In
contrast, within healthy brain tissue, microglial somata remain in
place, but during increased synaptic activity their processes
extend and retract at an increased rate (Nimmerjahn et al., 2005).
Since SD is preceded by a flurry of increased synaptic activity
(Brazier, 1963; Kruger et al., 1996; Kunkler and Kraig, 1998)
followed by a brief period of electrical silence during SD, and
then long afterwards, a persistent increase in synaptic
activity.
[0064] The inventors contemplate that immune cells are activated
(and influence other cells) by contact-mediated effects as well as
by paracrine signaling. The inventors probed for microglial cell
motion associated with SD using vital imaging of microglia in
mature rat hippocampal slice cultures. The results show that a
fraction of microglia in control slice cultures moved in a
stereotypic fashion consistent with Levy flights. Furthermore,
hours after SD, the number of microglia moving long distances was
significantly increased. The inventors asked whether this effect
could be mimicked by alterations in synaptic activity. Synaptic
activity increased by activation of microglia (with
lipopolyssacharide (LPS)) as well as neuronal activity increased by
chemical long-term potentiation (cLTP) significantly decreased the
number of microglia moving long distances. In contrast, blockade of
synaptic activity via exposure to tetrodotoxin (TTX) significantly
increased the number of microglia moving long distances and this
increase could be abrogated by co-incubation with glutamate and
adenosine triphosphate (ATP), two paracrine mediators released with
synaptic activity, for which microglia have receptors.
[0065] Recently the study of cell movements from the perspective of
a random walk has attracted great interest (Berg, 1993; Li et al.,
2008; Reynolds, 2010a; Reynolds, 2010b; Selmeczi et al., 2008;
Selmeczi et al., 2005; Takagi et al., 2008). These studies have
focused on the movements of cells in culture or over surfaces. The
inventors provide evidence to show that microglia travel via Levy
flights. Moreover, the inventors show that these movements
correspond to the type of Levy flight that has been associated with
an optimal random search pattern (Cabrera and Milton, 2004;
Viswanathan et al. 1999). The inventors contemplate that microglial
migration after SD is a means by which these cells influence a
wider expanse of brain either by contact or by paracrine signaling,
perhaps to increase regional susceptibility to SD, and by
extension, migraine.
[0066] In one aspect, novel therapeutics and therapeutic methods
are provided that prevent recurrent migraine and its transition to
chronic migraine.
[0067] Interferon-gamma (IFN-.gamma.) is a cytokine produced by
T-lymphocytes and natural killer cells, and is the only member of
the type II class of interferons. This interferon was originally
called macrophage-activating factor, a term now used to describe a
larger family of proteins to which IFN-.gamma. belongs. In humans,
the IFN-.gamma. protein is encoded by the IFNG gene. IFN-.gamma.
has been shown to interact with Interferon gamma receptor 1. An
example of an IFN-.gamma. amino acid sequence is found in GenBank
accession number AAB59534 (GI:184639), which is incorporated herein
by reference as of the filing date of this application. Certain
aspects are directed to isoforms and variants of IFN-.gamma. that
retain one or more functions of IFN-.gamma., particularly the
therapeutic effects described herein. IFN-.gamma. peptides or
polypeptides can comprise all or part of an amino acid sequence
similar to that provided in GenBank accession number AAB59534,
which is incorporated by reference and which is SEQ ID NO:1.
[0068] Interleukin 11 (IL-11) is a member of a family of growth
factors that includes growth hormone, granulocyte
colony-stimulating factor (G-CSF), and others. IL-11 is also a
member of a family of cytokines that includes IL-6, leukemia
inhibitory factor (LIF), oncostatin M (OSM), and ciliary
neurotrophic factor (CNTF), which all signal through a common
receptor subunit, gp130. IL-11 is naturally produced by bone marrow
stromal cells, and is a thrombopoietic growth factor that, in
conjunction with other factors, stimulates the proliferation of
hematopoietic stem cells and megakaryocytic progenitor cells and
induces maturation, resulting in increased platelet production.
IL-11 is also known under the names adipogenesis inhibitory factor
(AGIF) and oprelvekin. In humans, the IL-11 protein is encoded by
the IL11 gene. Interleukin 11 has been shown to interact with the
interleukin 11 receptor, in addition to gp130. An example of an
IL-11 amino acid sequence is found in GenBank accession number NP
000632 (GI:10834994), which is incorporated herein by reference as
of the filing date of this application. Certain aspects are
directed to isoforms and variants of IL-11 that retain one or more
functions of IL-11, particularly the therapeutic effects described
herein. IL-11 peptides or polypeptides can comprise all or part of
an amino acid sequence similar to that provided in GenBank
accession number NP 000632, which is incorporated by reference and
which is SEQ ID NO:2.
[0069] Insulin-like growth factor 1 (IGF-1) is also known as
somatomedin C or mechano growth factor, and has also been referred
to as a "sulfation factor" or "nonsuppressible insulin-like
activity" (NSILA). The insulin-like growth factor family includes
two ligands, IGF-1 and IGF-2, two cell membrane receptors, IGF-1R
and IGF-2R, and six IGF-1-binding proteins IGFBP1-6. In humans, the
IGF-1 protein is encoded by the IGF1 gene. Insulin-like growth
factor 1 has been shown to interact with the IGF-1 receptor
(IGF1R), and the insulin receptor. An example of an IGF-1 amino
acid sequence is found in GenBank accession number CAA01955
(GI:4529932), which is incorporated herein by reference as of the
filing date of this application. Certain aspects are directed to
isoforms and variants of IGF-1 that retain one or more functions of
IGF-1, particularly the therapeutic effects described herein. IGF-1
peptides or polypeptides can comprise all or part of an amino acid
sequence similar to that provided in GenBank accession number
CAA01955, which is incorporated by reference and which is SEQ ID
NO:3.
[0070] Insulin is a hormone central to regulating carbohydrate and
fat metabolism in the body. Insulin is synthesized in the pancreas
within the .beta.-cells of the islets of Langerhans. Insulin has
also been shown to be produced within the brain. The proinsulin
precursor of insulin is encoded by the INS gene. Insulin has been
shown to interact with the insulin receptor. An example of an
insulin amino acid sequence is found in GenBank accession number
AAA59172 (GI:386828), which is incorporated herein by reference as
of the filing date of this application. Certain aspects are
directed to isoforms and variants of insulin that retain one or
more functions of insulin, particularly the therapeutic effects
described herein. Insulin peptides or polypeptides can comprise all
or part of an amino acid sequence similar to that provided in
GenBank accession number AAA59172, which is incorporated by
reference and which is SEQ ID NO:4.
[0071] Peptides and/or polypeptides described herein may possess
deletions and/or substitutions of amino acids relative to the
native sequence. Sequences with amino acid substitutions are
contemplated, as are sequences with a deletion, and sequences with
a deletion and a substitution. In some embodiments, these
polypeptides may further include insertions or added amino
acids.
[0072] Polypeptides that may be administered include those that
have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,
103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,
116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,
129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,
142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,
168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,
181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,
194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,
207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,
220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232,
233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,
246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,
259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,
272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,
285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297,
298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,
311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323,
324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,
337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,
350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362,
363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375,
376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,
389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401,
402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,
415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427,
428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,
441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,
454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,
467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,
480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,
493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505,
506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518,
519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531,
532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544,
545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557,
558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570,
571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583,
584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596,
597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609,
610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622,
623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635,
636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648,
649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661,
662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674,
675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687,
688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700,
710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,
840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960,
970, 980, 990 or 1000 contiguous amino acids, or any range
derivable therein, of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ
ID NO:4. Alternatively, the polypeptide in compositions or methods
may be 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100%, or any range derivable
therein, identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ
ID NO:4.
[0073] The following is a discussion based upon changing of the
amino acids of a peptide and/or polypeptide to create a library of
molecules or a second-generation molecule. For example, certain
amino acids may be substituted for other amino acids in a
polypeptide without appreciable loss of function, such as ability
to interact with a target peptide sequence. Since it is the
interactive capacity and nature of a polypeptide that defines that
polypeptide's functional activity, certain amino acid substitutions
can be made in a polypeptide sequence and nevertheless produce a
polypeptide with like properties.
[0074] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive function on a protein is generally
understood in the art (Kyte and Doolittle, 1982). It is accepted
that the relative hydropathic character of the amino acid
contributes to the secondary structure of the resultant protein,
which in turn defines the interaction of the protein with other
molecules.
[0075] It also is understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with a biological property of the protein. As
detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity
values have been assigned to amino acid residues: arginine (+3.0);
lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine
(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine
(-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5);
cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5);
tryptophan (-3.4).
[0076] It is understood that an amino acid can be substituted for
another having a similar hydrophilicity value and still produce a
biologically equivalent protein. In such changes, the substitution
of amino acids whose hydrophilicity values are within .+-.2 is
preferred, those that are within .+-.1 are particularly preferred,
and those within .+-.0.5 are even more particularly preferred.
[0077] As outlined above, amino acid substitutions generally are
based on the relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity, hydrophilicity,
charge, size, and the like. However, in some aspects a
non-conservative substitution is contemplated. In certain aspects a
random substitution is also contemplated. Exemplary substitutions
that take into consideration the various foregoing characteristics
are well known to those of skill in the art and include: arginine
and lysine; glutamate and aspartate; serine and threonine;
glutamine and asparagine; and valine, leucine and isoleucine.
[0078] Pharmaceutical compositions described herein comprise an
effective amount of interferon-gamma, interleukin 11, insulin-like
growth factor 1, insulin or a combination thereof and/or additional
agents dissolved or dispersed in a pharmaceutically acceptable
carrier. The phrases "pharmaceutical" or "pharmacologically
acceptable" refers to molecular entities and compositions that do
not produce an adverse, allergic or other untoward reaction when
administered to an animal, such as, for example, a human, as
appropriate. The preparation of a pharmaceutical composition that
contains interferon-gamma, interleukin 11, insulin-like growth
factor 1, insulin or a combination thereof or additional active
ingredients will be known to those of skill in the art in light of
the present disclosure, as exemplified by Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,
incorporated herein by reference. Moreover, for animal (e.g.,
human) administration, it will be understood that preparations
should meet sterility, pyrogenicity, general safety and purity
standards as required by FDA Office of Biological Standards or
similar regulatory bodies.
[0079] In certain embodiments, the active compound, e.g.,
interferon-gamma, interleukin 11, insulin-like growth factor 1,
insulin or a combination thereof, may be formulated for intranasal
administration. Nasal administration of the present invention may
comprise the use of a nasal spray which uses water or salt
solutions as the liquid carrier with peptide or polypeptide being
dispersed or dissolved in the water in a therapeutically effective
amount. In another embodiment, a permeation enhancer is emulsified
in the aqueous phase that contains the active compound. The
emulsification may be effected through the use of one or more
suitable surfactants. Any suitable surfactant or mixture of
surfactants can be used in the practice of the present invention,
including, for example, anionic, cationic, and non-ionic
surfactants. Examples of non-ionic surfactants are PEG-60 corn
glycerides, PEG-20 sorbitan monostearate,
phenoxy-poly(ethyleneoxy)ethanol, sorbitan monooleate, and the
like. In general the surfactant is present in an amount less than
about 4, 3, 2, 1.5, 1, 0.5, 0.2% (w/w) the composition, including
all values and ranges there between. In another embodiment, the
surfactant may be present in amounts less than about 1.5% (w/w),
less than about 1.3% (w/w), less than about 1% (w/w), or less than
about 0.3% (w/w). For examples see PCT/US2009/046438, specifically
incorporated herein by reference in its entirety.
[0080] In certain embodiments, the pharmaceutical compositions may
be formulated as eye drops, intranasal sprays, inhalants, and/or as
other aerosols. Methods for delivering compositions directly to the
nasal passage or lungs via nasal aerosol sprays has been described
in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically
incorporated herein by reference in its entirety). Likewise, the
delivery of drugs using intranasal microparticle resins (Takenaga
et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat.
No. 5,725,871, specifically incorporated herein by reference in its
entirety) are also well-known in the pharmaceutical arts. Likewise,
transmucosal drug delivery in the form of a
polytetrafluoroetheylene support matrix is described in U.S. Pat.
No. 5,780,045 (specifically incorporated herein by reference in its
entirety).
[0081] The term aerosol refers to a colloidal system of finely
divided solid or liquid particles dispersed in a liquefied or
pressurized gas propellant. A typical aerosol for inhalation may
consist of a suspension of active ingredients in liquid propellant
or a mixture of liquid propellant and a suitable solvent. Suitable
propellants include hydrocarbons and hydrocarbon ethers. Suitable
containers may vary according to the pressure requirements of the
propellant. Administration of the aerosol may vary according to a
subject's age, weight and the severity and response of the
symptoms.
[0082] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, gels,
binders, excipients, disintegration agents, lubricants, sweetening
agents, flavoring agents, dyes, such like materials and
combinations thereof, as would be known to one of ordinary skill in
the art (see, for example, Remington's Pharmaceutical Sciences,
18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated
herein by reference). Except insofar as any conventional carrier is
incompatible with the active ingredient, its use in the
pharmaceutical compositions is contemplated.
[0083] Embodiments may comprise different types of carriers
depending on whether it is to be administered in solid, liquid or
aerosol form, and whether it needs to be sterile for such routes of
administration as injection. The present invention can be
administered intravenously, intradermally, transdermally,
intrathecally, intraarterially, intraperitoneally, intranasally,
intravaginally, intrarectally, topically, intramuscularly,
subcutaneously, mucosally, orally, topically, locally, inhalation
(e.g., aerosol inhalation), injection, infusion, continuous
infusion, localized perfusion bathing a target directly, via a
catheter, via a lavage, in cremes, in lipid compositions (e.g.,
liposomes), or by other method or any combination of the forgoing
as would be known to one of ordinary skill in the art (see, for
example, Remington's Pharmaceutical Sciences, 18th Ed. Mack
Printing Company, 1990, incorporated herein by reference).
[0084] Compounds may be formulated into a composition in a free
base, neutral or salt form. Pharmaceutically acceptable salts
include the acid addition salts, e.g., those formed with the free
amino groups of a proteinaceous composition, or which are formed
with inorganic acids such as for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic, tartaric
or mandelic acid. Salts formed with the free carboxyl groups can
also be derived from inorganic bases such as for example, sodium,
potassium, ammonium, calcium or ferric hydroxides; or such organic
bases as isopropylamine, trimethylamine, histidine or procaine.
Upon formulation, solutions may be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations may be easily
administered in a variety of dosage forms such as formulated for
parenteral administrations such as injectable solutions, or
aerosols, or formulated for alimentary administrations such as drug
release capsules and the like.
[0085] Methods and compositions that are suitable for
administration may be provided in a pharmaceutically acceptable
carrier with or without an inert diluent. The carrier should be
assimilable and includes liquid, semi-solid (i.e., pastes), or
solid carriers. Except insofar as any conventional media, agent,
diluent or carrier is detrimental to the recipient or to the
therapeutic effectiveness of the composition contained therein, its
use in an administrable composition for use in practicing the
methods of the present invention is appropriate. Examples of
carriers or diluents include fats, oils, water, saline solutions,
lipids, liposomes, resins, binders, fillers and the like, or
combinations thereof. The composition may also comprise various
antioxidants to retard oxidation of one or more component.
Additionally, the prevention of the action of microorganisms can be
brought about by preservatives such as various antibacterial and
antifungal agents, including but not limited to parabens (e.g.,
methylparabens, propylparabens), chlorobutanol, phenol, sorbic
acid, thimerosal or combinations thereof.
[0086] In some embodiments, the composition is combined with the
carrier in any convenient and practical manner (i.e., by solution,
suspension, emulsification, admixture, encapsulation, absorption
and the like). Such procedures are routine for those skilled in the
art.
[0087] In a further embodiment, the composition is combined or
mixed thoroughly with a semi-solid or solid carrier. The mixing can
be carried out in any convenient manner such as grinding.
Stabilizing agents can be also added in the mixing process in order
to protect the composition from loss of therapeutic activity (i.e.,
denaturation in the stomach). Examples of stabilizers for use in an
the composition include buffers, amino acids such as glycine and
lysine, carbohydrates such as dextrose, mannose, galactose,
fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
[0088] In further embodiments, methods may concern the use of
pharmaceutical lipid vehicle compositions that include
interferon-gamma, interleukin 11, insulin-like growth factor 1,
insulin or a combination thereof, one or more lipids, and an
aqueous solvent. As used herein, the term "lipid" will be defined
to include any of a broad range of substances that is
characteristically insoluble in water and extractable with an
organic solvent. This broad class of compounds is well known to
those of skill in the art, and as the term "lipid" is used herein,
it is not limited to any particular structure. Examples include
compounds which contain long-chain aliphatic hydrocarbons and their
derivatives. A lipid may be naturally occurring or synthetic (i.e.,
designed or produced by man). However, a lipid is usually a
biological substance. Biological lipids are well known in the art,
and include for example, neutral fats, phospholipids,
phosphoglycerides, steroids, terpenes, lysolipids,
glycosphingolipids, glycolipids, sulphatides, lipids with ether and
ester-linked fatty acids and polymerizable lipids, and combinations
thereof. Of course, compounds other than those specifically
described herein that are understood by one of skill in the art as
lipids are also encompassed by the compositions and methods
described herein.
[0089] One of ordinary skill in the art would be familiar with the
range of techniques that can be employed for dispersing a
composition in a lipid vehicle. For example, the composition
comprising interferon-gamma, interleukin 11, insulin-like growth
factor 1, insulin or a combination thereof may be dispersed in a
solution containing a lipid, dissolved with a lipid, emulsified
with a lipid, mixed with a lipid, combined with a lipid, covalently
bonded to a lipid, contained as a suspension in a lipid, contained
or complexed with a micelle or liposome, or otherwise associated
with a lipid or lipid structure by any means known to those of
ordinary skill in the art. The dispersion may or may not result in
the formation of liposomes.
[0090] The actual dosage amount of a composition that is
administered to a subject can be determined by physical and
physiological factors such as body weight, severity of condition,
the type of disease being treated, previous or concurrent
therapeutic interventions, idiopathy of the patient, and on the
route of administration. Depending upon the dosage and the route of
administration, the number of administrations of a preferred dosage
and/or an effective amount may vary according to the response of
the subject. The practitioner responsible for administration will,
in any event, determine the concentration of active ingredient(s)
in a composition and appropriate dose(s) for the individual
subject.
[0091] In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound.
In other embodiments, an active compound may comprise between about
2% to about 75% of the weight of the unit, or between about 25% to
about 60%, for example, about 2%, about 5%, about 10%, about 15%,
about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,
and any range derivable therein. Naturally, the amount of active
compound(s) in each therapeutically useful composition may be
prepared in such a way that a suitable dosage will be obtained in
any given unit dose of the compound. Factors such as solubility,
bioavailability, biological half-life, route of administration,
product shelf life, as well as other pharmacological considerations
will be contemplated by one skilled in the art of preparing such
pharmaceutical formulations, and as such, a variety of dosages and
treatment regimens may be desirable.
[0092] In other non-limiting examples, a dose may also comprise
from about 1 microgram/kg/body weight, about 5 microgram/kg/body
weight, about 10 microgram/kg/body weight, about 50
microgram/kg/body weight, about 100 microgram/kg/body weight, about
200 microgram/kg/body weight, about 350 microgram/kg/body weight,
about 500 microgram/kg/body weight, about 1 milligram/kg/body
weight, about 5 milligram/kg/body weight, about 10
milligram/kg/body weight, about 50 milligram/kg/body weight, about
100 milligram/kg/body weight, about 200 milligram/kg/body weight,
about 350 milligram/kg/body weight, about 500 milligram/kg/body
weight, to about 1000 mg/kg/body weight or more per administration,
and any range derivable therein. In non-limiting examples of a
derivable range from the numbers listed herein, a range of about 1,
5, 10, 20, 30, 40, 50, 60, 70, 80 mg/kg/body weight to about 50,
60, 70, 80, 90, 100, 150, 200 mg/kg/body weight, about 5
microgram/kg/body weight to about 500 milligram/kg/body weight,
etc., can be administered, based on the numbers described
above.
[0093] In some embodiments, the compositions of the present
invention are formulated to be administered via an alimentary
route. Alimentary routes include all possible routes of
administration in which the composition is in direct contact with
the alimentary tract. Specifically, the pharmaceutical compositions
disclosed herein may be administered orally, buccally, rectally, or
sublingually. As such, these compositions may be formulated with an
inert diluent or with an assimilable edible carrier, or they may be
enclosed in hard- or soft-shell gelatin capsule, or they may be
compressed into tablets, or they may be incorporated directly with
the food of the diet.
[0094] In certain embodiments, the active compounds may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et
al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792,451, each
specifically incorporated herein by reference in its entirety). The
tablets, troches, pills, capsules and the like may also contain the
following: a binder, e.g., gum tragacanth, acacia, cornstarch,
gelatin or combinations thereof; an excipient, e.g., dicalcium
phosphate, mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate or combinations thereof;
a disintegrating agent, e.g., corn starch, potato starch, alginic
acid or combinations thereof; a lubricant, e.g., magnesium
stearate; a sweetening agent, e.g., sucrose, lactose, saccharin or
combinations thereof; a flavoring agent, e.g., peppermint, oil of
wintergreen, cherry flavoring, orange flavoring, etc. When the
dosage unit form is a capsule, it may contain, in addition to
materials of the above type, a liquid carrier. Various other
materials may be present as coatings or to otherwise modify the
physical form of the dosage unit. For instance, tablets, pills, or
capsules may be coated with shellac, sugar, or both. When the
dosage form is a capsule, it may contain, in addition to materials
of the above type, carriers such as a liquid carrier. Gelatin
capsules, tablets, or pills may be enterically coated. Enteric
coatings prevent denaturation of the composition in the stomach or
upper bowel where the pH is acidic. See, U.S. Pat. No. 5,629,001.
Upon reaching the small intestines, the basic pH therein dissolves
the coating and permits the composition to be released and absorbed
by specialized cells, e.g., epithelial enterocytes and Peyer's
patch M cells. A syrup of elixir may contain the active compound
sucrose as a sweetening agent methyl and propylparabens as
preservatives, a dye and flavoring, such as cherry or orange
flavor. Of course, any material used in preparing any dosage unit
form should be pharmaceutically pure and substantially non-toxic in
the amounts employed. In addition, the active compounds may be
incorporated into sustained-release preparation and
formulations.
[0095] For oral administration the compositions of the present
invention may alternatively be incorporated with one or more
excipients in the form of a mouthwash, dentifrice, buccal tablet,
oral spray, or sublingual orally-administered formulation. For
example, a mouthwash may be prepared incorporating the active
ingredient in the required amount in an appropriate solvent, such
as a sodium borate solution (Dobell's Solution). Alternatively, the
active ingredient may be incorporated into an oral solution such as
one containing sodium borate, glycerin and potassium bicarbonate,
or dispersed in a dentifrice, or added in a
therapeutically-effective amount to a composition that may include
water, binders, abrasives, flavoring agents, foaming agents, and
humectants. Alternatively the compositions may be fashioned into a
tablet or solution form that may be placed under the tongue or
otherwise dissolved in the mouth.
[0096] Additional formulations which are suitable for other modes
of alimentary administration include suppositories. Suppositories
are solid dosage forms of various weights and shapes, usually
medicated, for insertion into the rectum. After insertion,
suppositories soften, melt or dissolve in the cavity fluids. In
general, for suppositories, traditional carriers may include, for
example, polyalkylene glycols, triglycerides or combinations
thereof. In certain embodiments, suppositories may be formed from
mixtures containing, for example, the active ingredient in the
range of about 0.5% to about 10%, and preferably about 1% to about
2%.
[0097] In further embodiments, the composition of the present
invention may be administered via a parenteral route. As used
herein, the term "parenteral" includes routes that bypass the
alimentary tract. Specifically, the pharmaceutical compositions
disclosed herein may be administered for example, but not limited
to intravenously, intradermally, intramuscularly, intraarterially,
intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos.
6,753,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and
5,399,363 (each specifically incorporated herein by reference in
its entirety).
[0098] Solutions of the active compounds as free base or
pharmacologically acceptable salts may be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms. The
pharmaceutical forms suitable for injectable use include sterile
aqueous solutions or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersions (U.S. Pat. No. 5,466,468, specifically incorporated
herein by reference in its entirety). In all cases the form must be
sterile and must be fluid to the extent that easy injectability
exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (i.e., glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), suitable mixtures thereof,
and/or vegetable oils. Proper fluidity may be maintained, for
example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0099] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous, and
intraperitoneal administration. In this connection, sterile aqueous
media that can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage may
be dissolved in isotonic NaCl solution and either added
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject. Moreover, for human administration, preparations should
meet sterility, pyrogenicity, general safety and purity standards
as required by FDA Office of Biologics standards or other
regulatory bodies.
[0100] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. A
powdered composition is combined with a liquid carrier such as,
e.g., water or a saline solution, with or without a stabilizing
agent.
[0101] In other embodiments, the active compound, i.e.,
interferon-gamma, interleukin 11, insulin-like growth factor 1,
insulin or a combination thereof, may be formulated for
administration via various miscellaneous routes, for example,
transdermal administration.
[0102] Pharmaceutical compositions for topical administration may
include the active compound formulated for a medicated application
such as an ointment, paste, cream or powder. Ointments include all
oleaginous, adsorption, emulsion and water-soluble based
compositions for topical application, while creams and lotions are
those compositions that include an emulsion base only. Topically
administered medications may contain a penetration enhancer to
facilitate absorption of the active ingredients through the skin.
Suitable penetration enhancers include glycerin, alcohols, alkyl
methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for
compositions for topical application include polyethylene glycol,
lanolin, cold cream and petrolatum as well as any other suitable
absorption, emulsion or water-soluble ointment base. Topical
preparations may also include emulsifiers, gelling agents, and
antimicrobial preservatives as necessary to preserve the active
ingredient and provide for a homogenous mixture. Transdermal
administration of the present invention may also comprise the use
of a "patch". For example, the patch may supply one or more active
substances at a predetermined rate and in a continuous manner over
a fixed period of time.
[0103] In certain embodiments, the compositions and methods of the
present invention involve a therapeutic composition comprising a
compound that reduces, ameliorates, or prevents migraine. These
compositions can be used in combination with a second therapy to
enhance the therapeutic effect of a first and/or second therapy.
These compositions would be provided in a combined amount effective
to achieve the desired effect. This process may involve providing
or administering a first therapy and a second therapy at the same
or different time. This may be achieved by administering one or
more compositions or pharmacological formulations that includes or
more of the agents, or by contacting the cell with two or more
distinct compositions or formulations, wherein one composition
provides (1) a first therapy comprising administering TNF-.alpha.
pathway effector, such as interferon-gamma, IL-11, IGF-1 insulin,
or a combination thereof; and/or (2) a second therapy. A second
therapy may be administered that includes analgesics, such as
aspirin, caffeine, vasoconstrictors, narcotics, 5HT1 receptor
agonist (e.g., sumatriptan, naratriptan, rizatriptan, zolmitriptan,
eletriptan, almotriptan and frovatriptan), other anti-migraine
drugs, and combinations thereof. Several antimigraine drugs are
known. See, e.g., U.S. Pat. Nos. 4,650,810, 4,914,125, 4,916,125,
4,994,483, 5,021,428, 5,200,413, 5,242,949, 5,248,684, 5,273,759,
5,317,103, 5,364,863, 5,399,574, 5,434,154, 5,441,969, 5,464,864,
5,466,699, 5,468,768, 5,491,148 and 5,494,910, each of which is
incorporated herein by reference in its entirety. Antimigraine
drugs most commonly used in treatment of migraine fall into the
following groups: ergot alkaloids, beta-blocking agents, calcium
channel blocking agents, antidepressants, selective 5-HT1 agonists,
sedatives, local anesthetics, adrenergic blocking agents and
mixtures of these.
[0104] It is contemplated that one may provide a patient with the
first therapy and the second therapy within about 12-24 h of each
other and, more preferably, within about 6-12 h of each other. In
some situations, it may be desirable to extend the time period for
treatment significantly, however, where several days (2, 3, 4, 5, 6
or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administrations.
[0105] In certain embodiments, a course of treatment (i.e., a first
therapy, or a first therapy in combination with a second therapy)
will last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90 days or more. It is contemplated that one or
more therapies may be given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and/or 90, any
combination thereof, and a second therapy can be given on day 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, and/or 90, or any combination thereof. Within a single day
(24-hour period), the subject may be given one or multiple
administrations of a first and/or second therapy. Moreover, after a
course of treatment, it is contemplated that there is a period of
time at which no treatment is administered. This time period may
last 1, 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 3, 4, 5 weeks, and/or
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, depending on
the condition of the patient, such as their prognosis, strength,
health, etc.
[0106] Various combinations may be employed, for example a first
therapy is "A" and a second therapy is "B":
TABLE-US-00001 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B
A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0107] Administration of any compound or therapy of the present
invention to a subject will follow general protocols for the
administration of such compounds or therapies, taking into account
the toxicity, if any, of the vector or any protein or other agent.
Therefore, in some embodiments there is a step of monitoring
toxicity that is attributable to combination therapy. It is
expected that the treatment cycles would be repeated as necessary.
It also is contemplated that various standard therapies may be
applied in combination with the described therapy.
[0108] Therapeutic agents of the invention can be administered in
doses of 0.01, 0.05, 0.01, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 200 pg, ng, or mg per dose or per
kilogram of subject body weight, including all values and ranges
there between.
[0109] Components and compounds of the invention can be provided to
a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50 or more times as part of a therapy or treatment. Moreover,
it is contemplated that there may be a course of therapy
prescribed, and that the course may be repeated, if necessary.
[0110] In other embodiments, components or compounds of the
invention are provided separately to the patient. It is
contemplated that subject is provided with first agent and a second
agent is provided or administered within 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours,
and/or 1, 2, 3, 4, 5, 6, 7 day and/or 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12 weeks, or any range derivable therein. Consequently, a
subject may take or be provided a first or second component or
compound of the invention 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20 or more times each, or any range
derivable therein, within a specified time period of being provided
a first or second component or compound.
[0111] In some embodiments a polypeptide, such as IL-11,
IFN-.gamma., IGF-1 or insulin is not provided directly to the
patient or to cells of the patient, and instead, the patient is
provided with an expression vector that comprises a nucleic acid
sequence encoding the polypeptide under the control of a promoter,
wherein the polypeptide is expressed in a cell containing the
vector. Consequently, embodiments involving polypeptides may be
implemented with an expression vector to achieve a treatment for
migraine patients. There are two basic approaches to such a
therapy, (i) ex vivo gene expression and (ii) in vivo gene
expression.
[0112] In ex vivo gene expression, cells are removed from a subject
and transfected with a desired gene in vitro. The genetically
modified cells are expanded and then implanted back into the
subject. Various methods of transfecting cells such as by
electroporation, calcium phosphate precipitation, liposomes,
microparticles, and other methods known to those skilled in the art
can be used in the practice of the present invention.
[0113] In in vivo gene expression, the desired gene is introduced
into cells of the recipient in vivo. This can be achieved by using
a variety of methods known to those skilled in the art. Such
methods include but are not limited to, direct injection of an
expression vector and introduction of an expression vector in a
carrier such as a virus, liposome, or exosome.
[0114] Various transduction processes can be used for the transfer
of nucleic acid into a cell using a DNA or RNA virus. In one aspect
of the present invention, a retrovirus is used to transfer nucleic
acid into a cell. Exogenous genetic material encoding a desired
gene product is contained within the retrovirus and is incorporated
into the genome of the transduced cell. In other aspects, exogenous
genetic material encoding a desired gene product is contained
within the virus and is maintained in the cytoplasm of the
transduced cell. The amount of gene product that is provided in
situ is regulated by various factors, such as the type of promoter
used, the gene copy number in the cell, the number of
transduced/transfected cells that are administered, and the level
of expression of the desired product. The expression vector of the
present invention may include a selection gene, for example, a
neomycin resistance gene, to facilitate selection of transfected or
transduced cells.
[0115] Expression vectors can be comprised in viruses, such as
retroviruses. Replication-deficient viruses are incapable of making
infectious particles. Genetically altered viral expression vectors
are useful for high-efficiency transduction of genes in cultured
cells and are also useful for the efficient transduction of genes
into cells in vivo. Standard protocols for the use of viruses to
transfer genetic material into cells are known to those skilled in
the art. For example, a standard protocol can be found in Kriegler
(1990) and Murray (1991).
[0116] The expression vector may also be in the form of a plasmid,
which can be transferred into the target cells using a variety of
standard methodologies, such as electroporation, microinjection,
calcium or strontium co-precipitation, lipid mediated delivery,
cationic liposomes, and other procedures known to those skilled in
the art.
[0117] The present invention also provides methods for in vivo gene
therapy. An expression vector carrying a heterologous gene product
is injected into a recipient. In particular, the method comprises
introducing a targeted expression vector, i.e., a vector which has
a cell- or tissue-specific promoter.
[0118] Embodiments also concern kits, such as therapeutic kits. For
example, a kit may comprise one or more pharmaceutical composition
as described herein and optionally instructions for their use. Kits
may also comprise one or more devices for accomplishing
administration of such compositions. For example, a subject kit may
comprise a pharmaceutical composition and device for accomplishing
nasal administration of a composition to a subject at risk of
developing, having, or beginning to have a migraine. In other
embodiments, a subject kit may comprise pre-filled ampules of a
peptide or other pharmaceutical composition, optionally formulated
as a lyophilized composition, for use with a delivery device.
[0119] Kits may comprise a container with a label. Suitable
containers include, for example, bottles, vials, and test tubes.
The containers may be formed from a variety of materials such as
glass or plastic. The container may hold a composition which
includes a peptide or polypeptide that is effective for therapeutic
or non-therapeutic applications, such as described above. The label
on the container may indicate that the composition is used for a
specific therapy or non-therapeutic application, and may also
indicate directions for in vivo or in vitro use, such as those
described herein. The kit of the invention will typically comprise
the container described above and one or more other containers
comprising materials desirable from a commercial and user
standpoint, including buffers, inhalers, cartridges, diluents,
filters, needles, syringes, and package inserts with instructions
for use.
EXAMPLES
[0120] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
[0121] IFN-.gamma. has detrimental and beneficial brain effects,
consistent with physiological conditioning hormesis. IFN-.gamma.
exacerbates demyelination from experimental autoimmune
encephalomyelitis (EAE), a model of multiple sclerosis. Yet,
low-level IFN-.gamma. before the onset of disease protects against
demyelination, an effect involving an oligodendrocyte oxidative
stress response (OSR). Also, spreading depression (SD) triggers a
transient (1 and 3 but not 7 day) drop in myelin basic protein
(MBP) in rat hippocampal slice cultures (HSC); and demyelination
increases SD susceptibility in vivo.
[0122] Since T-cells are present in hippocampal slice cultures
(HSC) and SD increases their production of IFN-.gamma., the
inventors examined how T-cells and IFN-.gamma. affect SD
susceptibility. Results were based on n.gtoreq.3-6/group and
comparisons made v. shams.
[0123] PCR arrays show a 3.61 fold increase in osteopontin and a
2.22 fold decrease in IL-10, which indicates an enhanced Th1 effect
from SD. Exposure to the Th1 cytokine IFN-.gamma. (500 U/mL)
triggers significantly increased susceptibility to SD at 1 day but,
importantly, resulted in a significantly reduced susceptibility at
3 days. Removal of IFN-.gamma. by depletion of T-cells by anti-CD4
prevented altered susceptibility to SD and prevented the SD-induced
demyelination, which otherwise triggered ruptured myelin sheaths,
shown via electron microscopy (EM). Neocortical SD in vivo
triggered a similar reduction in MBP a day later.
[0124] Three day treatment with IFN-.gamma. (500 U/mL)
significantly reduced reactive oxygen species generated from
chemical long-term potentiation (cLTP), a physiological means to
increase brain excitability like that seen hours after SD. This
beneficial effect of low-level IFN-.gamma. is supported by results
from rats. In rats, enrichment, which occurs with hippocampal
learning, led to a significant elevation in hippocampal T-cells,
IFN-.gamma. and MBP.
[0125] The results show SD acutely activates T-cells and overwhelms
brain OSR, resulting in increased susceptibility to SD and
demyelination. These effects can be prevented via treatment with
IFN-.gamma., which modulates immune parameters that favor a
Th1-skewed response extended over time. These results support the
use of IFN-.gamma. as a therapy for migraine.
Example 2
[0126] Cytokines are likely to be involved in spreading depression
(SD), a well-accepted model of migraine pathogenesis. Recurrent SD
significantly lowers subsequent SD threshold via TNF-.alpha.
signaling. Also, cold-preconditioning is neuroprotective via
low-level production of TNF-.alpha. from microglia. Importantly,
production of IL-11 from neurons dampens this protective effect.
The inventors focused on potential interrelations of oxidative
stress (OS) and IL-11, since SD generates OS and IL-11 has
antioxidant properties.
[0127] Experiments were performed in rat hippocampal slices
cultures and SD induced as previously described (Pusic et al.,
2011). Results were derived from n>3-6/group. Pretreatment with
minocycline significantly reduced SD susceptibility, further
supporting a role for microglia in SD. Importantly, both acute and
chronic (three-day) treatment with IL-11 (100 ng/mL) significantly
reduced SD susceptibility. Importantly, cLTP, a cellular model of
learning, significantly reduced SD susceptibility. Treatment of
slice cultures with IL-11 for 3 days significantly reduced OS
generated from physiological activation of the cultures via cLTP
compared to shams. This is consistent with the neural activity
increase seen after SD, which mimics the hyperexcitability evident
in migraine patients. IL-11 reduced SD susceptibility through
mechanisms that include OSR signaling. Furthermore, whole animal
experiments involving environmental enrichment (i.e., increased
volitional physical, intellectual, and social activity), which
occurs with increased brain activity, resulted in increased IL-11
mRNA and protein, supporting the notion that increased exercise may
help prevent migraine by altering neural OSR via IL-11. These
results support the use of IL-11 as a therapy for migraine.
Example 3
[0128] Environmental enrichment not only protects hippocampus, but
also reduces seizure susceptibility, both effects that occur with
increased learning. Such learning is known to occur with increased
CA3 area pyramidal neuron bursting. The inventors found that CA3
area pyramidal neuron bursting in hippocampal slice cultures (HSC)
reduced susceptibility to spreading depression (SD), the most
likely cause of migraine aura and perhaps migraine (Lauritzen and
Kraig, 2005; Benedict et al., 2004).
[0129] This prompted the inventors to examine whether insulin could
reduce SD susceptibility by increasing CA3 pyramidal neuron
bursting. The inventors induced SD trans-synaptically in rat HSC
using electrical stimuli (100 .mu.s pulses @ 10 Hz for 1 s) ranging
from 1 to 10,000 nC in order to determine SD threshold in sham and
experimental groups (Pusic et al., 2011). Results were based on
n>3-6/group. Insulin (400 .mu.g/mL) exposure for three days
triggered a significant reduction in SD susceptibility. This
insulin dose approximates the pharmacological levels seen in humans
treated with nasal insulin. However, the inventors found that IGF-1
was much more effective, producing results when administered at 40
ng/mL. The inventors hypothesized that the effect of insulin
occurred through IGF-1 receptor cross-talk. IGF-1 significantly
reduced SD susceptibility both acutely and after three days of
treatment. This effect of IGF-1 did not involve a change in
TNF-.alpha. mRNA levels two hours after SD evoked every 7-9 minutes
for an hour. In contrast, three day IGF-1 treatment leads to a
significant reduction in OS from cLTP, a physiological means to
increase brain excitability like that seen hours after SD. These
results support the use of IGF-1 and/or insulin as a therapy for
migraine.
Example 4
[0130] SD is the most likely cause of migraine aura and pain
(Lauritzen and Kraig, 2005). Neocortical and hippocampal SD
triggers a significant increase in nociceptive activation of the
caudal trigeminal nucleus (TGN). For the purposes of this example,
the inventors focused on hippocampal SD since it is known to occur
in uninjured human brain and it is the brain area most susceptible
to SD.
[0131] The paradigm for eliciting neocortical or hippocampal SD
consists of triggering SD by local KCl or electrical stimuli in
rostral neocortex or hippocampus respectively. The occurrence of SD
is confirmed with a DC potential recording electrode placed in the
caudal neocortex or hippocampus respectively.
[0132] Immunostaining for c-fos serves as a functional marker for
nociceptive activation in the TGN. c-fos positive cells in the TGN
can be detected after 2 hours of SD triggered every 9 min.
[0133] Experiments were performed in HSC since this in vitro
preparation closely parallels its in vivo counterpart. Cultures
were initially grown in a horse serum-based media (Kunkler and
Kraig, 1997) and after 18 days in vitro (DIV) some cultures were
transferred to a serum-free media based on Neurobasal and Gem-21
(Mitchell et al., 2010, Mitchell et al., 2011). SD was induced by
bipolar electrical stimulation in the dentate gyrus and SD
confirmed by recording its DC change in the CA3 area. SD showed the
typical DC changes. Baseline activity increased with a flurry of
activity after stimulation to trigger SD followed by a loss in
spontaneous or evoked activity before SD recovery.
[0134] SD occurs with increased cytokine production, which some
consider a consequence and not a cause of the malady. However,
microglial TNF-.alpha. increases AMPA and decreases GABA receptors,
which alters excitability. The mechanisms responsible for this are
not understood. Since vascular T-cells become activated in
migraineurs (Empl et al., 1999) and activated T-cells enter brain
(Engelhardt and Ransohoff, 2005), the inventors explored whether
T-cells may induce an upstream change that influences SD
susceptibility by signaling between neurons and glia. Since T-cells
can live up to a year (Empl et al., 1999) but perhaps less in
brain, the inventors probed for their presence in HSC (18-35 DIV)
where environmental conditions can be controlled and single cells
followed in space and time.
[0135] After an hour of SD, slices were allowed to recover for 2
hours and then harvested for total RNA isolation and PCR
amplification of cytokine targets. The extraction strategies
produced high quality intact RNA as determined via a gel that
showed sharp RNA bands. Next, dilution curve amplifications of
primers were checked to be sure primers produced uniform
amplification differences. For example, optimal amplifications show
a uniform increase in Ct thresholds. In contrast, amplifications
are not uniform with defective primers.
[0136] The inventors first probed for increased TNF-.alpha. mRNA
expression and found that SD induced a significant (P<0.001)
2-20 fold rise in TNF-.alpha. mRNA (n=6/group). These positive
samples were used for PCR array screening. Next, PCR array
technology was used as a means more sensitive than traditional gene
chips to look for evidence of T-cells in adult hippocampal slice
cultures. The gene expression screen for inflammatory cytokine
changes was completed by RT.sup.2 Profiler.TM. PCR Arrays.
[0137] Several cytokine targets suggested T-cells may be present in
mature slices. The inventors looked for the presence of T-cells
using CD6 labeling. The inventors found that .about.56 T-cells were
positive for CD6 in individual slice cultures. CD6 marked T-cells
can be visualized by via confocal microscopy, cell diameters are
about 5-6 .mu.m.
[0138] IFN-.gamma. is thought to be expressed predominantly, if not
soley, by T-cells in brain. Thus, the fact that IFN-.gamma. mRNA
could not be detect after SD seemed odd, especially since the
inventors previously detected a modest but significant change in
IFN-.gamma. protein (via bead-based ELISA assays) after SD (Kunkler
et al., 2004).
[0139] Since only .about.50 T-cells are found in a slice culture
(out of 100,000 total cells), the inventors used the SAB RT.sup.2
nano PreAMP cDNA Synthesis Kit as a means to detect potential
ultra-low level expression of IFN-.gamma.. This means of cDNA
preamplification provided a 12-fold increase in sensitivity to RNA
levels. The results illustrate the capacity of preamplification to
increase detection sensitivity. Ct threshold for the housekeeper,
Rpl13a, was 20.5 with initial amplification and this was increased
to 14.1 with use of the preamplification kit, a 12-fold increase
corresponding to 12 cycles of PCR amplification of cDNA. In
parallel, IFN-.gamma. in control cultures was not detectable but
was well within detection range with preamplification.
[0140] Next, the inventors probed for evidence of IFN-.gamma.
production after slice exposure to lipopolyssacharide (LPS) and
found it in 5-6 .mu.m cells consistent with a T-cell morphology.
Furthermore, the inventors found that IFN-.gamma. mRNA rose by 4.66
fold with 2 hours SD elicited every 9 min for an hour.
[0141] Goddard and coworkers (Goddard et al., 2007) show that MHC
expression, necessary for T-cell activation, changes with neural
activity on neurons. Furthermore, activated astrocytes and
especially microglia also present MHC. Thus, SD (or migraine) may
activate T-cells and their interactions with neural cell MHC may
initiate excitability changes directly or via hormetic immune
signaling (Kraig et al., 2010). If so, T-cells, and their
activation behavior, may be ideal targets for development of novel
therapeutics to mitigate episodic migraine and prevent chronic
migraine.
Example 5
[0142] Chronic migraine (CM) is a prevalent healthcare burden whose
pathogenesis remains incompletely defined. Evidence suggests that
central sensitization involving increased expression of
pro-inflammatory mediators and alterations in the periaqueductal
gray are involved (Aurora, 2009). However, increased migraine
frequency also correlates with the transformation of episodic
migraine (EM) to CM (Silberstein and Olesen, 2005)).
[0143] Central sensitization and alterations of the periaqueductal
gray may be "downstream" signaling phenomena of CM while recurrent
spreading depression (SD) is the "upstream" neural signaling
change. This conclusion is based on several facts. First, SD may
trigger both migraine aura and pain since it is sufficient for
nociceptive activation of the trigeminal nuclei and periaqueductal
gray. Second, SD can inhibit neuronal firing in the brainstem and
facilitate trigeminovascular activation, further suggesting its
involvement in CM. Third, SD initiates pro-inflammatory changes
within involved brain. Astrocytes and microglia show reactive
changes for weeks after recurrent SD. Importantly, SD also triggers
increased production of eicosanoids and innate cytokines, including
TNF-.alpha., Interleukin-1 beta (IL-1.beta.) and potentially
IFN-.gamma. principally involving microglia. These signaling
molecules are involved in somatosensory central sensitization and
thus may have a similar functional impact on SD.
[0144] Increasing evidence indicates that TNF-.alpha. has
particular involvement in activity-dependent signaling function
within brain. Malenka and coworkers show that physiological levels
of TNF-.alpha. increase AMPA receptor membrane expression and
reduce GABA receptor expression there. Our work and that of others
indicate that increased brain activity associated with
environmental enrichment (i.e., increased social, physical and
intellectual opportunities) occurs with increased expression of
TNF-.alpha.. Furthermore, the neuroprotective effects of
environmental enrichment requiring TNF-.alpha. can be amplified by
eicosanoids and involve activation of microglia, which generate the
TNF-.alpha.. In contrast to high-dose acute TNF-.alpha. effects
from disease which are toxic, these activity-dependent low-dose
physiological TNF-.alpha. changes improve brain function and
require time to develop.
[0145] The inventors contemplate that the greater TNF-.alpha.
change from SD (compared to that seen from learning), while
non-toxic, is sufficiently high to be maladaptive and over time
triggers the increased excitability seen after recurrent SD and
migraine. The latter occurs via alterations in AMPA and GABA
receptor membrane expression and function. Accordingly, the
inventors examine the degree to which microglia and TNF-.alpha.
expression alter brain excitability from recurrent SD.
[0146] Slice cultures were prepared and maintained as previously
described (Mitchell et al., 2010a). After 18 days in vitro (DIV),
slice cultures were transferred to serum-free media that included
additional calcium and magnesium. Cultures were screened at 21 DIV
to ensure no pyramidal neuron death was present. Standard
electrophysiological recordings were performed by placing a slice
culture insert in a PDMI recording chamber with controlled
temperature and pH (FIG. 1). The recording chamber was aerated with
5% CO2-balance air at 36.degree. C. and media flowing at 1.2 mL/min
or was held static around the slice culture insert. A recording
electrode was placed at the CA3 pyramidal cell layer and a
stimulating electrode was placed at the surface of the dentate
gyrus. Recordings were initiated prior to placing the stimulating
electrode on the culture to ensure no spreading depression was
triggered as a result.
[0147] Studies began with determination of the current needed to
maximize standard CA3 area field potential responses. This
documented the normalcy of evoked synaptic responses between
preparations. If a slice's field EPSPs were not at least 3-4 mV, it
was discarded.
[0148] Next, trans-synaptically evoked SD was determined. Here, the
inventors stimulated slices via the dentate gyrus with 100 .mu.s
pulses delivered at 10 Hz for 1 sec every 1-2 minutes at increasing
.mu.A current intensities until SD occurred. This proved to be a
most sensitive means to establish the threshold for synaptically
driven SD.
[0149] Considerable effort was expended in establishing a slice
culture media that provides healthy cultures to 35 days in vitro,
easily elicits SD, and allows detection of low-level immune
signaling. Slice cultures maintained in horse serum-based media are
almost always resistant to SD induction. The inventors found this
to also be true for a media that contained 20% horse serum,
Neurobasal A, B-27, insulin and ascorbate. A robust CA3 area field
potential response and ability to trigger SD was, however, rarely
seen. Instead, triggering pulses for SD commonly evoked
bursting.
[0150] The inventors tested SD susceptibility in cultures grown in
serum free media (based on Neurobasal and either B-27 or Gem-21
(Chen et al., 2008) that also included ascorbate. Evoked field
potentials were analogous to those in horse serum-based media. SD
could easily be stimulated in the serum free media. Furthermore, SD
significantly lowered the threshold for subsequent SD triggered
three days later.
[0151] The inventors determined that TNF-.alpha. was involved in SD
susceptibility (FIG. 2D). For example, exposure to TNF-.alpha. (100
ng/mL) for 3 days significantly lowered the threshold for SD
compared to that needed in other cultures on first exposure to SD
stimuli (FIG. 2D). Furthermore, the increased susceptibility to SD
triggered by previous SD could be abrogated by removal of
TNF-.alpha. signaling by treatment with soluble TNFR1 (200 ng/mL)
after the first day of SD (FIG. 2E).
[0152] Microglia are the predominant, if not the sole, source of
TNF-.alpha. in non-injured brain. Thus, taken together our results
demonstrate that SD, and therefore migraine, involves microglia and
their low-level production of the pro-inflammatory cytokine
TNF-.alpha..
[0153] SD and TNF-.alpha. are involved in improving brain function
via preconditioning neuroprotection. Thus, involvement of
TNF-.alpha. in increasing SD susceptibility may seem contradictory.
However, the basic tenets of hormetic signaling explain this
apparent contradiction of microglial-TNF-.alpha. signaling in
brain.
[0154] A hormetic (or U-shaped) dose response pattern consists of
low-level stimulation and high-level inhibition that involves two
basic tenets. First, an irritative (or stress) stimulus must be of
sufficient magnitude. Second, sufficient time must elapse for
adaptive changes to occur that result in a nutritive effect (FIG.
1).
[0155] FIG. 1 outlines our suggested pattern of increased brain
vitality versus neural activity (A and B). Irritative [(FIG. 2B);
e.g., TNF-.alpha., IL-11, IGF-1, insulin, or IFN-.gamma.)]stimuli
(1). Adaptive processes are (2). With recurrent activating stimuli
(3) and sufficient time (4), brain increases its resilience to
disease.
[0156] Maladaptive changes within brain may ensue when irritative
stimuli occur without sufficient time to allow for adaptive
nutritive changes (5) or become constant (6). The inventors
contemplate that such neuroimmune signaling from microglia and
TNF-.alpha. may be involved in the transformation of episodic
migraine to chronic migraine. Deciphering the SD-dependent innate
cytokine signaling within brain will identify therapeutic targets
to prevent episodic and chronic migraine.
Example 6
[0157] Neuronal activity necessarily increases brain TNF-.alpha.
(from microglia) and IFN-.gamma. (from T-cells) levels as well as
oxidative stress (OS) (from oxidative metabolism). In turn, these
small molecules and OS can increase neuronal activity. If neuronal
activity becomes excessive (i.e., as occurs after recurrent SD), SD
susceptibility will increase. Accordingly, the inventors define the
interrelated roles of TNF-.alpha., IFN-.gamma., and OS in promoting
SD susceptibility. The inventors use an SD model in rat hippocampal
slice cultures and rats in vivo. SD susceptibility is compared to
measurements of net OS, specific antioxidants, and critical OS
signaling system changes. OS-related gene expression changes are
assessed using PCR arrays and proteomic changes are assessed using
multiplexed-ELISAs and immunostaining. Cell-specificity of relevant
mRNA and protein changes are determined using laser dissection
microscopy and double-label immunostaining, respectively. The
inventors examine the impact of inhibiting critical OS signaling
points on SD susceptibility.
[0158] The inventors have established a highly reliable in vitro
model showing that SD-induced increased SD susceptibility depends
on TNF-.alpha. from activated microglia. (FIG. 2).
[0159] Using PCR array technology, the inventors discovered that
the slice cultures contain Th1 T-cells (Pusic and Kraig, 2010).
Since vascular T-cells become activated in migraineurs (Empl et
al., 1999) and activated T-cells enter brain (Engelhardt and
Ransohoff, 2005), the inventors began exploring whether T-cells
induce an upstream change that influences SD susceptibility. The
results show that T-cells become activated after SD in slice
cultures and increase their production of IFN-.gamma., which
acutely increases SD susceptibility (FIG. 3). Thus, slice cultures
are well-suited for study of T-cell signaling of SD.
[0160] IFN-.gamma. from T-cells is believed to play a crucial role
in immune-mediated demyelinating disease (Popko et al., 1997;
Imitola et al., 2005; Lees and Cross, 2007). SD triggers an acute,
but transient, disruption of myelin that is associated with a
significant reduction in myelin basic protein (MBP) after 1 and 3
days (Kunkler et al., 2006). Using immunostaining, the inventors
have confirmed that this drop in MBP is also seen in vivo 1-2 days
after SD. Furthermore, oligodendrocytes are highly sensitive to OS
(Lin et al., 2008; Juurlink et al., 1998) and SD increases OS, a
finding the inventors have confirmed occurs in slice cultures (FIG.
4). Finally, removal of T-cells by exposure to anti-CD4 (0.1 mg/mL)
at 7 days for 24 hours completely prevented SD-induced increased
susceptibility to SD versus control >21 days later in slice
cultures (p=0.964; n=6/ea. group).
[0161] Slice cultures are prepared, maintained, and induced to fire
SD as previously described using trans-synaptic excitation of CA3,
where SD is initiated (FIG. 4) (Kunkler et al., 2005; Pusic et al.,
2011). Though others assess changes in excitatory and inhibitory
synaptic drive via evoked field potential analyses, the inventors
agree with the Dudek laboratory that this approach is less reliable
(Waldbaum and Dudek, 2009; Shao and Dudek, 2009). Instead,
measurement of the relative neural circuit excitability (as used by
the inventors for SD susceptibility) is preferable.
[0162] The inventors compare SD susceptibility to innate cytokine,
OS, critical nodes of OS signaling, and antioxidant levels.
Determination of this seemingly wide array of parameters is now
comfortably accomplished using multiplexed assays. The inventors
use multiplexed PCR arrays (from SABiosciences) and multiplexed
flow cytometric assays [(MFCAs) for proteins; from Bio-Rad and
Millipore]. Laser dissection microscopy and double-label
immunostaining are used for specific cell-type-enhanced
measurements of RNA and protein, respectively.
[0163] Cytokine, IGF-1, and antioxidant screening is done first
using PCR arrays that contain wells for: (1) cytokines
(TNF-.alpha., IFN-.gamma.), their cognate receptors; (2) IGF-1 and
a principal IGF-1 binding protein involved in brain signaling,
IGFBP-3 (Jogie-Brahim et al., 2009; Carro et al., 2000); and (3)
antioxidants [MnSOD (manganese superoxide dismutase), CuSOD (copper
superoxide dismutase), catalase, GSH (glutathione), peroxiredoxin,
thioredoxin, NQO-1 (NAD(P)H dehydrogenase quinone 1), and HO-1
(heme oxygenase 1)]. Significant mRNA changes (i.e., >2-fold)
are verified by MFCAs and immunostaining for corresponding protein
production. In addition, MFCAs are used to assess changes in
phosphoprotein kinases (GSK3.beta. and eIF2a) and transcription
factors (ATF-4 and Nrf2) critical to OS signaling.
[0164] The inventors use double-label immunostaining and digital
image quantification procedures that are standard in their lab to
confirm the cellular origin of cytokine-OS signaling molecule
changes (Mitchell et al., 2010; Mitchell et al., 2011). Laser
dissection microscopy procedures follow those established in the
inventors laboratory (Hulse et al., 2008). Laser dissection mRNA
samples are prepared as previously described (Hulse et al., 2008).
However, inventors now utilize a nano-preamplification system from
SABiosciences that allows detection of low-expression RNAs (e.g.,
from .about.50 cells in 100,000) (Pusic et al., 2011). This
sensitivity will allow detection of cytokine-antioxidant mRNA
measurements from specific brain cell types, as the inventors have
done for T-cells in slice cultures (Pusic et al., 2011; Pusic and
Kraig, 2010).
[0165] OS signaling inhibition. Optimal doses of inhibitors are
determined by a paradigm successfully used by the inventors (Pusic
et al., 2011) and others (Romera et al., 2004). First, initial
doses will be derived from published in vitro studies or
manufacturer's recommendations. The inventors apply doses 10.times.
below and above these values to test for toxicity in slice
cultures. At least 1 day is necessary to allow for the adaptive
changes that require protein synthesis induced by ischemia
tolerance or enriched environment (EE) (Pusic et al., 2011; Kraig
et al., 2010). These effects persist for several days to weeks.
Thus, use of a 3-day adaptive paradigm allows for experiment
efficiency and is of sufficient duration to preclude any toxic
effects of signaling cascade modulation from chronic alterations.
This is consistent with the notion that phasic application of
EE-like signaling (e.g., from IL-11, IFN-.gamma., IGF-1, insulin)
is likely to be optimal. Three days are used here as a single cycle
of the phasic paradigm. For negative controls, heat inactivated
inhibitors are applied at previously determined doses (Hulse et
al., 2008). The inventors also modeled more prolonged EE-like
conditions using seven-day treatments of IGF-1 where IGF-1 was
given for only 12 hours daily as well as treating with IFN-.gamma.
for only 12 hours once a week. Sham controls consist of exposure to
vehicle alone. Sham controls consist of exposure to vehicle
alone.
[0166] The inventors will inhibit critical node OS signaling using
established pharmacological and siRNA strategies. To inhibit
eIF2.alpha. dephosphorylation, the inventors will apply sal003 (10
.mu.M, hippocampal slice culture) (Costa-Mattioli et al., 2007).
Pharmacological blockade of Nrf2 is accomplished by application of
brusatol (40 nM, cultured cell line) (Ren et al., 2011). Inhibition
of GSK3f3 can be accomplished through use of either lithium
chloride (LiCl, 2 mM, cultured hippocampal neurons) (Mendes et al.,
2009) or thiadiazolidinone-8 (TDZD-8, 1 .mu.M, primary brain
microvascular endothelial cells) (Ramirez et al., 2010). Accell
siRNA reagents targeting these genes in mouse and in rat are
commercially available from Thermo Scientific, and are used
according to the manufacturer's specifications (Pusic et al.,
2011). Accell Red Non-targeting siRNA are used to demonstrate
cellular transfection.
[0167] The inventors will analyze data using SigmaStat (v. 3.5)
software. All data will be tested for normality (P value to reject:
0.05) and equal variance (P value to reject: 0.05). Control data
will be normalized to 1.00 per experiment with related group
results scaled proportionally to facilitate inter-experiment
comparisons. All experimental groups will include sham controls.
Results are expressed as mean.+-.SEM. Power analysis is used to
determine adequacy of sample size. Two technical replicates are
used for mRNA and protein assays and 5 or more biological
replicates are used for all slice culture experiments. Behavioral
testing is completed with n=15/group. ANOVA with post hoc Holm
Sidak testing (or where applicable, Student's t-test) is used to
test for significance (i.e., p<0.05).
[0168] The stimulation paradigm is to trigger 1, 3 or 6 SDs and
measure threshold to SD responses 1, 3, 7 and 14 days later as
shown in FIG. 2. Sham controls consist of firing half-max field
potentials via 100 .mu.s single pulses instead of SD and then
assessing SD threshold 1, 3, 7 and 14 days later (FIG. 2).
[0169] In certain studies, the stimulation pattern is the same as
described above with measurements of (a) tissue and (b)
cell-type-specific OS changes 1, 3, 7 and 14 days after SD. The
same cultures can be used for both sets. Cell-specific measurements
are done using 20 .mu.m sections, confocal microscopy, and
double-label staining (FIG. 4, 13). Since oligodendrocytes are
highly sensitive to OS, and our work shows MBP falls shortly after
recurrent SD, the inventors will also measure MBP as a potential
functional marker of OS. In further studies the stimulation pattern
is to evoke 6 SDs over an hr with (a) multiplexed mRNA measurements
of cytokines, receptors, and antioxidants using PCR arrays. Again,
significant changes in tissue mRNA is confirmed by (b) laser
dissection microscopy for specific cell-enhanced mRNA changes and
(c) multiplexed ELISAs for confirmation of protein expression.
Double-label immunostaining is used for cell type identification.
(d) Changes in the phosphorylation state of tissue OS
signaling-related kinases and transcription factors are measured by
multiplexed ELISAs. (e) The cellular origin of significant changes
is verified with double-label immunostaining. Measurements of mRNA
species are made 2 hours after SD and proteins 1, 3, 7, and 14 days
after SD. Shams and controls (described above) are included.
[0170] In still further studies, the experimental paradigm is to
evoke 6 SDs over an hr and harvest tissue 3 days later. The
inventors will inhibit key points of the SD-OS signaling system and
measure resultant changes in (a) SD susceptibility, (b) OS levels,
and (c) antioxidant protein levels. Inhibition is accomplished at
the cytokine (TNF-.alpha. and IFN-.gamma.) level using sTNFR1 and
anti-CD4. Inhibition of key kinases (Nrf2, eIF2a, and GSK3.beta.)
is accomplished as described above. Inhibitors will be applied the
day before and maintained during SD, until harvest. Excessively
long inhibition of OS pathways can be deleterious, as can excessive
OS. Three day exposures [i.e., to TNF-.alpha., sTNFR1,
anti-IFN-.gamma., anti-CD4, IL-11, IFN-.gamma., IGF-1, as the
inventors show (FIG. 8)] are long enough for adaptive changes
(e.g., involving protein synthesis) to occur without triggering
irreversible cell injury and are short enough to be consistent with
phasic signaling of EE.
[0171] In certain studies the same paradigm as described above (1,
3, and 6 SD) is performed in vivo (hippocampus and neocortex) to
establish the next translational step in application of cytokine-OS
signaling for migraine therapeutics. After SD susceptibility
measurements 1, 3, 7, and 14 days later, brains are harvested for
measurement of OS using a carbonyl assay kit for oxidized proteins
(Shin et al., 2008). The paradigm producing the greatest change in
susceptibility is repeated, and tissue harvested a day later for
MBP quantification.
[0172] The inventors expect that the acute susceptibility change
for SD and associated OS from SD will show a linear or threshold
(and not hormetic) response. This conclusion is based on
preliminary data and the assumption that adequate time would not
have elapsed for adaptive production of increased antioxidant
proteins needed to dampen hyperexcitability of SD. The inventors
also expect that specific brain cell types will show differential
OS level changes, thus requiring measurements (preferably
multiplexed) at the level of specific brain cell types. Cellular
responses are varied and dose-dependent, with fewer cells
responding at a lower dose (Tay et al., 2010). Even within a
homogeneous culture, cells do not uniformly respond to TNF-.alpha.
activation. Accordingly, brain with four different principal cell
types (plus vascular endothelial cells), can be expected to show
similarly heterogeneous responses to cytokines (and OS), that are
likely to be interactive. Pretreatment with IFN-.gamma. protects
microglia from OS via upregulation of MnSOD (Chen et al., 2009) and
astrocytes prevent neuronal death from OS (Rohl et al., 2008).
Additionally, activated microglia influence the expression of
antioxidants in astrocytes (Correa et al., 2011). Finally, a recent
study shows that SD inversely correlates with cortical myelin
content (Merkler et al., 2009), indicating the importance of
oligodendrocytes in SD susceptibility. The inventors' data directly
extend this finding from a model of demyelinating disease to
showing that recurrent SD itself can be demyelinating.
[0173] OS signaling is also likely to show differential cellular
changes. Synaptic activity boosts intrinsic antioxidant activity in
neurons (Papadia et al., 2008) and eIF2a plays a critical role in
neuronal synaptic activity associated with memory and learning
(Costa-Mattioli et al., 2005; Costa-Mattioli et al., 2009; Gkogkas
et al., 2010) and OS signaling in oligodendrocytes (Lin et al.,
2008). Nrf2 plays an important role in responding to astrocytic OS
(Haskew-Layton et al., 2010). The role of these factors in
microglia is less clear and whether all (including GSK3.beta.) are
involved is unknown. The mechanism for how specific cell types are
responsible for production of certain antioxidants in response to
SD is unclear. The inventors expect OS (and related downstream
signaling and antioxidant production) will show neural cell type
heterogeneity, with perhaps the greatest change occurring in
microglia and astrocytes, compared to neurons and
oligodendrocytes.
[0174] Cytokines can also be expected to show differential cell
type changes. T-cells are the main, if not sole, source of
IFN-.gamma., while TNF-.alpha. is produced under physiological
conditions by microglia (Hulse et al., 2008), and IL-11 mostly by
neurons, and to a lesser extent, astrocytes (Mitchell et al.,
2011). IGF-1, on the other hand, often comes from the periphery
(Jogie-Brahim et al., 2009) but can also be produced by astrocytes
and neurons. Cognate receptors for these mediators are also
differentially expressed among brain cell types.
[0175] In vivo studies will parallel culture results since the
slices closely parallel the in vivo counterpart. The data support
this suggestion by showing that MBP decreases 1-2 days after 1 hr
of recurrent SD in animal neocortex, a finding first established in
slices. To show that measurements are not unique to hippocampus
parallel measurements will be made after neocortical SD.
Example 7
[0176] Enriched environment (EE) reduces hyperexcitability from
seizures (Kraig et al., 2010; Young et al., 1999), SD (Guedes et
al., 1996), and migraine (Darabaneanu et al., 2011) through
adaptive signaling. However, the EE signaling that prevents
development of increased susceptibility to SD is unknown. The
inventors contemplate that low levels of IL-11, IFN-.gamma., and
IGF-1, small molecules involved in neuroprotection from
preconditioning, are also involved in EE, and follow a hormetic
pattern. IL-11, IFN-.gamma., IGF-1, and OS have interrelated roles
in reducing SD susceptibility via EE. Studies using EE, EE+SD and
SD+EE are modeled in hippocampal slice cultures, with their results
confirmed in vivo using EE+SD in rats. The experimental endpoint of
altered SD susceptibility from EE is compared to measurements of
net OS, specific antioxidant levels, and changes in critical OS
signaling molecules. Measurements follow those described above,
extended to include confirmation via behavioral testing of EE
efficacy. The overall expected outcome is to define the signature
of small molecules critical for OS signaling by which EE reduces SD
susceptibility. This provides novel information on how EE enhances
naturally occurring means to prevent increased susceptibility to SD
from SD, and by extension, high frequency and chronic migraine
(HFCM).
[0177] Adaptive (i.e., hormetic) signaling requires that (a) an
initiating stimulus be sufficiently robust to evoke an adaptive
response and (b) sufficient time must elapse for adaptive responses
to occur. For our purposes, the initiating activity-dependent
stimuli (EE) are the pro-inflammatory cytokines TNF-.alpha. from
microglia (Kraig et al., 2010) and IFN-.gamma. from T-cells, which
trigger adaptive responses.
[0178] Adaptive signaling from EE includes production of IL-11,
IFN-.gamma., and IGF-1. IL-11 is an anti-inflammatory cytokine
localized to neurons, and to a lesser extent, astrocytes (Mitchell
et al., 2010; Mitchell et al., 2011). TNF-.alpha. stimulates the
production of IL-11, which in turn inhibits TNF-.alpha. production
(Mitchell et al., 2010; Mitchell et al., 2011). T-cells, the main
if not sole source of IFN-.gamma. under physiological brain
conditions, play a role in the nutritive effect of EE on brain.
Increased numbers of T-cells enter brain parenchyma with EE (Ziv et
al., 2006). Furthermore, T-cells are involved in the maintenance of
neurogenesis and spatial learning (Ziv et al., 2006), effects our
data show require IFN-.gamma.. Circulating IGF-1 mediates the
neuroprotective effects of exercise (Carro et al., 2000), likely
via activity-dependent entry of IGF-1 from the periphery (Nishijima
et al., 2010). However, ischemic brain injury triggers expression
of IGF-1 in neurons and astrocytes (Hwang et al., 2004), and these
cells may also be a source of IGF-1 from EE. Furthermore, IGF-1 can
increase brain excitability (Nunez et al., 2003; Ramsey et al.,
2005) and thus may play a role in the increased activity of EE.
Finally, IGF-1 impacts OS since GSK3.beta. is the main downstream
effector of IGF-1 signaling. In each case, these small molecules
(i.e., IL-11, IFN-.gamma., and IGF-1) stimulate the production of
antioxidants, which increase with neuronal activity (Papadia et
al., 2008). The inventors contemplate that activity-dependent
increased antioxidant levels are an important means by which EE can
reduce SD susceptibility (Guedes et al., 1996), and by extension
migraine (Darabaneanu et al., 2011). Our data support this
conclusion.
[0179] The inventors show that IL-11, IFN-.gamma., and IGF-1
significantly reduced SD susceptibility after 3 days of
pretreatment (FIG. 5), effects that reduce OS (FIGS.
4,9,10,13,15,16). Furthermore, EE (which increases MBP) and cLTP
(an in vitro model of learning used here) both significantly raise
the threshold for SD. EE in C57BL/10J mice triggered a significant
(2 and 2.2-fold, respectively) rise in IFN-.gamma. and IL-11 mRNA.
Similarly, cLTP in slices triggered an 89-fold rise in IL-11 mRNA 2
hours after stimulation.
[0180] Slice cultures are used for EE, EE+SD and SD+EE with groups
and measurements per experiment as described above, except as noted
below. The experimental paradigm will be to: evoke cLTP and then a
day later, trigger SD (i.e., 1, 3, or 6), followed by measurements
taken 1, 3, or 7 days later. Note: 14 day measurements after SD are
excluded since the cultures are used from 21-35 days in vitro. The
endpoints are to compare SD susceptibility after EE to innate
cytokine, OS, critical nodes of OS signaling, and antioxidant
levels.
[0181] The inventors use a well-accepted method for inducing cLTP
in slice cultures (Kraig et al., 2010; Otmakhov et al., 2004a;
Otmakhov et al., 2004b; Kopec et al., 2006). The protocol consists
of raising cAMP levels and increasing synaptic activity using
rolipram/forskolin applied in a Mg.sup.2+-- free Ringer's solution
(36.degree. C.) for 5 min, then allowing slices to recover in
normal media.
[0182] For EE, rats (12/cage) are housed in a Marlau-style
enrichment cage with free access to food and water, an array of
toys, running wheel, and socialization bowl that are changed weekly
for 4 weeks to provide increased volitional opportunities for
intellectual, physical, and social stimulation (i.e., EE). NE rats
are housed in single standard cages.
[0183] Hippocampal and neocortical SD will be induced in
isoflurane-anesthetized rats as previously described for rats using
nanoliter injections of 0.5 M KCl (or 0.5 M NaCl for sham controls)
every 9 minutes for 1 hr (Kunkler and Kraig, 2003) for acute SD, SD
after EE or SD after EE signaling mimics.
[0184] A visual recognition task is used to test
hippocampus-dependent memory, since it is non-stressful. Ability to
recognize a novel versus familiar object is a measure of
hippocampus-dependent memory (Clark et al., 2000; Gobbo and O'Mara,
2004; Mansuy et al., 1998; Mumby et al., 2002; Rampon et al., 2000;
Ruby et al., 2008; Rutten et al., 2008; Thuret et al., 2009).
[0185] Methods will follow those outlined above, except that SD
will be preceded by EE (i.e., cLTP). Certain studies will include
SD followed by EE. The inventors will mimic our targeted EE
signaling variables (i.e., IL-11, IFN-.gamma., and IGF-1) by
applying them for 3 days, as shown in FIG. 7. Initial
concentrations will be as previously described (FIG. 5) as well as
0.1.times. and 10.times. of those doses. The latter will also
include SD threshold measurements.
[0186] To ensure that EE enhanced hippocampus-dependent memory,
animals are behaviorally tested. The threshold for SD, OS, and
antioxidant levels will be measured 1, 3, 7, and 14 days later. The
behavioral testing paradigm should not interfere with cytokine and
OS signaling since it is non-stressful. Controls versus EE alone
will verify this.
[0187] While the process of reducing susceptibility to SD from
activity associated with EE is likely to be hormetic, the inventors
contemplate that the dose-response of SD susceptibility and OS
change from EE will show a linear or threshold dose-response
pattern that is higher than those seen in the absence of EE.
[0188] The inventors expect that specific brain cell types will
show differential OS level changes. However, such changes are
likely to be reduced as a result of EE. This suggests that the
numbers (or diversity) of cell types responding are reduced, as
illustrated by the differential responses of primary cultures to
lower doses of TNF-.alpha. (Tay et al., 2010).
[0189] In vivo results should parallel in vitro slice results. As
noted above, hippocampal slice cultures closely resemble the
structure and function of their in vivo counterpart. To-date, our
results of SD susceptibility, cellular responses to SD [e.g.,
astrogliosis, microgliosis, and now oligodendrocyte dysfunction
(reduced MBP)], and OS are comparable between preparations.
Example 8
[0190] Nasal insulin enters brain (Born et al., 2002) and improves
cognitive function (Stockhorst et al., 2004; Hallschmid et al.,
2008)--therefore it could be used as a mimetic of EE. If insulin
can effectively mimic EE, the inventors contemplate that other
small molecules may exert a similar effect. In fact, considerable
evidence demonstrates that nasally delivered IGF-1 enters brain
(Thorne et al., 2004) and significantly improves brain function
after injury (Liu et al., 2001). Since other small molecules have
also been shown to enter brain and mediate a therapeutic impact
(Akpan et al., 2011; De Rosa et al., 2005), the inventors expect
that IL-11 and IFN-.gamma. does as well. Accordingly, the inventors
define the degree to which their small molecules (IL-11,
IFN-.gamma., and IGF-1) enter brain and reduce SD susceptibility
and OS. The inventors used rats for hippocampal and neocortical SD
(FIGS. 6-7).
[0191] Rats (FIGS. 6 and 7) are anesthetized with inhalational
isoflurane and kept warm. While anesthetized, animals are placed in
a supine position and 50 .mu.L of sterile drug (i.e., IFN-.gamma.,
IGF-1, or IL-11) solution [or sterile saline vehicle (sham
control)] is administered nasally by delivering 5 .mu.L alternating
between the left and right naris every 2 min over 20 minutes.
Animals are given nasal drug treatments daily at the same time for
7 days before subsequent experiments. Doses for all 3 agents begin
with the effective doses noted in our work using slice cultures
(1.times.), then include others (0.1.times. and 10.times.). The
inventors base this strategy on the fact that the effective culture
dose for IGF-1 (40 ng/mL) closely approximates the dose used in
vivo (143 .mu.g/kg).
[0192] Inventors will detect delivery of EE-mimicking agents via
immunostaining. Human recombinant IFN-.gamma., and human
recombinant IGF-1 are identified via monoclonal antibodies
(Nishijima et al., 2010). Since there is no antibody specific to
human recombinant IL-11 that will not cross-react with mouse or
rat, biotinylated IL-11 is administered and detected with an
anti-biotin antibody.
[0193] In certain studies, agents will be delivered via nasal
administration daily for seven days before experiments. On the 8th
day, initial SD threshold will be determined and 1, 3, or 6 SDs
will be induced. This will be followed by measurement of SD
threshold [and OS] 1, 3, 7, and 14 days later. SD will be evoked
and measurements made separately in hippocampus and neocortex.
[0194] In other studies a high dose of antioxidant (100.times.
vitamin C) is administered for 4 weeks (i.e., "anti-EE"), then
measuring threshold to first SD, then elicit 1, 3, or 6 SDs
followed by measurements of SD threshold and OS 1, 3, 7, and 14
days later. Again, the studies are performed in hippocampus and
neocortex.
[0195] The small molecule agents (IL-11, IFN-.gamma., IGF-1) may
have equal impact on reducing SD susceptibility since nasal
delivery of IGF-1 comparably increases IGF-1 in various brain
regions such as hippocampus and neocortex (Thorne et al., 2004).
Furthermore, given the similar molecular size of the other agents,
their entry into brain should be comparable to that seen with
IGF-1. Given the comparable efficacy of these three agents in
preventing increased susceptibility to SD in vitro, they will show
similar efficacy in vivo.
[0196] A whole animal recording paradigm was developed to determine
the threshold for SD in neocortex and hippocampus from anesthetized
rat. FIG. 6 below illustrates this capacity.
[0197] This approach was applied to measurement of SD threshold
(FIG. 10) after treatment (FIG. 7) with nasally administered
IGF-1(150 .mu.g), IL-11 (1 .mu.g), IFN-.gamma. (50,000 units), and
insulin (20 .mu.g). In each case, nasal delivery of these agents
significantly (p<0.001) reduced susceptibility to SD in
neocortex plus hippocampus. Resistance against SD was always
greater in hippocampus compared to neocortex (n=4, controls; n=3,
acutely after IGF-1; n=6, one day after IGF-1; n=6, one day after
IL-11; n=6, one day after IFN-.gamma.; and n=3, acutely after
insulin).
[0198] Nasal administration followed by SD threshold evaluation in
vivo. Vehicle or human recombinant IGF-1 (150 .mu.g) is
administered intranasally. SD threshold is established by injection
of nanoliter volumes of 0.5 M KCl into neocortex or hippocampus
(-2.0 mm from Bregma and 1.5 mm lateral from midline at either 750
.mu.m or 2,800 .mu.m into brain, respectively) using a thin-walled
glass pipette and Picospritzer. Once a threshold is established, it
is confirmed two more times, with an exemplary record from a
vehicle-sham animal shown below. The injection volume is measured
by injecting, with the same pressure and duration, the amount of
0.5M KCl that led to the first SD, into a glass well filled with
light machine oil (a liquid of appropriate density to maintain a
sphere of the injected solution that does not sink). The diameter
of the injected sphere is then measured using a compound microscope
and used to calculate the injected volume, then moles of potassium.
Recordings are made at -6.0 mm from Bregma and 4.5 mm lateral to
the midline at either 750 .mu.m or 4,500 .mu.m in depth for
neocortex and hippocampus, respectively.
Example 9
[0199] Experiments using hippocampal slice cultures demonstrated
that phasic IGF-1 markedly protected against SD and this effect was
related to reduced oxidative stress.
[0200] Spreading depression (SD), the likely cause of migraine aura
and perhaps migraine, is triggered by widespread and unfettered
neuronal hyperexcitability. Migraine and the initiating
hyperexcitability of seizure, which involve oxidative stress (OS),
are likely interrelated. Environmental enrichment (EE) decreases
seizure and can reduce migraine. EE's well-characterized
neuroprotective effect involves insulin-like growth factor-1
(IGF-1). Accordingly, the inventors asked if IGF-1 could mitigate
the hyperexcitability that initiates SD using rat hippocampal slice
cultures. The inventors demonstrate that IGF-1 significantly
decreased SD susceptibility and related OS. The inventors mimicked
OS of SD and observed that IGF-1 abolished hyperexcitability from
OS. Application of an antioxidant significantly decreased SD
susceptibility and co-administration of an antioxidant with IGF-1
produced no additive effect, whereas an oxidizer significantly
increased SD, and this effect was abrogated by IGF-1. Moreover,
IGF-1 significantly decreased baseline OS, despite seemingly
paradoxically increasing CA3 bursting. These results suggest that
IGF-1 increased endogenous antioxidants to levels sufficient to
buffer against the OS of SD. Insulin similarly mitigated SD
susceptibility, but required a far greater dose. Since brain IGF-1
increases with EE, and, like insulin, independently functions as an
EE mimetic, the inventors suggest that EE mimetics are a novel
source of therapeutics for SD, and by extension, migraine.
[0201] IGF-1 (and insulin) significantly increased SD threshold.
Hippocampal slices were exposed to IGF-1 either acutely (i.e.,
15-30 min), for 3 days, or for 7 days prior to assessing the SD
threshold. The 7-day IGF-1 exposure was performed phasically to
better mimic anticipated effects of EE [i.e., exercise-rest
intervals (Will et al., 2004; Kraig et al., 2010)], where slices
were exposed to IGF-1-supplemented media in the day and returned to
regular media at night. Acute, 3-day, and 7-day exposure to IGF-1
all significantly increased SD threshold compared to control by 24,
75, and 22-fold (FIG. 8). Furthermore, 3-day exposure to insulin
[(400 .mu.g/mL); but not lower insulin doses, i.e., 6, 12, and 100
.mu.g/mL (n=3-9/group)] resulted in a significantly (p=0.03) higher
SD threshold versus control [i.e., 22.60.+-.9.60 (n=8) and
1.00.+-.0.20 (n=9), respectively]. However, the insulin dose needed
for this protective effect was 15 500-fold higher than IGF-1 (i.e.,
70 .mu.M versus 4.5 or 10 nM), suggesting that IGF-1 has greater
therapeutic utility against SD. Accordingly, the inventors focused
our subsequent work on IGF-1.
[0202] IGF-1 significantly reduced OS from SD Since SD may increase
OS (Viggiano et al. 2011), OS can enhance brain excitability
(Muller et al. 1993; Gulati et al.2005; Waldbaum and Patel 2010),
and IGF-1 is involved in antioxidant signaling, the inventors next
tested whether IGF-1 treatment altered SD-induced OS. Results show
that acute, 3-day, and 7-day treatment with IGF-1 significantly
reduced OS from SD (FIG. 9). Seven-day exposure was again phasic,
as described for SD threshold studies above. While acute treatment
with IGF-1 led to a 20% decrease in OS from SD, 3-day exposure to
IGF-1 afforded an even greater level of protection, with a 30%
decrease in OS from SD, and 7 days offered a 73% decrease in OS
from SD.
[0203] SD susceptibility is modulated by OS. Slices were exposed to
either ascorbic acid or hydrogen peroxide and SD threshold was
assessed. Ascorbate (2 mM) significantly increased the SD
threshold, while hydrogen peroxide (50 .mu.M) significantly
decreased the SD threshold (FIG. 14). Co-exposure to IGF-1 and a
higher dose of hydrogen peroxide (200 .mu.M) led to a significant
decrease in the SD threshold when compared with IGF-1 alone.
However, 50 .mu.M hydrogen peroxide co-exposed with IGF-1 was an
insufficient oxidant stress to overwhelm the protective effect of
IGF-1 on SD susceptibility (FIG. 10). Finally, coincubation of
slice cultures with ascorbate and IGF-1 (n=8) did not significantly
raise the threshold for SD versus IGF-1 alone (n=7; p=0.28 with
relative SD threshold levels of 7.39.+-.6.16 and 1.00.+-.0.31,
respectively).
[0204] IGF-1 eliminated effects of SD-mimetics on excitability and
OS. The inventors further assessed the ability of IGF-1 to reduce
slice culture excitability by decreasing OS. First, the inventors
mimicked OS from SD by application of hydrogen peroxide. This
exogenously induced OS significantly increased evoked slice
hyperexcitability (FIG. 11), like that seen from SD (Mitchell et
al. 2010a). Both 3-day and acute exposure to IGF-1 abrogated this
hydrogen peroxide-induced hyperexcitability. Second, the inventors
additionally mimicked OS from SD by slice exposure to menadione
(FIG. 11). As expected, this treatment triggered a significant
increase in slice OS, an effect that was abrogated by acute and
3-day exposure to IGF-1. In fact, 3-day exposure to IGF-1 alone
could significantly reduce baseline OS from control levels.
Furthermore, 7-day exposure to IGF-1 also significantly reduced
baseline OS levels by 26% when compared with controls (p=0.001;
n=11 and 9 for controls and 7-day IGF-1, respectively). The latter
is important because exposure to IGF-1 alone, which led to the
significant reductions in baseline OS (FIG. 11), triggered a
significant increase in spontaneous CA3 bursting (FIG. 12).
Example 10
[0205] Oxidative stress from SD preferentially rises in astrocytes
and microglia, with the latter effect mitigated by IGF-1 (FIG. 13).
The inventors have recently shown that spreading depression (SD),
the most likely cause of migraine aura and perhaps migraine
(Lauritzen and Kraig, 2005), occurs with increased oxidative stress
(OS) and that OS, in turn, increases SD susceptibility (Grinberg et
al., 2012). Reactive oxygen and nitrogen species that cause OS have
both autocrine and paracrine signaling capacities that can affect
SD susceptibility by altering excitability (Kishida and Klann,
2007). Accordingly, the inventors looked for the cellular origin of
OS from SD. Here the inventors used hippocampal slice cultures
(HSC) to probe for cell-specific changes in OS from SD. SD was
induced trans-synaptically in rat HSCs using bipolar electrical
stimuli at the dentate gyrus (Pusic et al., 2011). Six SDs were
induced every 7-9 min over an hr, followed by 24 hr incubation in
CellROX.TM., a fixable fluorogenic probe for measuring OS (Grinberg
et al., 2012). HSCs were then fixed in 10% buffered formalin
phosphate. Other fixatives (PLP, 4% paraformaldehyde) prevented
detection of OS change. Tissue was then labeled for neurons
(anti-NeuN), oligodendrocytes (anti-RIP), astrocytes (anti-GFAP),
or for microglia (isolectin GS-M4). Using confocal microscopy,
followed by MetaMorph analysis of cell-specific fluorescence
intensity, the inventors found that OS from SD significantly
increased in astrocytes (p=0.019) and microglia (p=0.003) but not
in neurons or oligodendrocytes, when compared to sham controls
(n=3-6/group).
[0206] Since the environmental enrichment mimetic insulin-like
growth factor-1 (IGF-1) mitigates tissue OS from SD (Grinberg et
al., 2012), the inventors next looked for the cell types
responsible for this effect. The inventors applied IGF-1 (100
ng/mL) for three days and observed that the OS from SD seen in
microglia was significantly (p=0.018) decreased by IGF-1, but
astrocytic OS from SD was unchanged. The finding that astrocytes
but not neurons show increased OS from SD provides physiologic
evidence that extends recent work indicating astrocytes have a
higher oxidative metabolism potential (Lovatt et al., 2007).
However, the increased astrocytic OS was surprising given their
expected high antioxidant potential (Belanger et al., 2011).
Furthermore, SD triggers reactive microgliosis (Caggiano and Kraig,
1996), a change that can be expected to occur with increased OS.
The results confirm this, but importantly, show IGF-1 selectively
abrogated microglial OS. Since OS promotes SD (Grinberg et al.,
2012), this work points to microglia and their associated OS as
potential therapeutic targets in novel high-frequency and chronic
migraine therapeutics.
Example 11
[0207] The inventors noted above that IFN-.gamma. has detrimental
and beneficial effects on oligodendrocytes (e.g., myelin),
contrasting responses that mutually involve oxidative stress (OS).
Loss of myelin and impaired remyelination in multiple sclerosis,
and its animal model, experimental autoimmune allergic
encephalomyelitis, involve increased IFN-.gamma. and OS signaling
concomitant with disease. Conversely, if occurring prior to disease
onset and allowed adequate time for adaptation, elevated
IFN-.gamma. and associated OS reduce the degree of demyelination
otherwise seen in animal models of multiple sclerosis. While the
mechanisms for IFN-.gamma./OS effects on myelin are incompletely
defined, recent evidence suggests involvement of neural activity
driven anti-oxidant production.
[0208] Environmental enrichment [(EE) i.e., volitionally increased
intellectual, social, and physical activity] occurs with enhanced
learning and memory from phasically increased neural activity and
lessened subsequent injury from a wide array of neurodegenerative
disorders including demyelinating diseases. In addition, EE
increased T cell trafficking in the brain, expression of
IFN-.gamma., production of myelin, and reduced OS. Importantly,
enhanced neuronal activity leads to elevated production of
anti-oxidants, including glutathione. Furthermore, glutathione
inhibits demyelination by blocking sphingomyelinase, and
antioxidants stimulate expression of genes associated with
myelination.
[0209] As noted above, the inventors probed for further evidence of
these potential IFN-.gamma./OS--antioxidant interactions on brain
myelin using mature hippocampal slice cultures, since T cells are
present and the tissue shows a rise in IFN-.gamma. after SD. SD is
a benign perturbation of brain that is thought to be the most
likely cause of migraine aura, and perhaps migraine. When
recurrent, SD may also play a role in the conversion of episodic to
high frequency and chronic migraine. Furthermore, SD increases OS
and experimental demyelination increases susceptibility to SD.
[0210] Latest results confirm that when IFN-.gamma. is pulsed onto
hippocampal slice cultures OS is reduced, SD susceptibility is
reduced, and importantly MBP rises above baseline, effects that
appear to be due to increased anti-oxidant production, including
glutathione. Reduced myelin basic protein (MBP) from SD depends on
IFN-.gamma./T-cell activation and sphingomylinase (FIG. 18). In
contrast, physiological and transient elevation of IFN-.gamma.
triggers completely opposite effects (FIG. 19).
[0211] Since stimulated immune cells release exosomes that are
capable of reducing OS in recipient cells, the inventors next
tested whether IFN-.gamma. stimulated slice cultures (and possibly
their microglia) to release exosomes that mimicked the effects of
IFN-.gamma.. The inventor's results confirm this hypothesis (FIGS.
15-17). IFN-.gamma., when pulsed onto slice cultures for 12 hours
triggers the release of nutritive exosomes that mimic the effect of
pulsed exposure to IFN-.gamma. (16, 17). Since pulsed-exposure to
IFN-.gamma. reduces OS and glutathione is a naturally occurring
inhibitor of neutral sphingomyelinase, the inventors probed for an
IFN-.gamma.-induced rise in slice culture glutathione using Thiol
Tracker.TM., a fluorescent indicator of glutathione (FIG. 17).
[0212] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
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Sequence CWU 1
1
41166PRTHomo sapiens 1Met Lys Tyr Thr Ser Tyr Ile Leu Ala Phe Gln
Leu Cys Ile Val Leu1 5 10 15Gly Ser Leu Gly Cys Tyr Cys Gln Asp Pro
Tyr Val Lys Glu Ala Glu 20 25 30Asn Leu Lys Lys Tyr Phe Asn Ala Gly
His Ser Asp Val Ala Asp Asn 35 40 45Gly Thr Leu Phe Leu Gly Ile Leu
Lys Asn Trp Lys Glu Glu Ser Asp 50 55 60Arg Lys Ile Met Gln Ser Gln
Ile Val Ser Phe Tyr Phe Lys Leu Phe65 70 75 80Lys Asn Phe Lys Asp
Asp Gln Ser Ile Gln Lys Ser Val Glu Thr Ile 85 90 95Lys Glu Asp Met
Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg 100 105 110Asp Asp
Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val 115 120
125Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu Ser
130 135 140Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu
Phe Arg145 150 155 160Gly Arg Arg Ala Ser Gln 1652199PRTHomo
sapiens 2Met Asn Cys Val Cys Arg Leu Val Leu Val Val Leu Ser Leu
Trp Pro1 5 10 15Asp Thr Ala Val Ala Pro Gly Pro Pro Pro Gly Pro Pro
Arg Val Ser 20 25 30Pro Asp Pro Arg Ala Glu Leu Asp Ser Thr Val Leu
Leu Thr Arg Ser 35 40 45Leu Leu Ala Asp Thr Arg Gln Leu Ala Ala Gln
Leu Arg Asp Lys Phe 50 55 60Pro Ala Asp Gly Asp His Asn Leu Asp Ser
Leu Pro Thr Leu Ala Met65 70 75 80Ser Ala Gly Ala Leu Gly Ala Leu
Gln Leu Pro Gly Val Leu Thr Arg 85 90 95Leu Arg Ala Asp Leu Leu Ser
Tyr Leu Arg His Val Gln Trp Leu Arg 100 105 110Arg Ala Gly Gly Ser
Ser Leu Lys Thr Leu Glu Pro Glu Leu Gly Thr 115 120 125Leu Gln Ala
Arg Leu Asp Arg Leu Leu Arg Arg Leu Gln Leu Leu Met 130 135 140Ser
Arg Leu Ala Leu Pro Gln Pro Pro Pro Asp Pro Pro Ala Pro Pro145 150
155 160Leu Ala Pro Pro Ser Ser Ala Trp Gly Gly Ile Arg Ala Ala His
Ala 165 170 175Ile Leu Gly Gly Leu His Leu Thr Leu Asp Trp Ala Val
Arg Gly Leu 180 185 190Leu Leu Leu Lys Thr Arg Leu 1953119PRTHomo
sapiens 3Met Ala Leu Cys Leu Leu Thr Phe Thr Ser Ser Ala Thr Ala
Gly Pro1 5 10 15Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln
Phe Val Cys 20 25 30Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr Gly
Tyr Gly Ser Ser 35 40 45Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp
Glu Cys Cys Phe Arg 50 55 60Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr
Cys Ala Pro Leu Lys Pro65 70 75 80Ala Lys Ser Ala Arg Ser Val Arg
Ala Gln Arg His Thr Asp Met Pro 85 90 95Lys Thr Gln Lys Glu Val His
Leu Lys Asn Ala Ser Arg Gly Ser Ala 100 105 110Gly Asn Lys Asn Tyr
Arg Met 1154110PRTHomo sapiens 4Met Ala Leu Trp Met Arg Leu Leu Pro
Leu Leu Ala Leu Leu Ala Leu1 5 10 15Trp Gly Pro Asp Pro Ala Ala Ala
Phe Val Asn Gln His Leu Cys Gly 20 25 30Ser His Leu Val Glu Ala Leu
Tyr Leu Val Cys Gly Glu Arg Gly Phe 35 40 45Phe Tyr Thr Pro Lys Thr
Arg Arg Glu Ala Glu Asp Leu Gln Val Gly 50 55 60Gln Val Glu Leu Gly
Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu65 70 75 80Ala Leu Glu
Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys 85 90 95Thr Ser
Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 100 105 110
* * * * *