U.S. patent application number 11/893100 was filed with the patent office on 2008-04-17 for compositions and methods for neuroprotectin.
This patent application is currently assigned to The Johns Hopkins University. Invention is credited to Norman Haughey, Avindra Nath, Joseph P. Steiner.
Application Number | 20080090897 11/893100 |
Document ID | / |
Family ID | 39082698 |
Filed Date | 2008-04-17 |
United States Patent
Application |
20080090897 |
Kind Code |
A1 |
Steiner; Joseph P. ; et
al. |
April 17, 2008 |
Compositions and methods for neuroprotectin
Abstract
Disclosed herein are neuroprotective compounds. Methods for the
preparation of such compounds are disclosed. Also disclosed are
pharmaceutical compositions that include the compounds. Methods of
using the compounds disclosed, alone or in combination with other
therapeutic agents, for the treatment of neurodegenerative
conditions are provided.
Inventors: |
Steiner; Joseph P.; (Mount
Airy, MD) ; Nath; Avindra; (Ellicott City, MD)
; Haughey; Norman; (Baltimore, MD) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP;Client: JHU
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
The Johns Hopkins
University
Baltimore
MD
|
Family ID: |
39082698 |
Appl. No.: |
11/893100 |
Filed: |
August 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60837365 |
Aug 11, 2006 |
|
|
|
Current U.S.
Class: |
514/453 ;
514/454; 514/456; 514/460 |
Current CPC
Class: |
A61P 25/18 20180101;
A61P 25/28 20180101; A61K 31/366 20130101 |
Class at
Publication: |
514/453 ;
514/456; 514/460; 514/454 |
International
Class: |
A61K 31/366 20060101
A61K031/366 |
Claims
1. A pharmaceutical composition, comprising at least one compound
having the structure of Formula I: ##STR65## wherein each compound
of Formula I is in a substantially purified form; and Y.sub.1 is O
and Y.sub.2 is OH or O-alkyl; or Y.sub.1 and Y.sub.2 together form
a furan group; R.sub.1 and R.sub.2 together form a substituted
cycloalkyl or cycloalkenyl group; and is selected from , or
##STR66## wherein X.sub.4 and R.sub.2 together form a substituted
heteroalicyclic group provided that R.sub.1 is H; or
pharmaceutically acceptable salts, prodrugs, or metabolites
thereof; or ester derivatives, saccharide derivatives, or
--(CH.sub.2CH.sub.2O).sub.nCH.sub.3 derivatives thereof, where n is
1 to 100; and a pharmaceutically acceptable excipient.
2. The pharmaceutical composition of claim 1, wherein the at least
one compound of Formula I has a structure selected from:
##STR67##
3. The pharmaceutical composition of claim 2, wherein the at least
one compound of Formula I has the structure: ##STR68## wherein
R.sub.4 and R.sub.5 together form a substituted cycloalkyl or
cycloalkenyl group.
4. The pharmaceutical composition of claim 3, wherein the at least
one compound of Formula I has the structure: ##STR69## wherein
R.sub.6 and R.sub.7 together form a substituted cycloalkyl or
cycloalkenyl group; and X.sub.1 is selected from H, oxo, OH,
O-alkyl, or O--C(O)-alkyl.
5. The pharmaceutical composition of claim 4, wherein the at least
one compound of Formula I has a structure selected from: ##STR70##
wherein R.sub.8 and R.sub.9 are independently H or alkyl; X.sub.2
and X.sub.3 are independently selected from H, oxo, OH, O-alkyl, or
O--C(O)-alkyl; and is selected from or ##STR71##
6. The pharmaceutical composition of claim 2, wherein the at least
one compound of Formula I has the structure: ##STR72## wherein
R.sub.4 and R.sub.5 together form a substituted cycloalkyl or
cycloalkenyl group.
7. The pharmaceutical composition of claim 6, wherein the at least
one compound of Formula I has the structure: ##STR73## wherein
R.sub.6 and R.sub.7 together form a substituted cycloalkyl or
cycloalkenyl group; and X.sub.1 is selected from H, oxo, OH,
O-alkyl, or O--C(O)-alkyl.
8. The pharmaceutical composition of claim 7, wherein the at least
one compound of Formula I has a structure selected from: ##STR74##
wherein R.sub.8 and R.sub.9 are independently H or alkyl; X.sub.2
and X.sub.3 are independently selected from H, oxo, OH, O-alkyl, or
O--C(O)-alkyl; and is selected from or ##STR75##
9. The pharmaceutical composition of claim 2, wherein the at least
one compound of Formula I has the structure: ##STR76## X.sub.4 and
R.sub.10 together form a substituted heteroalicyclic group.
10. The pharmaceutical composition of claim 2, wherein the at least
one compound of Formula I has the structure: ##STR77## wherein
R.sub.11 and R.sub.12 together form a substituted cycloalkyl or
cycloalkenyl group.
11. The pharmaceutical composition of claim 2, wherein the at least
one compound of Formula I has the structure: ##STR78## wherein
R.sub.13 and R.sub.14 are independently H or alkyl; X.sub.5 is
selected from H, oxo, OH, O-alkyl, or O--C(O)-alkyl; and R.sub.15
is alkyl-C(O)O-alkyl.
12. A method for treating or reducing the risk of a
neurodegenerative condition in a subject in need thereof,
comprising administering to the subject a therapeutically effective
amount of the pharmaceutical composition of claim 1.
13. The method of claim 12, wherein, at a concentration of 10
.mu.M, the compound provides at least about 20% protection against
3 mM 3-nitropropionic acid to rat mixed hippocampal cultures.
14. The method of claim 12, wherein the subject in need thereof is
diagnosed as suffering from the neurodegenerative condition prior
to the administration.
15. The method of claim 12, wherein the administration is
parenteral, intravenous, subcutaneous, intra-muscular, trans-nasal,
intra-arterial, transdermal, or respiratory.
16. The method of claim 12, wherein the neurodegenerative condition
is a chronic neurodegenerative condition.
17. The method of claim 16, wherein the chronic neurodegenerative
condition is selected from Alzheimer's Disease, multiple sclerosis,
Huntington's Disease, or Parkinson's Disease.
18. The method of claim 16, wherein the chronic neurodegenerative
condition is AIDS related dementia.
19. The method of claim 16, wherein the chronic neurodegenerative
condition is Amyotrophic Lateral Sclerosis.
20. The method of claim 16, wherein the chronic neurodegenerative
condition is a retinal disease.
21. The method of claim 16, wherein the chronic neurodegenerative
condition is epilepsy.
22. The method of claim 12, wherein the neurodegenerative condition
is an acute neurodegenerative condition.
23. The method of claim 22, wherein the acute neurodegenerative
condition is stroke.
24. The method of claim 23, wherein the stroke is selected from an
acute thromboembolic stroke, a focal ischemia, a global ischemia,
or a transient ischemic attack.
25. The method of claim 22, wherein the acute neurodegenerative
condition is an ischemia resulting from a surgical technique
involving prolonged halt of blood flow to the brain.
26. The method of claim 22, wherein the acute neurodegenerative
condition is selected from head trauma, spinal trauma, optic nerve
stroke, anterior ischemic optic neuropathy, or traumatic optic
neuropathy.
27. The method of claim 16, wherein the chronic neurodegenerative
condition is glaucoma, optic neuritis, compressive optic
neuropathy, or a hereditary neuropathy.
28. The method of claim 16, wherein the chronic neurodegenerative
condition is schizophrenia.
29. A method for treating multiple sclerosis in a subject in need
thereof, comprising administering to the subject a composition
comprising a therapeutically effective amount of the compound of
claim 1.
30. A method for treating AIDS-related dementia in a subject in
need thereof, comprising administering to the subject a composition
comprising a therapeutically effective amount of the compound of
claim 1.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/837,365, entitled "Role of Liminoid
Compounds as Neuroprotective Agents," filed Aug. 11, 2006, the
contents of which are incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] Neurodegenerative conditions such as Alzheimer's Disease,
multiple sclerosis, AIDS-related dementia, Huntington's Disease,
stroke, and spinal cord trauma are characterized by extensive loss
of neurons or glia.
SUMMARY OF THE INVENTION
[0003] Described herein are neuroprotective compounds. Also
described herein are pharmaceutical formulations comprising such
neuroprotective compounds, and methods for using such
neuroprotective compounds in the prophylaxis and treatment of
neurodegenerative conditions.
[0004] Accordingly, in one aspect provided herein is a
pharmaceutical composition comprising at least one compound having
the structure of Formula I: ##STR1## wherein each compound of
Formula I is in a substantially purified form; and Y.sub.1 is O and
Y.sub.2 is OH, O-alkyl, O-(hydroxyalkyl) or O-(alkoxyalkyl); or
Y.sub.1 and Y.sub.2 together form a furan group; R.sub.1 and
R.sub.2 together form a substituted cycloalkyl or cycloalkenyl
group; and is selected from , ##STR2## wherein X.sub.4 and R.sub.2
together form a substituted heteroalicyclic group provided that
R.sub.1 is H; or pharmaceutically acceptable salts, esters,
prodrugs, or metabolites thereof; or ester derivatives, saccharide
derivatives, or --(CH.sub.2CH.sub.2O).sub.nCH.sub.3 derivatives
thereof, where n is 1 to 100; and a pharmaceutically acceptable
excipient.
[0005] In a further embodiment, the at least one compound of
Formula I has a structure selected from: ##STR3##
[0006] In a further embodiment, the at least one compound of
Formula I has the structure: ##STR4## wherein R.sub.4 and R.sub.5
together form a substituted cycloalkyl or cycloalkenyl group.
[0007] In a further embodiment, the at least one compound of
Formula I has the structure: ##STR5## wherein R.sub.6 and R.sub.7
together form a substituted cycloalkyl or cycloalkenyl group; and
X.sub.1 is selected from H, oxo, OH, O-alkyl, O-(hydroxyalkyl),
O-(alkoxyalkyl), or O--C(O)-alkyl.
[0008] In a further embodiment, the at least one compound of
Formula I has a structure selected from: ##STR6## wherein R.sub.8
and R.sub.9 are independently H or alkyl; X.sub.2 and X.sub.3 are
independently selected from H, oxo, OH, O-alkyl, O-(hydroxyalkyl),
O-(alkoxyalkyl), or O--C(O)-alkyl; and is selected from or
##STR7##
[0009] In a further embodiment, the at least one compound of
Formula I has the structure: ##STR8## wherein R.sub.4 and R.sub.5
together form a substituted cycloalkyl or cycloalkenyl group.
[0010] In a further embodiment, the at least one compound of
Formula I has the structure: ##STR9## wherein R.sub.6 and R.sub.7
together form a substituted cycloalkyl or cycloalkenyl group; and
X.sub.1 selected from H, oxo, OH, O-alkyl, O-(hydroxyalkyl),
O-(alkoxyalkyl), or O--C(O)-alkyl.
[0011] In a further embodiment, the at least one compound of
Formula I has a structure selected from: ##STR10## wherein R.sub.8
and R.sub.9 are independently H or alkyl; X.sub.2 and X.sub.3 are
independently selected from H, oxo, OH, O-alkyl, O-(hydroxyalkyl),
O-(alkoxyalkyl), or O--C(O)-alkyl; and is selected from or
##STR11##
[0012] In a further embodiment, the at least one compound of
Formula I has the structure: ##STR12## X.sub.4 and R.sub.10
together form a substituted heteroalicyclic group.
[0013] In a further embodiment, the at least one compound of
Formula I has the structure: ##STR13## wherein R.sub.11 and
R.sub.12 together form a substituted cycloalkyl or cycloalkenyl
group.
[0014] In a further embodiment, the at least one compound of
Formula I has the structure: ##STR14## wherein R.sub.13 and
R.sub.14 are independently H or alkyl; X.sub.5 is selected from H,
oxo, OH, O-alkyl, O-(hydroxyalkyl), O-(alkoxyalkyl), or
O--C(O)-alkyl; and R.sub.15 is alkyl-C(O)O-alkyl.
[0015] In another aspect provided herein is a pharmaceutical
composition comprising at least one compound having the structure
of Formula II: ##STR15## wherein Het.sub.o is a substituted or
unsubstituted oxygen-containing aromatic or non-aromatic
heterocycle; L is a bond or an alkylene group; each is
independently selected from , ##STR16## or ##STR17## provided that
no two adjacent groups are adjacent or ##STR18## groups; each
X.sub.20, X.sub.21, X.sub.22, X.sub.23, and X.sub.24 is
independently selected from H, oxo, OH, OC(O)-alkyl,
O-(hydroxyalkyl), O-(alkoxyalkyl), or O-alkyl; each R.sub.20,
R.sub.21, and R.sub.22 is selected from H or alkyl; or any two of
X.sub.20, X.sub.21, X.sub.22, X.sub.23, X.sub.24, R.sub.20,
R.sub.21, or R.sub.22 can form an optionally substituted
oxygen-containing heterocycle; or pharmaceutically acceptable
salts, esters, prodrugs, or metabolites thereof; or ester
derivatives, saccharide derivatives, or
--(CH.sub.2CH.sub.2O).sub.nCH.sub.3 derivatives thereof, where n is
1 to 100; and a pharmaceutically acceptable excipient.
[0016] In a further embodiment, the Het.sub.o is an unsubstituted
furanyl group. In a further or alternative embodiment, L is a
bond.
[0017] In a further or alternative embodiment, R.sub.21, and
R.sub.22 are CH.sub.3. In a further embodiment at least one of
groups is a . In a further or alternative embodiment, at least one
of groups is a ##STR19## In a further or alternative embodiment, at
least one of groups is a ##STR20## In a further or alternative
embodiment, X.sub.20 is an oxo group. In a further or alternative
embodiment, the compound of Formula II is selected from:
##STR21##
[0018] In a further or alternative embodiment, the pharmaceutical
composition has a therapeutically effective amount of a compound
presented in Tables 2 or 3, along with pharmaceutically acceptable
excipients. In a further or alternative embodiment, the
pharmaceutical composition has a therapeutically effective amount
of a compound isolated from the plant families of order Rutales,
including in Maliaceae and Rutaceae. In a further or alternative
embodiment, the pharmaceutical composition has a therapeutically
effective amount of a compound derived from a
4,4,8-trimethyl-17-furanylsteroid skeleton. In a further or
alternative embodiment, the pharmaceutical composition has a
therapeutically effective amount of a compound is a
tetranortriterpenoid.
[0019] In another aspect provided herein is a method for treating
or reducing the risk of a neurodegenerative condition in a subject
in need thereof by administering to the subject a therapeutically
effective amount of any of the above-described neuroprotective
compound compositions. In some embodiments, the sole active
ingredient in the pharmaceutical composition administered to the
subject is a neuroprotective compound disclosed herein. In some
embodiments, the composition to be administered to the subject
comprises a neuroprotective compound that at a concentration of 10
.mu.M provides at least 6% (e.g., at least 20%, 50%, or 70%)
protection against 3 mM 3-nitropropionic acid to rat mixed
hippocampal cultures. In some embodiments, the subject is diagnosed
as suffering from the neurodegenerative condition prior to
administration of the composition. In some embodiments,
administration of the composition to the subject is parenteral,
intravenous, subcutaneous, intra-muscular, trans-nasal,
intra-arterial, transdermal, or respiratory. In some embodiments,
the subject to be treated is administered, in addition to one of
the above-described compositions, a composition comprising a
therapeutically effective amount of a polyphenol (e.g., resveratrol
or epigallocatechin 3-gallate) or an antioxidant compound (e.g.,
Vitamin C or Vitamin E).
[0020] In a related aspect, the subject to be treated is suffering
from a chronic neurodegenerative condition. In some embodiments,
the chronic neurodegenerative condition is Alzheimer's disease. In
some embodiments, in addition to administering one of the
above-described neuroprotective compositions to a subject suffering
from Alzheimer's Disease, the level of one or more Alzheimer's
Disease prognostic biomarkers is determined in a biological sample
from the subject. In some embodiments, one or more Alzheimer's
Disease prognostic biomarkers to be assayed comprise tau protein,
phospho-tau protein, .beta.-amyloid.sub.1-42 peptide,
.beta.-amyloid.sub.1-40 peptide, C1q protein, IL-6 protein, ApoE
protein, .alpha.-1-antichymotrypsin protein, oxysterol,
isoprostane, 3-nitrotyrosine, or any combination thereof. In some
embodiments, the subject to be treated is suffering from multiple
sclerosis. In some embodiments, the subject to be treated is
suffering from Huntington's disease. In some embodiments, the
subject to be treated is suffering from AIDS-related dementia. In
some embodiments, the subject to be treated is suffering from
schizophrenia. In some embodiments, the subject to be treated is
suffering from Amyotrophic Lateral Sclerosis. In some embodiments,
the subject to be treated is suffering from a retinal disease. In
some embodiments, the subject to be treated is suffering from
glaucoma, optic neuritis, compressive optic neuropathy, or a
hereditary neuropathy. In some embodiments, the subject to be
treated is suffering from epilepsy.
[0021] In a related aspect, the subject to be treated is suffering
from an acute neurodegenerative condition. In some embodiments, the
subject to be treated is suffering from a stroke (e.g., an acute
thromboembolic stroke, a focal ischemia, a global ischemia, or a
transient ischemic attack). In some embodiments, the subject to be
treated is suffering from ischemia resulting from a surgical
technique involving prolonged halt of blood flow to the brain. In
some embodiments, the subject to be treated is suffering from head
trauma. In some embodiments, the subject to be treated is suffering
from spinal trauma. In some embodiments, the subject to be treated
is suffering from an optic nerve stroke, anterior ischemic optic
neuropathy, or traumatic optic neuropathy.
[0022] In another aspect provided herein is a method for treating
multiple sclerosis in a subject in need thereof by administering to
the subject a therapeutically effective amount of any of the
above-described neuroprotective compound compositions.
[0023] In a further aspect provided herein is a method for treating
AIDS-related dementia in a subject in need thereof by administering
to the subject a therapeutically effective amount of any of the
above-described neuroprotective compound compositions.
INCORPORATION BY REFERENCE
[0024] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a bar graph showing the results of an in vitro
screen for neuroprotective compounds performed on a library of
compounds (Spectrum Collection from MicroSource Discovery Systems,
Inc.) containing FDA approved compounds, natural products, and
other bioactive compounds.
[0026] FIG. 2 shows the chemical structures of five of the
neuroprotective compounds as described herein.
[0027] FIG. 3 is a bar graph showing the effect of a series of
3-nitropropionic acid (3-NP) concentrations (0.5-10 mM) on survival
of mixed hippocampal cell cultures in a
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]
(MTT) viability assay. Absorbance at 595 nm (corresponding to the
absorbance of the reduced MTT formazen product) is directly
proportional to cell viability.
[0028] FIG. 4 is a set of bar graphs showing the results of an MTT
viability assay of mixed hippocampal cell cultures in which (A) the
effect of 3-NP (3 mM) is tested alone or in the presence of
neuroprotective antioxidants GPI 1046 (1 .mu.M) or Resveratrol (25
.mu.M); and (B) the effect of HIV Tat protein (500 nM) is tested
alone or in the GPI 1046 (10 .mu.M) or Resveratrol (10 .mu.M). The
statistical significance of data compared to either 3-NP or Tat
treatment (right panel) is indicated by ANOVA, with Newman-Keuls
post hoc comparisons. ** indicates p<0.01; *** indicates
p<0.001 (n=8 per treatment). Each experiment was performed in
triplicate.
[0029] FIG. 5 is a set of bar graphs showing the results of an MTT
viability assay of mixed hippocampal cell culture in which the
effect of 3-NP (3 mM) is tested alone or in the presence of (A)
Khivorin (10 .mu.M); (B) Isogedunin (10 .mu.M); (C) Angolensic
acid, ME; and (D) Odoratone. The statistical significance of data
compared to 3-NP treatment is indicated by ANOVA, with Newman-Keuls
post hoc comparisons. ** indicates p<0.01; *** p<0.001.
[0030] FIG. 6 is a set of bar graphs showing the results of an MTT
viability assay of mixed hippocampal cell cultures in which the
effect of 3-NP (3 mM) is tested alone or in the presence of a range
of concentrations of modified terpenoid compounds (A) Khivorin
(0.1-10 .mu.M); (B) Isogedunin (0.1-10 .mu.M); (C) Nomilin (0.5-10
.mu.M); and (D) Limonin (0.1-10 .mu.M).
[0031] FIG. 7 is a set of bar graphs showing the results of an MTT
viability assay of mixed hippocampal cell cultures in which the
effect of the neurotoxic HIV Tat protein (500 nM) is tested alone
or in the presence of a series of concentrations of (A) Isogedunin
(0.5-10 .mu.M) and (B) Limonin (0.1-10 .mu.M).
[0032] FIG. 8 is a set of bar graphs showing the results of an MTT
viability assay of mixed hippocampal cell cultures in which the
effect of (A) NMDA (100 .mu.M) or (B) 6-OHDA (100 .mu.M) is tested
alone or in the presence of a range of concentrations of Limonin
(0.1-10 .mu.M).
[0033] FIG. 9 is a set of bar graphs showing the results of an MTT
viability assay of human fetal neuron cultures in which the effect
of (A) 3-NP (3 mM) or (B) 6-OHDA (100 .mu.M) is tested alone or in
the presence of a range of concentrations of Limonin (0.1-10
.mu.M).
DETAILED DESCRIPTION OF THE INVENTION
[0034] The appended claims particularly point out features set
forth herein. A better understanding of the features and advantages
of the present disclosure will be obtained by reference to the
following detailed description that sets forth illustrative
embodiments, in which the principles described herein are
utilized.
[0035] Disclosed herein are neuroprotective compounds that decrease
induced or spontaneous neuronal or glial cell death, compositions
that include the neuroprotective compounds, and methods of their
use in treating a neurodegenerative condition. The neuroprotective
compounds described herein are shown to promote survival of neurons
and glia in response to cytotoxic challenges, e.g., oxidative
stress. Cytotoxic challenges are associated with a number of
neurodegenerative conditions (see, e.g., Lin et al. (2006), Nature,
443(7113):787-795); contact with neurotoxic viral proteins such as
HIV Tat (see King et al. (2006), Microbes Infect 2006,
8(5):1347-1357); and hypoxia (see Won et al. (2002), J Biochem Mol
Biol 2002, 35(1):67-86). Accordingly, the neuroprotective
compounds, compositions, methods described herein can be used to
treat a variety of neurodegenerative conditions.
[0036] In some embodiments, the methods described herein are used
to treat a chronic neurodegenerative disease, which includes, but
is not limited to, Alzheimer's Disease, Multiple Sclerosis,
HIV-associated dementia, Schizophrenia, Huntington's Disease,
Parkinson's Disease, Amyotrophic Lateral Sclerosis, Multiple System
Atrophy, degenerative retinal disease (e.g., macular degeneration),
optic neuropathies (e.g., glaucoma, optic nerve stroke, optic
neuritis, anterior ischemic optic neuropathy, traumatic optic
neuropathy, compressive optic neuropathy, or hereditary
neuropathies, such as Leber's hereditary optic neuropathy),
Schizophrenia, Pick's disease, Alexander disease, Alper's disease,
Ataxia telangiectasia, Batten disease (also known as
Spielmeyer-Vogt-Sjogren-Batten disease), Canavan disease, Cockayne
syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,
Kennedy's disease, Krabbe disease, Lewy body dementia,
Machado-Joseph disease (Spinocerebellar ataxia type 3),
Neuroborreliosis, Pelizaeus-Merzbacher Disease, Primary lateral
sclerosis, Prion diseases, Refsum's disease, Sandhoff disease,
Schilder's disease, Spielmeyer-Vogt-Sjogren-Batten disease (also
known as Batten disease), Spinocerebellar ataxia (multiple types
with varying characteristics), Spinal muscular atrophy,
Steele-Richardson-Olszewski disease, Tabes dorsalis, or any
combination thereof.
[0037] In some embodiments, the methods described herein can be
used to treat acute neurodegenerative conditions, which include,
but are not limited to stroke (e.g., thromboembolic stroke, focal
ischemia, global ischemia, or transient ischemic attack), ischemia
resulting from a surgical technique involving prolonged halt of
blood flow to the brain, head trauma, spinal trauma, or any
combination thereof.
[0038] Symptoms, diagnostic tests, and prognostic tests for each of
the above-mentioned conditions are described in, e.g., the
Diagnostic and Statistical Manual of Mental Disorders, 4.sup.th
ed., 1994, Am. Psych. Assoc.; and Harrison's Principles of Internal
Medicine.COPYRGT.," 16th ed., 2004, The McGraw-Hill Companies,
Inc.
[0039] For example, where the subject is at risk of or is suffering
from multiple sclerosis, a set of standard criteria, such as the
"McDonald Criteria" can be used for prognosis/diagnosis. See
McDonald et al. (2001), Ann Neurol, 50(1):121-127. Magnetic
resonance imaging (MRI) of the brain and spine can be used to
evaluate individuals with suspected multiple sclerosis. MRI shows
areas of demyelination as bright lesions on T2-weighted images or
FLAIR (fluid attenuated inversion recovery) sequences. Gadolinium
contrast is used to demonstrate active plaques on T1-weighted
images. Further, a prognostic biomarker assay of cerebrospinal
fluid (CSF) obtained by lumbar puncture can provide evidence of
chronic inflammation of the central nervous system. Specifically,
CSF is tested for oligoclonal bands, which are immunoglobulins
found in 85% to 95% of people with definite MS, albeit not
exclusively in MS patients. Additional criteria for diagnosis of
multiple sclerosis include, e.g., a reduction in visual evoked
potentials and somatosensory evoked potentials, which are
indicative of demyelination.
[0040] Where a neurodegenerative disorder affects a cognitive
ability, a subject can be diagnosed by any one of a number of
standardized cognitive assays, e.g., the Mini-Mental State
Examination, the Blessed Information Memory Concentration assay, or
the Functional Activity Questionnaire. See, e.g., Adelman et al.
(2005), Am. Family Physician, 71(9):1745-1750. Indeed, in some
cases a subject can also be diagnosed as having a high risk of
developing a chronic neurodegenerative condition (e.g., Alzheimer's
disease), even in the absence of overt symptoms. For example, the
risk of Alzheimer's disease in a subject can be determined by
detecting a decrease in the volumes of the subject's hippocampus
and amygdala, using magnetic resonance imaging. See, e.g., den
Heijer et al. (2006), Arch Gen Psychiatry, 63(1):57-62. Assay of
prognostic biomarkers in a sample from a subject are also useful in
prognosis or diagnosis of a chronic neurodegenerative condition.
For example, where the chronic neurodegenerative condition is
Alzheimer's disease, prognostic biomarkers include, but are not
limited to, total tau protein, phospho-tau protein,
.beta.-amyloid.sub.1-42 peptide, .beta.-amyloid.sub.1-40 peptide,
complement component 1, q subcomponent (C1q) protein, interleukin 6
(IL-6) protein, apolipoprotein E (APOE) protein,
.alpha.-1-antichymotrypsin protein, oxysterol (e.g.,
24S-hydroxycholesterol), isoprostane (e.g., an F2-isoprostane),
3-nitrotyrosine, homocysteine, or cholesterol, or any combination
thereof, e.g., the ratio of .beta.-amyloid.sub.1-42 peptide to
.beta.-amyloid.sub.1-40 peptide.
[0041] The type of biological sample utilized in prognostic
Alzheimer's biomarker assays will vary depending on the prognostic
biomarker to be measured. Further, the relationship between the
level of a prognostic biomarker and Alzheimer's risk varies
depending on the particular biomarker, as well as on the biological
sample in which the level of the biomarker is determined. In other
words, the level of the biomarker in a biological sample may be
directly correlated or inversely correlated with the risk of
Alzheimer's Disease, as summarized in Table 1. TABLE-US-00001 TABLE
1 ALZHEIMER'S DISEASE PROGNOSTIC BIOMARKERS Biological Correlation
to Biomarker Sample Type Dementia Risk Reference tau protein
cerebrospinal increased Hampel et al. (2004), Mol Psychiatry, fluid
(CSF) 9: 705-710 phospho-tau protein CSF increased Hampel et al.
(2004), Arch Gen Psychiatry, 61: 95-102 Hansson et al. (2006),
Lancet Neurol, 5(3): 228-234 .beta.-amyloid.sub.1-42 peptide CSF
decreased Hampel et al. (2004), Mol Psychiatry, 9: 705-710 Ratio of
.beta.-amyloid.sub.1-42 plasma decreased Graff-Radford et al.
(2007), Arch Neurol, peptide to .beta.-amyloid.sub.1-40 64(3):
354-362; peptide CSF decreased Hansson et al. (2007), Dement
Geriatr Cogn Disord, 23(5): 316-20 C1q protein CSF decreased Smyth
et al. (1994), Neurobiol Aging, 15(5): 609-614 IL-6 protein plasma
increased Licastro et al. (2000), J Neuroimmunol, 103: 97-102; CSF
increased Sun et al. (2003), Dement Geriatr Cogn Disord, 16(3):
136-44 APOE protein CSF increased Fukuyama et al. (2000), Eur
Neurol, 43(3): 161-169 .alpha.-1-antichymotrypsin plasma increased
Dik et al. (2005), Neurology, 64(8): protein 1371-1377. oxysterol
CSF increased Papassotiropoulos et al. (2002), J Psychiatr Res,
36(1): 27-32 isoprostane CSF increased Montine et al. (2005),
Antioxid Redox Signal, 7(1-2): 269-275 3-nitrotyrosine CSF
increased Tohgi et al. (1999), Neurosci Lett, 269(1): 52-54
homocysteine plasma increased Seshadri et al. (2002), N Engl J Med,
346(7): 476-83 cholesterol plasma increased Panza et al. (2006),
Neurobiol Aging, 27(7): 933-940
[0042] Animal models are useful for establishing a range of
therapeutically effective doses of neuroprotective compounds for
treating any of the foregoing diseases. For example, animal models
of chronic neurodegenerative conditions have been established,
e.g., for Alzheimer's disease, multiple sclerosis, amyotrophic
lateral sclerosis, multiple system atrophy, and Huntington's
disease. See, e.g., Spires et al. (2005), NeuroRx., 2(3):447-464;
Gold et al. (2006), Brain, 129(8): 1953-1971; Wong et al. (2002),
Nat. Neurosci., 5(7):633-639, Stefanova et al. (2005), Am J Pathol,
166(3): 869-876, Tadros et al. (2005), Pharmacol Biochem Behav;
82(3):574-582. These animal models develop a chronic
neurodegenerative condition that is manifested behaviorally by
impaired learning, memory, or locomotion. Cognitive abilities, as
well as motor functions in non-human animals suffering from a
chronic neurodegenerative condition can be assessed using a number
of behavioral tasks. Well-established sensitive learning and memory
assays include the Morris Water Maze (MWM), context-dependent fear
conditioning, cued-fear conditioning, and context-dependent
discrimination. See, e.g., Anger (1991), Neurotoxicology,
12(3):403-413. Locomotor behavior, e.g., following spinal trauma,
is commonly assessed using a 21-point open field locomotion score
assay developed by Basso, Beattie, and Bresnahan (BBB) (Basso, et
al. (1995), J Neurotrauma, 12(1): 1-21). Such animal models are
suitable for testing effective dose ranges for the neuroprotective
compounds and Compositions described herein as well as for
identifying additional neuroprotective compounds.
Certain Chemical Terminology
[0043] All patents, patent applications, published materials
referred to throughout the entire disclosure herein, unless noted
otherwise, are incorporated by reference in their entirety. In the
event that there are a plurality of definitions for terms herein,
those in this section prevail. Where reference is made to a URL or
other such identifier or address, it is understood that such
identifiers can change and particular information on the internet
can come and go, but equivalent information can be found by
searching the internet. Reference thereto evidences the
availability and public dissemination of such information.
[0044] It is to be understood that the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of any subject matter
claimed. In this application, the use of the singular includes the
plural unless specifically stated otherwise. It must be noted that,
as used in the specification and the appended claims, the singular
forms "a," "an" and "the" include plural referents unless the
context clearly dictates otherwise. In this application, the use of
"or" means "and/or" unless stated otherwise. Furthermore, use of
the term "including" as well as other forms, such as "include,"
"includes," and "included," is not limiting. The section headings
used herein are for organizational purposes only and are not to be
construed as limiting the subject matter described. All documents,
or portions of documents, cited in the application including, but
not limited to, patents, patent applications, articles, books,
manuals, and treatises are hereby expressly incorporated by
reference in their entirety for any purpose.
[0045] Definition of standard chemistry terms may be found in
reference works, including Carey and Sundberg "ADVANCED ORGANIC
CHEMISTRY 4.sup.TH ED." Vols. A (2000) and B (2001), Plenum Press,
New York. Unless otherwise indicated, methods of mass spectroscopy,
NMR, HPLC, protein chemistry, biochemistry, recombinant DNA
techniques and pharmacology are employed. Standard techniques can
be used for chemical syntheses, chemical analyses, pharmaceutical
preparation, formulation, and delivery, and treatment of patients.
Standard techniques can be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection).
[0046] As used herein, C.sub.1-C.sub.x includes C.sub.1-C.sub.2,
C.sub.1-C.sub.3 . . . C.sub.1-C.sub.x.
[0047] An "alkyl" group refers to a straight-chained, branched or
cyclic aliphatic hydrocarbon group. The "alkyl" moiety may have 1
to 10 carbon atoms (whenever it appears herein, a numerical range
such as "1 to 10" refers to each integer in the given range; e.g.,
"1 to 10 carbon atoms" means that the alkyl group may have 1 carbon
atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10
carbon atoms, although the present definition also covers the
occurrence of the term "alkyl" where no numerical range is
designated). The alkyl group of the compounds described herein may
be designated as "C.sub.1-C.sub.4 alkyl" or similar designations.
By way of example only, "C.sub.1-C.sub.4 alkyl" indicates that
there are one to four carbon atoms in the alkyl chain, i.e., the
alkyl chain is selected from among methyl, ethyl, propyl,
iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Thus
C.sub.1-C.sub.4 alkyl includes C.sub.1-C.sub.2 alkyl and
C.sub.1-C.sub.3 alkyl. Alkyl groups can be substituted or
unsubstituted. Typical alkyl groups include, but are in no way
limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,
tertiary butyl, pentyl, hexyl, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, and the like.
[0048] An "alkoxy" group refers to a (alkyl)O-- group, where alkyl
is as defined herein.
[0049] "Hydroxyalkyl" refers to an alkyl radical, as defined
herein, substituted with at least one hydroxy group. Non-limiting
examples of a hydroxyalkyl include, but are not limited to,
hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl,
1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl,
4-hydroxybutyl, 2,3-dihydroxypropyl,
1-(hydroxymethyl)-2-hydroxyethyl, 2,3-dihydroxybutyl,
3,4-dihydroxybutyl and 2-(hydroxymethyl)-3-hydroxypropyl,
2-hydroxyethyl, 2,3-dihydroxypropyl, and
1-(hydroxymethyl)-2-hydroxyethyl.
[0050] "Alkoxyalkyl" refers to an alkyl radical, as defined herein,
substituted with at least one alkoxy group, as defined herein.
[0051] An "amide" is a chemical moiety with the formula --C(O)NHR
or --NHC(O)R, where R is selected from among alkyl, cycloalkyl,
aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic
(bonded through a ring carbon). An amide moiety may form a linkage
between an amino acid or a peptide molecule and a compound
described herein, thereby forming a prodrug. Any amine, or carboxyl
side chain on the compounds described herein can be amidified. The
procedures and specific groups to make such amides can be found in
Greene and Wuts, Protective Groups in Organic Synthesis, 3.sup.rd
Ed., John Wiley & Sons, New York, N.Y., 1999, which is
incorporated herein by reference in its entirety.
[0052] The term "bond" or "single bond" refers to a chemical bond
between two atoms, or two moieties when the atoms joined by the
bond are considered to be part of larger substructure.
[0053] The term "carbocyclic" or "carbocycle" refers to a compound
which contains one or more covalently closed ring structures, and
that the atoms forming the backbone of the ring are all carbon
atoms. The term thus distinguishes carbocyclic from heterocyclic
rings in which the ring backbone contains at least one atom which
is different from carbon. Carbocycles include cycloalkyls and
aryls.
[0054] The term "cycloalkyl" refers to a monocyclic or polycyclic
radical that contains only carbon and hydrogen, and may be
saturated, partially unsaturated, or fully unsaturated. Cycloalkyl
groups include groups having from 3 to 10 ring atoms. Illustrative
examples of cycloalkyl groups include the following moieties:
##STR22## and the like. Depending on the structure, an cycloalkyl
group can be a monoradical or a diradical (e.g., an cycloalkylene
group).
[0055] "Cycloalkylalkyl" means an alkyl radical, as defined herein,
substituted with a cycloalkyl group. Non-cycloalkylalkyl limiting
cycloalkylalkyl groups include cyclopropylmethyl, cyclobutylmethyl,
cyclopentylethyl, cyclohexylmethyl, and the like.
[0056] The term "ester" refers to a chemical moiety with formula
--COOR, where R is selected from among alkyl, cycloalkyl, aryl,
heteroaryl (bonded through a ring carbon) and heteroalicyclic
(bonded through a ring carbon). Any hydroxy, or carboxyl side chain
on the compounds described herein can be esterified. The procedures
and specific groups to make such esters can be found in Greene and
Wuts, Protective Groups in Organic Synthesis, 3.sup.rd Ed., John
Wiley & Sons, New York, N.Y., 1999, which is incorporated
herein by reference in its entirety.
[0057] The term "halo" or, alternatively, "halogen" or "halide"
means fluoro, chloro, bromo or iodo.
[0058] The terms "haloalkyl," "haloalkenyl," "haloalkynyl" and
"haloalkoxy" include alkyl, alkenyl, alkynyl and alkoxy structures
in which at least one hydrogen is replaced with a halogen atom. In
certain embodiments in which two or more hydrogen atoms are
replaced with halogen atoms, the halogen atoms are all the same as
one another. In other embodiments in which two or more hydrogen
atoms are replaced with halogen atoms, the halogen atoms are not
all the same as one another. The terms "fluoroalkyl" and
"fluoroalkoxy" include haloalkyl and haloalkoxy groups
respectively, in which the halo is fluorine. In certain
embodiments, haloalkyls are optionally substituted.
[0059] As used herein, the terms "heteroalkyl" "heteroalkenyl" and
"heteroalkynyl" include optionally substituted alkyl, alkenyl and
alkynyl radicals in which one or more skeletal chain atoms are
selected from an atom other than carbon, e.g., oxygen, nitrogen,
sulfur, silicon, phosphorus or combinations thereof.
[0060] The term "heteroatom" refers to an atom other than carbon or
hydrogen. Heteroatoms are typically independently selected from
among oxygen, sulfur, nitrogen, silicon and phosphorus, but are not
limited to these atoms. In embodiments in which two or more
heteroatoms are present, the two or more heteroatoms can all be the
same as one another, or some or all of the two or more heteroatoms
can each be different from the others.
[0061] As used herein, the term "ring" refers to any covalently
closed structure. Rings include, for example, carbocycles (e.g.,
aryls and cycloalkyls), heterocycles (e.g., heteroaryls and
non-aromatic heterocycles), aromatics (e.g. aryls and heteroaryls),
and non-aromatics (e.g., cycloalkyls and non-aromatic
heterocycles). Rings can be optionally substituted. Rings can be
monocyclic or polycyclic.
[0062] As used herein, the term "ring system" refers to one, or
more than one ring.
[0063] As used herein, the term "non-aromatic heterocycle",
"heterocycloalkyl" or "heteroalicyclic" refers to a non-aromatic
ring wherein one or more atoms forming the ring is a heteroatom. A
"non-aromatic heterocycle" or "heterocycloalkyl" group refers to a
cycloalkyl group that includes at least one heteroatom selected
from nitrogen, oxygen and sulfur. The radicals may be fused with an
aryl or heteroaryl. Heterocycloalkyl rings can be formed by three,
four, five, six, seven, eight, nine, or more than nine atoms.
Heterocycloalkyl rings can be optionally substituted. In certain
embodiments, non-aromatic heterocycles contain one or more carbonyl
or thiocarbonyl groups such as, for example, oxo- and
thio-containing groups. Examples of heterocycloalkyls include, but
are not limited to, lactams, lactones, cyclic imides, cyclic
thioimides, cyclic carbamates, tetrahydrothiopyran, 4H-pyran,
tetrahydropyran, piperidine, 1,3-dioxin, 1,3-dioxane, 1,4-dioxin,
1,4-dioxane, piperazine, 1,3-oxathiane, 1,4-oxathiin,
1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide,
succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine,
hydantoin, dihydrouracil, morpholine, trioxane,
hexahydro-1,3,5-triazine, tetrahydrothiophene, tetrahydrofuran,
pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazoline,
pyrazolidine, imidazoline, imidazolidine, 1,3-dioxole,
1,3-dioxolane, 1,3-dithiole, 1,3-dithiolane, isoxazoline,
isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline,
thiazolidine, and 1,3-oxathiolane. Illustrative examples of
heterocycloalkyl groups, also referred to as non-aromatic
heterocycles, include: ##STR23## the like. The term heteroalicyclic
also includes all ring forms of the carbohydrates, including but
not limited to the monosaccharides, the disaccharides and the
oligosaccharides. Depending on the structure, a heterocycloalkyl
group can be a monoradical or a diradical (i.e., a
heterocycloalkylene group).
[0064] "Heterocycloalkylalkyl" refers to an alkyl group, as defined
herein, substituted with a heterocycloalkyl, as defined herein.
[0065] The term "heterocycle" refers to heteroaromatic and
heteroalicyclic groups containing one to four heteroatoms each
selected from O, S and N, wherein each heterocyclic group has from
4 to 10 atoms in its ring system, and with the proviso that the
ring of said group does not contain two adjacent O or S atoms.
Herein, whenever the number of carbon atoms in a heterocycle is
indicated (e.g., C.sub.1-C.sub.6 heterocycle), at least one other
atom (the heteroatom) must be present in the ring. Designations
such as "C.sub.1-C.sub.6 heterocycle" refer only to the number of
carbon atoms in the ring and do not refer to the total number of
atoms in the ring. It is understood that the heterocylic ring can
have additional heteroatoms in the ring. Designations such as "4-6
membered heterocycle" refer to the total number of atoms that are
contained in the ring (i.e., a four, five, or six membered ring, in
which at least one atom is a carbon atom, at least one atom is a
heteroatom and the remaining two to four atoms are either carbon
atoms or heteroatoms). In heterocycles that have two or more
heteroatoms, those two or more heteroatoms can be the same or
different from one another. Heterocycles can be optionally
substituted. Binding to a heterocycle can be at a heteroatom or via
a carbon atom. Non-aromatic heterocyclic groups include groups
having only 4 atoms in their ring system, but aromatic heterocyclic
groups must have at least 5 atoms in their ring system. The
heterocyclic groups include benzo-fused ring systems. An example of
a 4-membered heterocyclic group is azetidinyl (derived from
azetidine). An example of a 5-membered heterocyclic group is
thiazolyl. An example of a 6-membered heterocyclic group is
pyridyl, and an example of a 10-membered heterocyclic group is
quinolinyl. Examples of non-aromatic heterocyclic groups are
pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,
tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl,
piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl,
azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl,
thiepanyl, oxazepinyl, diazepinyl, thiazepinyl,
1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl,
2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl,
dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,
dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,
3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl
and quinolizinyl. Examples of aromatic heterocyclic groups are
pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl,
pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,
oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl,
indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl,
indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl,
pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl,
benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl,
quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The
foregoing groups, as derived from the groups listed above, may be
C-attached or N-attached where such is possible. For instance, a
group derived from pyrrole may be pyrrol-1-yl (N-attached) or
pyrrol-3-yl (C-attached). Further, a group derived from imidazole
may be imidazol-1-yl or imidazol-3-yl (both N-attached) or
imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-attached). The
heterocyclic groups include benzo-fused ring systems and ring
systems substituted with one or two oxo (.dbd.O) moieties such as
pyrrolidin-2-one. Depending on the structure, a heterocycle group
can be a monoradical or a diradical (i.e., a heterocyclene group).
Phosphorous-containing rings include, but are not limited to,
1-oxo-phospholanyl, 1-methyl-1-oxo-phosphinan-4-yl,
1-phenyl-1-oxo-phosphinan-4-yl,
1-(cyclopropylmethyl)-1-oxo-phosphinan-4-yl,
4-methyl-4-oxo-[1,4]azaphosphinan-1-yl,
4-phenyl-4-oxo-[1,4]azaphosphinan-1-yl, and
4-(cyclopropylmethyl)-4-oxo-[1,4]azaphosphinan-1-yl.
[0066] The term "membered ring" can embrace any cyclic structure.
The term "membered" is meant to denote the number of skeletal atoms
that constitute the ring. Thus, for example, cyclohexyl, pyridine,
pyran and thiopyran are 6-membered rings and cyclopentyl, pyrrole,
furan, and thiophene are 5-membered rings.
[0067] The term "moiety" refers to a specific segment or functional
group of a molecule. Chemical moieties are often recognized
chemical entities embedded in or appended to a molecule.
[0068] As used herein, the term "O-carboxy" or "acyloxy" refers to
a group of formula RC(.dbd.O)O--.
[0069] "Alkylcarbonyloxy" refers to a (alkyl)-C(.dbd.O)O--
group.
[0070] As used herein, the term "alkoxycarbonyl" refers to a group
of formula --C(.dbd.O)OR.
[0071] "Carboxy" means a --C(O)OH radical.
[0072] As used herein, the term "acetyl" refers to a group of
formula --C(.dbd.O)CH.sub.3.
[0073] "Acyl" refers to the group --C(O)R.
[0074] The term "optionally substituted" or "substituted" means
that the referenced group may be substituted with one or more
additional group(s) individually and independently selected from
alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy,
alkoxy, aryloxy, alkylthio, arylthio, alkylsulfoxide,
arylsulfoxide, alkylsulfone, arylsulfone, cyano, halo, carbonyl,
isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl,
fluoroalkyl, silyl, and amino, including mono- and di-substituted
amino groups, and the protected derivatives thereof. By way of
example an optional substituents may be L.sub.sR.sub.s, wherein
each L.sub.s is independently selected from a bond, --O--,
--C(.dbd.O)--, --S--, --S(.dbd.O)--, --S(.dbd.O).sub.2--, --NH--,
--NHC(O)--, --C(O)NH--, S(.dbd.O).sub.2NH--, --NHS(.dbd.O).sub.2,
--OC(O)NH--, --NHC(O)O--, -(substituted or unsubstituted
C.sub.1-C.sub.6 alkyl), or -(substituted or unsubstituted
C.sub.2-C.sub.6 alkenyl); and each R.sub.5 is independently
selected from H, (substituted or unsubstituted
C.sub.1-C.sub.4alkyl), (substituted or unsubstituted
C.sub.3-C.sub.6cycloalkyl), heteroaryl, or heteroalkyl.
[0075] The compounds presented herein may possess one or more
stereocenters and each center may exist in the R or S
configuration. The compounds presented herein include all
diastereomeric, enantiomeric, and epimeric forms as well as the
appropriate mixtures thereof. Stereoisomers, if desired, may be
obtained, for example, by the separation of stereoisomers by chiral
chromatographic columns.
[0076] The methods and formulations described herein include the
use of N-oxides, crystalline forms (also known as polymorphs), or
pharmaceutically acceptable salts of compounds described herein, as
well as active metabolites of these compounds having the same type
of activity. In some situations, compounds may exist as tautomers.
All tautomers are included within the scope of the compounds
presented herein. In addition, the compounds described herein can
exist in unsolvated as well as solvated forms with pharmaceutically
acceptable solvents such as water, ethanol, and the like. The
solvated forms of the compounds presented herein are also
considered to be disclosed herein.
Neuroprotective Compounds
[0077] The neuroprotective compounds for use in the pharmaceutical
compositions and methods described herein are compounds of Formula
I, Formula II, and/or compounds found in Tables 2 and 3.
[0078] Neuroprotective compounds suitable for the methods described
herein also can come from a variety of sources including both
natural (e.g., plant extracts) and synthetic. For example,
neuroprotective compounds falling within the class of triterpenes
can be extracted from plants of the order Rutales, e.g., from
extracts of seeds, oils, kernels, leaves and bark of various plants
from the Meliaceae family, including Neem and Mahogany. See, e.g.,
Roy et al. (2006), Biol Pharm Bull, 29(2):191-201.
[0079] The neuroprotective compounds described herein are
identified or characterized in an in vitro cellular assay, e.g., an
assay that determines the viability of neurons or glia in the
presence of a cytotoxic challenge and one or more concentrations of
a candidate neuroprotective compound. Such assays are useful for
identifying and testing the in vitro neuroprotective potency of
candidate neuroprotective compounds.
[0080] For example, a cell viability assay can be used in a
preliminary screen on a large series of compounds each of which is
tested at a fixed concentration. In some embodiments, mixed
hippocampal cell cultures are prepared as described in, e.g.,
Haughey et al. (1999), J Neurochem, 73(4):1363-1374, and Haughey et
al (2004), J Neurosci, 24(1):257-268. Subsequently, the cultures
are pretreated for one hour in the presence of a candidate
neuroprotective agent and then incubated with a cytotoxic agent for
about 18 hours. Examples of cytotoxic agents include, but are not
limited to, oxidative stressors (e.g., 3-nitropropionic acid (3-NP)
or H.sub.2O.sub.2), excitatory amino acids (e.g., kainate), or
neurotoxic proteins (e.g., Hiv Tat, or A.beta. peptides). If the
survival of cell cultures in the presence of the candidate compound
and the cytotoxic agent (e.g., 3-NP) is significantly greater than
that of cultures incubated with the cytotoxic agent alone, the
candidate test compound is considered to have neuroprotective
activity. The level of protection ("percent protection") provided
by the test compound at a given test concentration in vitro can be
expressed as: [ ( Cell .times. .times. Viability .times. .times.
with .times. .times. Cytoxic .times. .times. agent + Test .times.
.times. Compound ) - ( Cell .times. .times. Viability .times.
.times. with .times. .times. Cytoxic .times. .times. agent .times.
.times. alone ) ] [ ( Control .times. .times. Cell .times. .times.
Viability .times. .times. ( medium .times. .times. alone ) - ( Cell
.times. .times. Viability .times. .times. with .times. .times.
Cytotoxic .times. .times. agent .times. .times. alone ) ] .times.
100 ##EQU1##
[0081] In some embodiments, at a concentration of 10 .mu.M, a
neuroprotective compound described herein, provides at least 6%
protection against a cytotoxic agent, i.e., at least 7%, 15%, 18%,
19%, 20%, 23%, 26%, 28%, 32%, 33%, 36%, 38%, 39%, 42%, 50%, 54%,
56%, 58%, 61%, 65%, 69%, 70%, 77%, 82%, 84%, 97%, or any other
percent from at least 6% to 100% protection against the cytotoxic
agent.
[0082] In some embodiments, at a concentration of 10 .mu.M, a
neuroprotective compound described herein, provides greater than
100% protection, which signifies that, in addition to blocking
cytotoxic agent-induced cell death, the neuroprotective compound
also inhibits spontanteous cell death in the control cell cultures
(i.e., cultures exposed to medium alone). In some embodiments, a
neuroprotective compound described herein provides, e.g., 101,
128%, 129, or 151% protection.
[0083] A variety of methods for determining cell viability may be
used. In some embodiments, cell viability is assessed based on the
"MTT" assay method described in Mosmann (1983), J Immunol Methods,
65(1-2):55-63, or a variant thereof. This assay is based on the
ability of a mitochondrial dehydrogenase enzyme from viable cells
to cleave the tetrazolium rings of the pale yellow MTT and form
dark blue formazan crystals, which are trapped in cells and readily
quantified by ELISA spectrophotometry.
[0084] High throughput cell viability assays may be used, and are
particularly useful for screening, with routine effort, a great
number of candidate neuroprotective compounds or structural
variants of identified neuroprotective compounds, e.g., structural
variants of Formula I or Formula II described herein. See, e.g., J
Biomol Screen, 9(6):506-515; Carrier et al. (2006), J Neurosci
Methods, 154(1-2):239-244; See, e.g., In addition, high throughput
screening systems are commercially available (see, e.g., Zymark
Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio;
Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems,
Inc., Natick, Mass., etc.). These systems typically automate entire
procedures including all sample and reagent pipetting, liquid
dispensing, timed incubations, and final readings of the microplate
in detector(s) appropriate for the assay. Automated systems thereby
allow the identification and characterization of a large number of
neuroprotective compounds of Formula I or Formula II without undue
effort.
[0085] The neuroprotective compounds described herein, e.g., those
of Formula I or Formula II, can be used for the manufacture of a
medicament for treating any of the foregoing neurodegenerative
conditions (e.g., multiple sclerosis, Alzheimer's disease, stroke,
or spinal cord injury).
[0086] In some embodiments, a neuroprotective compound used for the
methods described herein in vitro ED.sub.50 for neuroprotection of
less than 100 .mu.M (e.g., less than 10 .mu.M, less than 5 .mu.M,
less than 4 .mu.M, less than 3 .mu.M, less than 1 .mu.M, less than
0.8 .mu.M, less than 0.6 .mu.M, less than 0.5 .mu.M, less than 0.4
.mu.M, less than 0.3 .mu.M, less than less than 0.2 .mu.M, less
than 0.1 .mu.M, less than 0.08 .mu.M, less than 0.06 .mu.M, less
than 0.05 .mu.M, less than 0.04 .mu.M, less than 0.03 .mu.M, less
than less than 0.02 .mu.M, less than 0.01 .mu.M, less than 0.0099
.mu.M, less than 0.0098 .mu.M, less than 0.0097 .mu.M, 0.0096
.mu.M, less than 0.0095 .mu.M, less than 0.0094 .mu.M, less than
0.0093 .mu.M, less than 0.00092, or less than 0.0090 .mu.M).
Examples of Pharmaceutical Compositions and Methods of
Administration
[0087] The pharmaceutical solid dosage forms described herein can
include a compound of Formula I or Formula II, and one or more
pharmaceutically acceptable additives such as a compatible carrier,
binder, filling agent, suspending agent, flavoring agent,
sweetening agent, disintegrating agent, dispersing agent,
surfactant, lubricant, colorant, diluent, solubilizer, moistening
agent, plasticizer, stabilizer, penetration enhancer, wetting
agent, anti-foaming agent, antioxidant, preservative, or one or
more combination thereof. In still other aspects, using standard
coating procedures, such as those described in Remington's
Pharmaceutical Sciences, 20th Edition (2000), a film coating is
provided around the formulation of the compound of Formula I or
Formula II. In one embodiment, some or all of the particles of the
compound of Formula I or Formula II are coated. In another
embodiment, some or all of the particles of the compound of Formula
I or Formula II are microencapsulated. In still another embodiment,
the particles of the compound of Formula I or Formula II are not
microencapsulated and are uncoated.
[0088] Pharmacological techniques include, e.g., one or a
combination of methods: (1) dry mixing, (2) direct compression, (3)
milling, (4) dry or non-aqueous granulation, (5) wet granulation,
or (6) fusion. See, e.g., Lachman et al., "The Theory and Practice
of Industrial Pharmacy" (1986). Other methods include, e.g., spray
drying, pan coating, melt granulation, granulation, fluidized bed
spray drying or coating (e.g., wurster coating), tangential
coating, top spraying, tableting, extruding and the like.
[0089] Provided herein are pharmaceutical compositions that include
one or more neuroprotective compounds described herein and a
pharmaceutically acceptable diluent(s), excipient(s), or
carrier(s). In addition, the compounds described herein can be
administered as pharmaceutical compositions in which compounds
described herein are mixed with other active ingredients, as in
combination therapy. In some embodiments, the pharmaceutical
compostions may include other medicinal or pharmaceutical agents,
carriers, adjuvants, such as preserving, stabilizing, wetting or
emulsifying agents, solution promoters, salts for regulating the
osmotic pressure, and/or buffers. In addition, the pharmaceutical
compositions can also contain other therapeutically valuable
substances.
[0090] In certain embodiments, compositions may also include one or
more pH adjusting agents or buffering agents, including acids such
as acetic, boric, citric, lactic, phosphoric and hydrochloric
acids; bases such as sodium hydroxide, sodium phosphate, sodium
borate, sodium citrate, sodium acetate, sodium lactate and
tris-hydroxymethylaminomethane; and buffers such as
citrate/dextrose, sodium bicarbonate and ammonium chloride. Such
acids, bases and buffers are included in an amount required to
maintain pH of the composition in an acceptable range.
[0091] In other embodiments, compositions may also include one or
more salts in an amount required to bring osmolality of the
composition into an acceptable range. Such salts include those
having sodium, potassium or ammonium cations and chloride, citrate,
ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or
bisulfite anions; suitable salts include sodium chloride, potassium
chloride, sodium thiosulfate, sodium bisulfite and ammonium
sulfate.
[0092] A pharmaceutical composition, as used herein, refers to a
mixture of a compound described herein, such as, for example,
compounds of Formula I or Formula II with other chemical
components, such as carriers, stabilizers, diluents, dispersing
agents, suspending agents, thickening agents, and/or excipients.
The pharmaceutical composition facilitates administration of the
compound to an organism. In practicing the methods of treatment or
use provided herein, therapeutically effective amounts of compounds
described herein are administered in a pharmaceutical composition
to a mammal having a neurodegenerative condition, disease, or
disorder to be treated. Preferably, the mammal is a human. A
therapeutically effective amount can vary widely depending on the
severity and stage of the of the condition, the age and relative
health of the subject, the potency of the compound used and other
factors. The compounds can be used singly or in combination with
one or more therapeutic agents as components of mixtures.
[0093] The pharmaceutical formulations described herein can be
administered to a subject by multiple administration routes,
including but not limited to, oral, parenteral (e.g., intravenous,
subcutaneous, intramuscular), intranasal, buccal, topical, rectal,
or transdermal administration routes. The pharmaceutical
formulations described herein include, but are not limited to,
aqueous liquid dispersions, self-emulsifying dispersions, solid
solutions, liposomal dispersions, aerosols, solid dosage forms,
powders, immediate release formulations, controlled release
formulations, fast melt formulations, tablets, capsules, pills,
delayed release formulations, extended release ions, pulsatile
release formulations, multiparticulate formulations, and mixed
immediate and controlled release formulations.
[0094] Pharmaceutical compositions including a compound described
herein may be manufactured, by way of example only, by means of
mixing, dissolving, granulating, dragee-making, levigating,
emulsifying, encapsulating, entrapping or compression
processes.
[0095] The pharmaceutical compositions will include at least one
compound described herein, such as, for example, a compound of
Formula I or Formula II, as an active ingredient in free-acid or
free-base form, or in a pharmaceutically acceptable salt form. In
addition, the methods and pharmaceutical compositions described
herein include the use of N-oxides, crystalline forms (also known
as polymorphs), as well as active metabolites of these compounds
having the same type of activity. In some situations, compounds may
exist as tautomers. All tautomers are included within the scope of
the compounds presented herein. Additionally, the compounds
described herein can exist in unsolvated as well as solvated forms
with pharmaceutically acceptable solvents such as water, ethanol,
and the like. The solvated forms of the compounds presented herein
are also considered to be disclosed herein.
[0096] "Antioxidants" include, for example, butylated
hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium
metabisulfite and tocopherol. In certain embodiments, antioxidants
enhance chemical stability where required.
[0097] In certain embodiments, compositions provided herein may
also include one or more preservatives to inhibit microbial
activity. Suitable preservatives include mercury-containing
substances such as merfen and thiomersal; stabilized chlorine
dioxide; and quaternary ammonium compounds such as benzalkonium
chloride, cetyltrimethylammonium bromide and cetylpyridinium
chloride.
[0098] Formulations described herein may benefit from antioxidants,
metal chelating agents, thiol containing compounds and other
general stabilizing agents. Examples of such stabilizing agents,
include, but are not limited to: (a) about 0.5% to about 2% w/v
glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1%
to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM
EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to
about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v.
polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k)
cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m)
divalent cations such as magnesium and zinc; or (n) combinations
thereof.
[0099] "Binders" impart cohesive qualities and include, e.g.,
alginic acid and salts thereof; cellulose derivatives such as
carboxymethylcellulose, methylcellulose (e.g., Methocel.RTM.),
hydroxypropylmethylcellulose, hydroxyethylcellulose,
hydroxypropylcellulose (e.g., Klucel.RTM.), ethylcellulose (e.g.,
Ethocel.RTM.), and microcrystalline cellulose (e.g., Avicel.RTM.);
microcrystalline dextrose; amylose; magnesium aluminum silicate;
polysaccharide acids; bentonites; gelatin;
polyvinylpyrrolidone/vinyl acetate copolymer; crosspovidone;
povidone; starch; pregelatinized starch; tragacanth, dextrin, a
sugar, such as sucrose (e.g., Dipac.RTM.), glucose, dextrose,
molasses, mannitol, sorbitol, xylitol (e.g., Xylitab.RTM.), and
lactose; a natural or synthetic gum such as acacia, tragacanth,
ghatti gum, mucilage of isapol husks, polyvinylpyrrolidone (e.g.,
Polyvidone.RTM. CL, Kollidon.RTM. CL, Polyplasdone.RTM. XL-10),
larch arabogalactan, Veegum.RTM., polyethylene glycol, waxes,
sodium alginate, and the like.
[0100] "Bioavailability" refers to the percentage of the weight of
compounds disclosed herein, such as, compounds of Formula I or
Formula II, that is delivered into the general circulation of the
animal or human being studied. The total exposure (AUC(0-.infin.))
of a drug when administered intravenously is usually defined as
100% bioavailable (F %). "Oral bioavailability" refers to the
extent to which compounds disclosed herein, such as, compounds of
Formula I or Formula II that are absorbed into the general
circulation when the pharmaceutical composition is taken orally as
compared to intravenous injection.
[0101] "Blood plasma concentration" refers to the concentration of
compounds disclosed herein, such as, compounds of Formula I or
Formula II, in the plasma component of blood of a subject. It is
understood that the plasma concentration of compounds of Formula I
or Formula II may vary significantly between subjects, due to
variability with respect to metabolism and/or possible interactions
with other therapeutic agents. In accordance with one embodiment
disclosed herein, the blood plasma concentration of the compounds
of Formula I or Formula II may vary from subject to subject.
Likewise, values such as maximum plasma concentration (Cmax) or
time to reach maximum plasma concentration (Tmax), or total area
under the plasma concentration time curve (AUC(0-.infin.)) may vary
from subject to subject. Due to this variability, the amount
necessary to constitute "a therapeutically effective amount" of a
compound of Formula I or Formula II may vary from subject to
subject.
[0102] "Carrier materials" include any commonly used excipients in
pharmaceutics and should be selected on the basis of compatibility
with compounds disclosed herein, such as, compounds of Formula I or
Formula II, and the release profile properties of the desired
dosage form. Exemplary carrier materials include, e.g., binders,
suspending agents, disintegration agents, filling agents,
surfactants, solubilizers, stabilizers, lubricants, wetting agents,
diluents, and the like. "Pharmaceutically compatible carrier
materials" may include, but are not limited to, acacia, gelatin,
colloidal silicon dioxide, calcium glycerophosphate, calcium
lactate, maltodextrin, glycerine, magnesium silicate,
polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,
sodium caseinate, soy lecithin, taurocholic acid,
phosphotidylcholine, sodium chloride, tricalcium phosphate,
dipotassium phosphate, cellulose and cellulose conjugates, sugars
sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride,
pregelatinized starch, and the like. See, e.g., Remington: The
Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack
Publishing Company, 1995); Hoover, John E., Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975;
Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,
Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage
Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams
& Wilkins 1999).
[0103] "Dispersing agents," and/or "viscosity modulating agents"
include materials that control the diffusion and homogeneity of a
drug through liquid media or a granulation method or blend method.
In some embodiments, these agents also facilitate the effectiveness
of a coating or eroding matrix. Exemplary diffusion
facilitators/dispersing agents include, e.g., hydrophilic polymers,
electrolytes, Tween.RTM. 60 or 80, PEG, polyvinylpyrrolidone (PVP;
commercially known as Plasdone.RTM.), and the carbohydrate-based
dispersing agents such as, for example, hydroxypropyl celluloses
(e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcelluloses
(e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),
carboxymethylcellulose sodium, methylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose phthalate,
hydroxypropylmethylcellulose acetate stearate (HPMCAS),
noncrystalline cellulose, magnesium aluminum silicate,
triethanolamine, polyvinyl alcohol (PVA), vinyl pyrrolidone/vinyl
acetate copolymer (S630), 4-(1,1,3,3-tetramethylbutyl)-phenol
polymer with ethylene oxide and formaldehyde (also known as
tyloxapol), poloxamers (e.g., Pluronics F68.RTM., F88.RTM., and
F108.RTM., which are block copolymers of ethylene oxide and
propylene oxide); and poloxamines (e.g., Tetronic 908.RTM., also
known as Poloxamine 908.RTM., which is a tetrafunctional block
copolymer derived from sequential addition of propylene oxide and
ethylene oxide to ethylenediamine (BASF Corporation, Parsippany,
N.J.)), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17,
polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30,
polyvinylpyrrolidone/vinyl acetate copolymer (S-630), polyethylene
glycol, e.g., the polyethylene glycol can have a molecular weight
of about 300 to about 6000, or about 3350 to about 4000, or about
7000 to about 5400, sodium carboxymethylcellulose, methylcellulose,
polysorbate-80, sodium alginate, gums, such as, e.g., gum
tragacanth and gum acacia, guar gum, xanthans, including xanthan
gum, sugars, cellulosics, such as, e.g., sodium
carboxymethylcellulose, methylcellulose, sodium
carboxymethylcellulose, polysorbate-80, sodium alginate,
polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan
monolaurate, povidone, carbomers, polyvinyl alcohol (PVA),
alginates, chitosans and combinations thereof. Plasticizcers such
as cellulose or triethyl cellulose can also be used as dispersing
agents. Dispersing agents particularly useful in liposomal
dispersions and self-emulsifying dispersions are dimyristoyl
phosphatidyl choline, natural phosphatidyl choline from eggs,
natural phosphatidyl glycerol from eggs, cholesterol and isopropyl
myristate.
[0104] The term "diluent" refers to chemical compounds that are
used to dilute the compound of interest prior to delivery. Diluents
can also be used to stabilize compounds because they can provide a
more stable environment. Salts dissolved in buffered solutions
(which also can provide pH control or maintenance) are utilized as
diluents, including, but not limited to a phosphate buffered saline
solution. In certain embodiments, diluents increase bulk of the
composition to facilitate compression or create sufficient bulk for
homogenous blend for capsule filling. Such compounds include e.g.,
lactose, starch, mannitol, sorbitol, dextrose, microcrystalline
cellulose such as Avicel.RTM.; dibasic calcium phosphate, dicalcium
phosphate dihydrate; tricalcium phosphate, calcium phosphate;
anhydrous lactose, spray-dried lactose; pregelatinized starch,
compressible sugar, such as Di-Pac.RTM. (Amstar); mannitol,
hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate
stearate, sucrose-based diluents, confectioner's sugar; monobasic
calcium sulfate monohydrate, calcium sulfate dihydrate; calcium
lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose;
powdered cellulose, calcium carbonate; glycine, kaolin; mannitol,
sodium chloride; inositol, bentonite, and the like.
[0105] The term "disintegrate" includes both the dissolution and
dispersion of the dosage form when contacted with gastrointestinal
fluid. "Disintegration agents or disintegrants" facilitate the
breakup or disintegration of a substance. Examples of
disintegration agents include a starch, e.g., a natural starch such
as corn starch or potato starch, a pregelatinized starch such as
National 1551 or Amijel.RTM., or sodium starch glycolate such as
Promogel.RTM. or Explotab.RTM., a cellulose such as a wood product,
methylcrystalline cellulose, e.g., Avicel.RTM., Avicel.RTM. PH101,
Avicel.RTM. PH102, Avicel.RTM. PH105, Elcema.RTM. P100,
Emcocel.RTM., Vivacel.RTM., Ming Tia.RTM., and Solka-Floc.RTM.,
methylcellulose, croscarmellose, or a cross-linked cellulose, such
as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol.RTM.),
cross-linked carboxymethylcellulose, or cross-linked
croscarmellose, a cross-linked starch such as sodium starch
glycolate, a cross-linked polymer such as crosspovidone, a
cross-linked polyvinylpyrrolidone, alginate such as alginic acid or
a salt of alginic acid such as sodium alginate, a clay such as
Veegum.RTM. HV (magnesium aluminum silicate), a gum such as agar,
guar, locust bean, Karaya, pectin, or tragacanth, sodium starch
glycolate, bentonite, a natural sponge, a surfactant, a resin such
as a cation-exchange resin, citrus pulp, sodium lauryl sulfate,
sodium lauryl sulfate in combination starch, and the like.
[0106] "Drug absorption" or "absorption" typically refers to the
process of movement of drug from site of administration of a drug
across a barrier into a blood vessel or the site of action, e.g., a
drug moving from the gastrointestinal tract into the portal vein or
lymphatic system.
[0107] The term "effective amount," as used herein, refers to a
sufficient amount of an agent or a compound being administered
which will relieve to some extent one or more of the symptoms of
the disease or condition being treated. The result can be reduction
and/or alleviation of the signs, symptoms, or causes of a disease,
or any other desired alteration of a biological system. An
appropriate "effective" amount in any individual case may be
determined using techniques, such as a dose escalation study.
[0108] An "enteric coating" is a substance that remains
substantially intact in the stomach but dissolves and releases the
drug in the small intestine or colon. Generally, the enteric
coating comprises a polymeric material that prevents release in the
low pH environment of the stomach but that ionizes at a higher pH,
typically a pH of 6 to 7, and thus dissolves sufficiently in the
small intestine or colon to release the active agent therein.
[0109] "Filling agents" include compounds such as lactose, calcium
carbonate, calcium phosphate, dibasic calcium phosphate, calcium
sulfate, microcrystalline cellulose, cellulose powder, dextrose,
dextrates, dextran, starches, pregelatinized starch, sucrose,
xylitol, lactitol, mannitol, sorbitol, sodium chloride,
polyethylene glycol, and the like.
[0110] "Flavoring agents" and/or "sweeteners" useful in the
formulations described herein, include, e.g., acacia syrup,
acesulfame K, alitame, anise, apple, aspartame, banana, Bavarian
cream, berry, black currant, butterscotch, calcium citrate,
camphor, caramel, cherry, cherry cream, chocolate, cinnamon, bubble
gum, citrus, citrus punch, citrus cream, cotton candy, cocoa, cola,
cool cherry, cool citrus, cyclamate, cylamate, dextrose,
eucalyptus, eugenol, fructose, fruit punch, ginger,
glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit,
honey, isomalt, lemon, lime, lemon cream, monoammonium
glyrrhizinate (MagnaSweet.RTM.), maltol, mannitol, maple,
marshmallow, menthol, mint cream, mixed berry, neohesperidine DC,
neotame, orange, pear, peach, peppermint, peppermint cream,
Prosweet.RTM. Powder, raspberry, root beer, rum, saccharin,
safrole, sorbitol, spearmint, spearmint cream, strawberry,
strawberry cream, stevia, sucralose, sucrose, sodium saccharin,
saccharin, aspartame, acesulfame potassium, mannitol, talin,
sylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine,
thaumatin, tutti fruitti, vanilla, walnut, watermelon, wild cherry,
wintergreen, xylitol, or any combination of these flavoring
ingredients, e.g., anise-menthol, cherry-anise, cinnamon-orange,
cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime,
lemon-mint, menthol-eucalyptus, orange-cream, vanilla-mint, and
mixtures thereof.
[0111] "Lubricants" and "glidants" are compounds that prevent,
reduce or inhibit adhesion or friction of materials. Exemplary
lubricants include, e.g., stearic acid, calcium hydroxide, talc,
sodium stearyl fumerate, a hydrocarbon such as mineral oil, or
hydrogenated vegetable oil such as hydrogenated soybean oil
(Sterotex.RTM.), higher fatty acids and their alkali-metal and
alkaline earth metal salts, such as aluminum, calcium, magnesium,
zinc, stearic acid, sodium stearates, glycerol, talc, waxes,
Stearowet.RTM., boric acid, sodium benzoate, sodium acetate, sodium
chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a
methoxypolyethylene glycol such as Carbowax.TM., sodium oleate,
sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium
or sodium lauryl sulfate, colloidal silica such as Syloid.TM.,
Cab-O-Sil.RTM., a starch such as corn starch, silicone oil, a
surfactant, and the like.
[0112] A "measurable serum concentration" or "measurable plasma
concentration" describes the blood serum or blood plasma
concentration, typically measured in mg, .mu.g, or ng of
therapeutic agent per ml, dl, or l of blood serum, absorbed into
the bloodstream after administration. As used herein, measurable
plasma concentrations are typically measured in ng/ml or
.mu.g/ml.
[0113] The terms "neuroprotective," "neuroprotection," or
"neuroprotectant," as used herein refer to the ability of an agent
(e.g., a compound described herein) to significantly prevent or
reduce the occurrence of spontaneous or induced death (e.g., by
apoptosis) of neurons or glia in vitro or in vivo, relative to the
ability of a control reagent (e.g., cell culture medium, or a drug
vehicle such as DMSO).
[0114] A "prognostic biomarker," as referred to herein, is any
molecular, biochemical, metabolic, cellular, or structural entity
(or a ratio of such entities), the presence or level of which
relates to a likelihood of developing, suffering from, or having a
relapse of a pathological condition.
[0115] "Pharmacodynamics" refers to the factors which determine the
biologic response observed relative to the concentration of drug at
a site of action.
[0116] "Pharmacokinetics" refers to the factors which determine the
attainment and maintenance of the appropriate concentration of drug
at a site of action.
[0117] "Plasticizers" are compounds used to soften the
microencapsulation material or film coatings to make them less
brittle. Suitable plasticizers include, e.g., polyethylene glycols
such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800,
stearic acid, propylene glycol, oleic acid, triethyl cellulose and
triacetin. In some embodiments, plasticizers can also function as
dispersing agents or wetting agents.
[0118] The term "prophylactically effective amount," as used
herein, refers that amount of a composition applied to a patient
which will relieve to some extent one or more of the symptoms of a
disease, condition or disorder being treated. In such prophylactic
applications, such amounts may depend on the patient's state of
health, weight, and the like.
[0119] "Solubilizers" include compounds such as triacetin,
triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl
sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide,
N-methylpyrrolidone, N-hydroxyethylpyrrolidone,
polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl
cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol,
bile salts, polyethylene glycol 200-600, glycofurol, transcutol,
propylene glycol, and dimethyl isosorbide and the like.
[0120] "Stabilizers" include compounds such as any antioxidation
agents, buffers, acids, preservatives and the like.
[0121] "Steady state," as used herein, is when the amount of drug
administered is equal to the amount of drug eliminated within one
dosing interval resulting in a plateau or constant plasma drug
exposure.
[0122] The term "subject" as used herein, refers to an animal which
is the object of treatment, observation or experiment. By way of
example only, a subject may be, but is not limited to, a mammal
including, but not limited to, a human.
[0123] "Suspending agents" include compounds such as
polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12,
polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or
polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer
(S630), polyethylene glycol, e.g., the polyethylene glycol can have
a molecular weight of about 300 to about 6000, or about 3350 to
about 4000, or about 7000 to about 5400, sodium
carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, hydroxymethylcellulose acetate
stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate,
gums, such as, e.g., gum tragacanth and gum acacia, guar gum,
xanthans, including xanthan gum, sugars, cellulosics, such as,
e.g., sodium carboxymethylcellulose, methylcellulose, sodium
carboxymethylcellulose, hydroxypropylmethylcellulose,
hydroxyethylcellulose, polysorbate-80, sodium alginate,
polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan
monolaurate, povidone and the like.
[0124] "Surfactants" include compounds such as sodium lauryl
sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E
TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate,
polysorbates, polaxomers, bile salts, glyceryl monostearate,
copolymers of ethylene oxide and propylene oxide, e.g.,
Pluronic.RTM. (BASF), and the like. Some other surfactants include
polyoxyethylene fatty acid glycerides and vegetable oils, e.g.,
polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene
alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol
40. In some embodiments, surfactants may be included to enhance
physical stability or for other purposes.
[0125] "Viscosity enhancing agents" include, e.g., methyl
cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl
cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl
cellulose acetate stearate, hydroxypropylmethyl cellulose
phthalate, carbomer, polyvinyl alcohol, alginates, acacia,
chitosans and combinations thereof.
[0126] "Wetting agents" include compounds such as oleic acid,
glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate,
triethanolamine oleate, polyoxyethylene sorbitan monooleate,
polyoxyethylene sorbitan monolaurate, sodium docusate, sodium
oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween
80, vitamin E TPGS, ammonium salts and the like.
[0127] The compositions described herein can be formulated for
administration to a subject via any means including, but not
limited to, oral, parenteral (e.g., intravenous, subcutaneous, or
intramuscular), buccal, intranasal, rectal or transdermal
administration routes. As used herein, the term "subject" is used
to mean an animal, preferably a mammal, including a human or
non-human. The terms patient and subject may be used
interchangeably.
[0128] Moreover, the pharmaceutical compositions described herein,
which include a compound of Formula I or Formula II, can be
formulated into any suitable dosage form, including but not limited
to, aqueous oral dispersions, liquids, gels, syrups, elixirs,
slurries, suspensions and the like, for oral ingestion by a patient
to be treated, solid oral dosage forms, aerosols, controlled
release formulations, fast melt formulations, effervescent
formulations, lyophilized formulations, tablets, powders, pills,
dragees, capsules, delayed release formulations, extended release
formulations, pulsatile release formulations, multiparticulate
formulations, and mixed immediate release and controlled release
formulations.
[0129] Pharmaceutical preparations for oral use can be obtained by
mixing one or more solid excipient with one or more of the
compounds described herein, optionally grinding the resulting
mixture, and processing the mixture of granules, after adding
suitable auxiliaries, if desired, to obtain tablets or dragee
cores. Suitable excipients include, for example, fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol;
cellulose preparations such as, for example, maize starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth,
methylcellulose, microcrystalline cellulose,
hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or
others such as: polyvinylpyrrolidone (PVP or povidone) or calcium
phosphate. If desired, disintegrating agents may be added, such as
the cross linked croscarmellose sodium, polyvinylpyrrolidone, agar,
or alginic acid or a salt thereof such as sodium alginate.
[0130] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol
gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0131] Pharmaceutical preparations which can be used orally include
push fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration.
[0132] In some embodiments, the solid dosage forms disclosed herein
may be in the form of a tablet, (including a suspension tablet, a
fast-melt tablet, a bite-disintegration tablet, a
rapid-disintegration tablet, an effervescent tablet, or a caplet),
a pill, a powder (including a sterile packaged powder, a
dispensable powder, or an effervescent powder) a capsule (including
both soft or hard capsules, e.g., capsules made from animal-derived
gelatin or plant-derived HPMC, or "sprinkle capsules"), solid
dispersion, solid solution, bioerodible dosage form, controlled
release formulations, pulsatile release dosage forms,
multiparticulate dosage forms, pellets, granules, or an aerosol. In
other embodiments, the pharmaceutical formulation is in the form of
a powder. In still other embodiments, the pharmaceutical
formulation is in the form of a tablet, including but not limited
to, a fast-melt tablet. Additionally, pharmaceutical formulations
of the present invention may be administered as a single capsule or
in multiple capsule dosage form. In some embodiments, the
pharmaceutical formulation is administered in two, or three, or
four, capsules or tablets.
[0133] In some embodiments, solid dosage forms, e.g., tablets,
effervescent tablets, and capsules, are prepared by mixing
particles of a compound of Formula I or Formula II, with one or
more pharmaceutical excipients to form a bulk blend composition.
When referring to these bulk blend compositions as homogeneous, it
is meant that the particles of the compound of Formula I or Formula
II, are dispersed evenly throughout the composition so that the
composition may be readily subdivided into equally effective unit
dosage forms, such as tablets, pills, and capsules. The individual
unit dosages may also include film coatings, which disintegrate
upon oral ingestion or upon contact with diluent. These
formulations can be manufactured by pharmacological techniques.
[0134] In another aspect, dosage forms may include
microencapsulated formulations. In some embodiments, one or more
other compatible materials are present in the microencapsulation
material. Exemplary materials include, but are not limited to, pH
modifiers, erosion facilitators, anti-foaming agents, antioxidants,
flavoring agents, and carrier materials such as binders, suspending
agents, disintegration agents, filling agents, surfactants,
solubilizers, stabilizers, lubricants, wetting agents, and
diluents.
[0135] Materials useful for the microencapsulation described herein
include materials compatible with compounds of Formula I or Formula
II, which sufficiently isolate the compound of Formula I or Formula
II from other non-compatible excipients. Materials compatible with
compounds of Formula I or Formula II are those that delay the
release of the compounds of Formula I or Formula II in vivo.
[0136] Exemplary microencapsulation materials useful for delaying
the release of the formulations including compounds of Formula I or
Formula II, include, but are not limited to, hydroxypropyl
cellulose ethers (HPC) such as Klucel.RTM. or Nisso HPC,
low-substituted hydroxypropyl cellulose ethers (L-HPC),
hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC,
Pharmacoat.RTM., Metolose SR, Methocel.RTM.-E, Opadry YS, PrimaFlo,
Benecel MP824, and Benecel MP843, methylcellulose polymers such as
Methocel.RTM.-A, hydroxypropylmethylcellulose acetate stearate
Aqoat (HF-LS, HF-LG, HF-MS) and Metolose.RTM., Ethylcelluloses (EC)
and mixtures thereof such as E461, Ethocel.RTM., Aqualon.RTM.-EC,
Surelease.RTM., Polyvinyl alcohol (PVA) such as Opadry AMB,
hydroxyethylcelluloses such as Natrosol.RTM.,
carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC)
such as Aqualon.RTM.-CMC, polyvinyl alcohol and polyethylene glycol
co-polymers such as Kollicoat IR.RTM., monoglycerides (Myverol),
triglycerides (KLX), polyethylene glycols, modified food starch,
acrylic polymers and mixtures of acrylic polymers with cellulose
ethers such as Eudragit.RTM. EPO, Eudragit.RTM. L30D-55,
Eudragit.RTM. FS 30D Eudragit.RTM. L100-55, Eudragit.RTM. L100,
Eudragit.RTM. S100, Eudragit.RTM. RD100, Eudragit.RTM. E100,
Eudragit.RTM. L12.5, Eudragit.RTM. S112.5, Eudragit.RTM. NE30D, and
Eudragit.RTM. NE 40D, cellulose acetate phthalate, sepifilms such
as mixtures of HPMC and stearic acid, cyclodextrins, and mixtures
of these materials.
[0137] In still other embodiments, plasticizers such as
polyethylene glycols, e.g., PEG 300, PEG 400, PEG 600, PEG 1450,
PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid,
and triacetin are incorporated into the microencapsulation
material. In other embodiments, the microencapsulating material
useful for delaying the release of the pharmaceutical compositions
is from the USP or the National Formulary (NF). In yet other
embodiments, the microencapsulation material is Klucel. In still
other embodiments, the microencapsulation material is methocel.
[0138] Microencapsulated compounds of Formula I or Formula II may
be formulated. Such methods include, e.g., spray drying processes,
spinning disk-solvent processes, hot melt processes, spray chilling
methods, fluidized bed, electrostatic deposition, centrifugal
extrusion, rotational suspension separation, polymerization at
liquid-gas or solid-gas interface, pressure extrusion, or spraying
solvent extraction bath. In addition to these, several chemical
techniques, e.g., complex coacervation, solvent evaporation,
polymer-polymer incompatibility, interfacial polymerization in
liquid media, in situ polymerization, in-liquid drying, and
desolvation in liquid media could also be used. Furthermore, other
methods such as roller compaction, extrusion/spheronization,
coacervation, or nanoparticle coating may also be used.
[0139] In one embodiment, the particles of compounds of Formula I
or Formula II are microencapsulated prior to being formulated into
one of the above forms. In still another embodiment, some or most
of the particles are coated prior to being further formulated by
using standard coating procedures, such as those described in
Remington's Pharmaceutical Sciences, 20th Edition (2000). In other
embodiments, the solid dosage formulations of the compounds of
Formula I or Formula II are plasticized (coated) with one or more
layers. Illustratively, a plasticizer is generally a high boiling
point solid or liquid. Suitable plasticizers can be added from
about 0.01% to about 50% by weight (w/w) of the coating
composition. Plasticizers include, but are not limited to, diethyl
phthalate, citrate esters, polyethylene glycol, glycerol,
acetylated glycerides, triacetin, polypropylene glycol,
polyethylene glycol, triethyl citrate, dibutyl sebacate, stearic
acid, stearol, stearate, and castor oil.
[0140] The pharmaceutical solid oral dosage forms including
formulations described herein, which include a compound of Formula
I or Formula II, can be further formulated to provide a controlled
release of the compound of Formula I or Formula II. Controlled
release refers to the release of the compound of Formula Formula I
or Formula II from a dosage form in which it is incorporated
according to a desired profile over an extended period of time.
Controlled release profiles include, for example, sustained
release, prolonged release, pulsatile release, and delayed release
profiles. In contrast to immediate release compositions, controlled
release compositions allow delivery of an agent to a subject over
an extended period of time according to a predetermined profile.
Such release rates can provide therapeutically effective levels of
agent for an extended period of time and thereby provide a longer
period of pharmacologic response while minimizing side effects as
compared to conventional rapid release dosage forms. Such longer
periods of response provide for many inherent benefits that are not
achieved with the corresponding short acting, immediate release
preparations.
[0141] In some embodiments, the solid dosage forms described herein
can be formulated as enteric coated delayed release oral dosage
forms, i.e., as an oral dosage form of a pharmaceutical composition
as described herein which utilizes an enteric coating to affect
release in the small intestine of the gastrointestinal tract. The
enteric coated dosage form may be a compressed or molded or
extruded tablet/mold (coated or uncoated) containing granules,
powder, pellets, beads or particles of the active ingredient and/or
other composition components, which are themselves coated or
uncoated. The enteric coated oral dosage form may also be a capsule
(coated or uncoated) containing pellets, beads or granules of the
solid carrier or the composition, which are themselves coated or
uncoated.
[0142] The term "delayed release" as used herein refers to the
delivery so that the release can be accomplished at some generally
predictable location in the intestinal tract more distal to that
which would have been accomplished if there had been no delayed
release alterations. In some embodiments the method for delay of
release is coating. Any coatings should be applied to a sufficient
thickness such that the entire coating does not dissolve in the
gastrointestinal fluids at pH below about 5, but does dissolve at
pH about 5 and above. It is expected that any anionic polymer
exhibiting a pH-dependent solubility profile can be used as an
enteric coating in the practice of the present invention to achieve
delivery to the lower gastrointestinal tract. In some embodiments
the polymers for use in the present invention are anionic
carboxylic polymers.
[0143] In other embodiments, the formulations described herein,
which include a compound of Formula I or Formula II, are delivered
using a pulsatile dosage form. A pulsatile dosage form is capable
of providing one or more immediate release pulses at predetermined
time points after a controlled lag time or at specific sites.
Pulsatile dosage forms including the formulations described herein,
which include a compound of Formula I or Formula II, may be
administered using a variety of pulsatile formulations. For
example, such formulations include, but are not limited to, those
described in U.S. Pat. Nos. 5,011,692, 5,017,381, 5,229,135, and
5,840,329. Other pulsatile release dosage forms suitable for use
with the present formulations include, but are not limited to, for
example, U.S. Pat. Nos. 4,871,549, 5,260,068, 5,260,069, 5,508,040,
5,567,441 and 5,837,284. In one embodiment, the controlled release
dosage form is pulsatile release solid oral dosage form including
at least two groups of particles, (i.e. multiparticulate) each
containing the formulation described herein. The first group of
particles provides a substantially immediate dose of the compound
of Formula I or Formula II upon ingestion by a mammal. The first
group of particles can be either uncoated or include a coating
and/or sealant. The second group of particles includes coated
particles, which includes from about 2% to about 75%, preferably
from about 2.5% to about 70%, and more preferably from about 40% to
about 70%, by weight of the total dose of the compound of Formula I
or Formula II in said formulation, in admixture with one or more
binders. The coating includes a pharmaceutically acceptable
ingredient in an amount sufficient to provide a delay of from about
2 hours to about 7 hours following ingestion before release of the
second dose. Suitable coatings include one or more differentially
degradable coatings such as, by way of example only, pH sensitive
coatings (enteric coatings) such as acrylic resins (e.g.,
Eudragit.RTM. EPO, Eudragit.RTM.& L30D-55, Eudragit.RTM. FS 30D
Eudragit.RTM. L100-55, Eudragit.RTM. L100, Eudragit.RTM. S100,
Eudragit.RTM. RD100, Eudragit.RTM. E100, Eudragit.RTM. L12.5,
Eudragit.RTM. S12.5, and Eudragit.RTM. NE30D, Eudragit.RTM. NE
40D.RTM.) either alone or blended with cellulose derivatives, e.g.,
ethylcellulose, or non-enteric coatings having variable thickness
to provide differential release of the formulation that includes a
compound of Formula I or Formula II.
[0144] Additional examples of controlled release delivery systems
include, e.g., polymer-based systems, such as polylactic and
polyglycolic acid, plyanhydrides and polycaprolactone; porous
matrices, nonpolymer-based systems that are lipids, including
sterols, such as cholesterol, cholesterol esters and fatty acids,
or neutral fats, such as mono-, di- and triglycerides; hydrogel
release systems; silastic systems; peptide-based systems; wax
coatings, bioerodible dosage forms, compressed tablets using
binders and the like. See, e.g., Liberman et al., Pharmaceutical
Dosage Forms, 2 Ed., Vol. 1, pp. 209-214 (1990); Singh et al.,
Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 751-753
(2002); U.S. Pat. Nos. 4,327,725, 4,624,848, 4,968,509, 5,461,140,
5,456,923, 5,516,527, 5,622,721, 5,686,105, 5,700,410, 5,977,175,
6,465,014 and 6,932,983.
[0145] Liquid formulation dosage forms for oral administration can
be aqueous suspensions selected from the group including, but not
limited to, pharmaceutically acceptable aqueous oral dispersions,
emulsions, solutions, elixirs, gels, and syrups. See, e.g., Singh
et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp.
754-757 (2002). In addition to the particles of compound of Formula
I or Formula II, the liquid dosage forms may include additives,
such as: (a) disintegrating agents; (b) dispersing agents; (c)
wetting agents; (d) at least one preservative, (e) viscosity
enhancing agents, (f) at least one sweetening agent, and (g) at
least one flavoring agent. In some embodiments, the aqueous
dispersions can further include a crystalline inhibitor.
[0146] The aqueous suspensions and dispersions described herein can
remain in a homogenous state, as defined in The USP Pharmacists'
Pharmacopeia (2005 edition, chapter 905), for at least 4 hours. The
homogeneity should be determined by a sampling method consistent
with regard to determining homogeneity of the entire composition.
In one embodiment, an aqueous suspension can be re-suspended into a
homogenous suspension by physical agitation lasting less than 1
minute. In another embodiment, an aqueous suspension can be
re-suspended into a homogenous suspension by physical agitation
lasting less than 45 seconds. In yet another embodiment, an aqueous
suspension can be re-suspended into a homogenous suspension by
physical agitation lasting less than 30 seconds. In still another
embodiment, no agitation is necessary to maintain a homogeneous
aqueous dispersion.
[0147] Examples of disintegrating agents for use in the aqueous
suspensions and dispersions include, but are not limited to, a
starch, e.g., a natural starch such as corn starch or potato
starch, a pregelatinized starch such as National 1551 or
Amijel.RTM., or sodium starch glycolate such as Promogel.RTM. or
Explotab.RTM.; a cellulose such as a wood product,
methylcrystalline cellulose, e.g., Avicel.RTM., Avicel.RTM. PH101,
Avicel.RTM. PH102, Avicel.RTM. PH 105, Elcema.RTM. P100,
Emcocel.RTM., Vivacel.RTM., Ming Tia.RTM., and Solka-Floc.RTM.,
methylcellulose, croscarmellose, or a cross-linked cellulose, such
as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol.RTM.),
cross-linked carboxymethylcellulose, or cross-linked
croscarmellose; a cross-linked starch such as sodium starch
glycolate; a cross-linked polymer such as crospovidone; a
cross-linked polyvinylpyrrolidone; alginate such as alginic acid or
a salt of alginic acid such as sodium alginate; a clay such as
Veegum.RTM. HV (magnesium aluminum silicate); a gum such as agar,
guar, locust bean, Karaya, pectin, or tragacanth; sodium starch
glycolate; bentonite; a natural sponge; a surfactant; a resin such
as a cation-exchange resin; citrus pulp; sodium lauryl sulfate;
sodium lauryl sulfate in combination starch; and the like.
[0148] In some embodiments, the dispersing agents suitable for the
aqueous suspensions and dispersions include, for example,
hydrophilic polymers, electrolytes, Tween.RTM. 60 or 80, PEG,
polyvinylpyrrolidone (PVP; commercially known as Plasdone.RTM.),
and the carbohydrate-based dispersing agents such as, for example,
hydroxypropylcellulose and hydroxypropyl cellulose ethers (e.g.,
HPC, HPC-SL, and HPC-L), hydroxypropyl methylcellulose and
hydroxypropyl methylcellulose ethers (e.g. HPMC K100, HPMC K4M,
HPMC K15M, and HPMC K100M), carboxymethylcellulose sodium,
methylcellulose, hydroxyethylcellulose,
hydroxypropylmethyl-cellulose phthalate,
hydroxypropylmethyl-cellulose acetate stearate, noncrystalline
cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl
alcohol (PVA), polyvinylpyrrolidone/vinyl acetate copolymer
(Plasdone.RTM., e.g., S-630), 4-(1,1,3,3-tetramethylbutyl)-phenol
polymer with ethylene oxide and formaldehyde (also known as
tyloxapol), poloxamers (e.g., Pluronics F68.RTM., F88.RTM., and
F108.RTM., which are block copolymers of ethylene oxide and
propylene oxide); and poloxamines (e.g., Tetronic 908.RTM., also
known as Poloxamine 908.RTM., which is a tetrafunctional block
copolymer derived from sequential addition of propylene oxide and
ethylene oxide to ethylenediamine (BASF Corporation, Parsippany,
N.J.)). In other embodiments, the dispersing agent is selected from
a group not comprising one of the following agents: hydrophilic
polymers; electrolytes; Tween.RTM. 60 or 80; PEG;
polyvinylpyrrolidone (PVP); hydroxypropylcellulose and
hydroxypropyl cellulose ethers (e.g., HPC, HPC-SL, and HPC-L);
hydroxypropyl methylcellulose and hydroxypropyl methylcellulose
ethers (e.g. HPMC K100, HPMC K4M, HPMC K15M, HPMC K100M, and
Pharmacoat.RTM. USP 2910 (Shin-Etsu)); carboxymethylcellulose
sodium; methylcellulose; hydroxyethylcellulose;
hydroxypropylmethyl-cellulose phthalate;
hydroxypropylmethyl-cellulose acetate stearate; non-crystalline
cellulose; magnesium aluminum silicate; triethanolamine; polyvinyl
alcohol (PVA); 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with
ethylene oxide and formaldehyde; poloxamers (e.g., Pluronics
F68.RTM., F88, and F108, which are block copolymers of ethylene
oxide and propylene oxide); or poloxamines (e.g., Tetronic
908.RTM., also known as Poloxamine 908.RTM.).
[0149] Wetting agents suitable for the aqueous suspensions and
dispersions include, but are not limited to, cetyl alcohol,
glycerol monostearate, polyoxyethylene sorbitan fatty acid esters
(e.g., the commercially available Tweens.RTM. such as e.g., Tween
20.RTM. and Tween 80.RTM. (ICI Specialty Chemicals)), and
polyethylene glycols (e.g., Carbowaxs 3350.RTM. and 1450.RTM., and
Carbopol 934.RTM. (Union Carbide)), oleic acid, glyceryl
monostearate, sorbitan monooleate, sorbitan monolaurate,
triethanolamine oleate, polyoxyethylene sorbitan monooleate,
polyoxyethylene sorbitan monolaurate, sodium oleate, sodium lauryl
sulfate, sodium docusate, triacetin, vitamin E TPGS, sodium
taurocholate, simethicone, phosphotidylcholine and the like
[0150] Suitable preservatives for the aqueous suspensions or
dispersions described herein include, for example, potassium
sorbate, parabens (e.g., methylparaben and propylparaben), benzoic
acid and its salts, other esters of parahydroxybenzoic acid such as
butylparaben, alcohols such as ethyl alcohol or benzyl alcohol,
phenolic compounds such as phenol, or quaternary compounds such as
benzalkonium chloride. Preservatives, as used herein, are
incorporated into the dosage form at a concentration sufficient to
inhibit microbial growth.
[0151] Suitable viscosity enhancing agents for the aqueous
suspensions or dispersions described herein include, but are not
limited to, methyl cellulose, xanthan gum, carboxymethyl cellulose,
hydroxypropyl cellulose, hydroxypropylmethyl cellulose,
Plasdon.RTM. S-630, carbomer, polyvinyl alcohol, alginates, acacia,
chitosans and combinations thereof. The concentration of the
viscosity enhancing agent will depend upon the agent selected and
the viscosity desired.
[0152] Examples of sweetening agents suitable for the aqueous
suspensions or dispersions described herein include, for example,
acacia syrup, acesulfame K, alitame, anise, apple, aspartame,
banana, Bavarian cream, berry, black currant, butterscotch, calcium
citrate, camphor, caramel, cherry, cherry cream, chocolate,
cinnamon, bubble gum, citrus, citrus punch, citrus cream, cotton
candy, cocoa, cola, cool cherry, cool citrus, cyclamate, cylamate,
dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger,
glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit,
honey, isomalt, lemon, lime, lemon cream, monoammonium
glyrrhizinate (MagnaSweet.RTM.), maltol, mannitol, maple,
marshmallow, menthol, mint cream, mixed berry, neohesperidine DC,
neotame, orange, pear, peach, peppermint, peppermint cream,
Prosweet.RTM. Powder, raspberry, root beer, rum, saccharin,
safrole, sorbitol, spearmint, spearmint cream, strawberry,
strawberry cream, stevia, sucralose, sucrose, sodium saccharin,
saccharin, aspartame, acesulfame potassium, mannitol, talin,
sucralose, sorbitol, swiss cream, tagatose, tangerine, thaumatin,
tutti fruitti, vanilla, walnut, watermelon, wild cherry,
wintergreen, xylitol, or any combination of these flavoring
ingredients, e.g., anise-menthol, cherry-anise, cinnamon-orange,
cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime,
lemon-mint, menthol-eucalyptus, orange-cream, vanilla-mint, and
mixtures thereof. In one embodiment, the aqueous liquid dispersion
can comprise a sweetening agent or flavoring agent in a
concentration ranging from about 0.001% to about 1.0% the volume of
the aqueous dispersion. In another embodiment, the aqueous liquid
dispersion can comprise a sweetening agent or flavoring agent in a
concentration ranging from about 0.005% to about 0.5% the volume of
the aqueous dispersion. In yet another embodiment, the aqueous
liquid dispersion can comprise a sweetening agent or flavoring
agent in a concentration ranging from about 0.01% to about 1.0% the
volume of the aqueous dispersion.
[0153] In addition to the additives listed above, the liquid
formulations can also include inert diluents such as water or other
solvents, solubilizing agents, and emulsifiers. Exemplary
emulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate,
ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol,
1,3-butyleneglycol, dimethylformamide, sodium lauryl sulfate,
sodium doccusate, cholesterol, cholesterol esters, taurocholic
acid, phosphotidylcholine, oils, such as cottonseed oil, groundnut
oil, corn germ oil, olive oil, castor oil, and sesame oil,
glycerol, tetrahydrofturfuryl alcohol, polyethylene glycols, fatty
acid esters of sorbitan, or mixtures of these substances, and the
like.
[0154] In some embodiments, the pharmaceutical formulations
described herein can be self-emulsifying drug delivery systems
(SEDDS). Emulsions are dispersions of one immiscible phase in
another, usually in the form of droplets. Generally, emulsions are
created by vigorous mechanical dispersion. SEDDS, as opposed to
emulsions or microemulsions, spontaneously form emulsions when
added to an excess of water without any external mechanical
dispersion or agitation. An advantage of SEDDS is that only gentle
mixing is required to distribute the droplets throughout the
solution. Additionally, water or the aqueous phase can be added
just prior to administration, which ensures stability of an
unstable or hydrophobic active ingredient. Thus, the SEDDS provides
an effective delivery system for oral and parenteral delivery of
hydrophobic active ingredients. SEDDS may provide improvements in
the bioavailability of hydrophobic active ingredients. Methods of
producing self-emulsifying dosage forms include, but are not
limited to, for example, U.S. Pat. Nos. 5,858,401, 6,667,048, and
6,960,563.
[0155] It is to be appreciated that there is overlap between the
above-listed additives used in the aqueous dispersions or
suspensions described herein, since a given additive is often
classified differently by different practitioners in the field, or
is commonly used for any of several different functions. Thus, the
above-listed additives should be taken as merely exemplary, and not
limiting, of the types of additives that can be included in
formulations described herein.
[0156] Intranasal formulations of a compound of Formula I or
Formula II can be prepared by adapting the methods described in
U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452. Formulations
that include a compound of Formula I or Formula II, which are
prepared according to these and other techniques are prepared as
solutions in saline, employing benzyl alcohol or other suitable
preservatives, fluorocarbons, and/or other solubilizing or
dispersing agents. See, for example, Ansel, H. C. et al.,
Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed.
(1995). Preferably these compositions and formulations are prepared
with suitable nontoxic pharmaceutically acceptable ingredients.
These ingredients can be found in REMINGTON: THE SCIENCE AND
PRACTICE OF PHARMACY, 21 st edition, 2005, a standard reference in
the field. The choice of suitable carriers is highly dependent upon
the exact nature of the nasal dosage form desired, e.g., solutions,
suspensions, ointments, or gels. Nasal dosage forms generally
contain large amounts of water in addition to the active
ingredient. Minor amounts of other ingredients such as pH
adjusters, emulsifiers or dispersing agents, preservatives,
surfactants, gelling agents, or buffering and other stabilizing and
solubilizing agents may also be present. Preferably, the nasal
dosage form should be isotonic with nasal secretions.
[0157] For administration by inhalation, the compounds of Formula
Formula I or Formula II described herein may be in a form as an
aerosol, a mist or a powder. Pharmaceutical compositions described
herein are conveniently delivered in the form of an aerosol spray
presentation from pressurized packs or a nebuliser, with the use of
a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol, the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of, such as, by way of
example only, gelatin for use in an inhaler or insufflator may be
formulated containing a powder mix of the compound described herein
and a suitable powder base such as lactose or starch.
[0158] Buccal formulations that include compounds of Formula I or
Formula II may be administered using a variety of formulations. For
example, such formulations include, but are not limited to, U.S.
Pat. Nos. 4,229,447, 4,596,795, 4,755,386, and 5,739,136. In
addition, the buccal dosage forms described herein can further
include a bioerodible (hydrolysable) polymeric carrier that also
serves to adhere the dosage form to the buccal mucosa. The buccal
dosage form is fabricated so as to erode gradually over a
predetermined time period, wherein the delivery of the compound of
Formula I or Formula II, is provided essentially throughout. Buccal
drug delivery avoids the disadvantages encountered with oral drug
administration, e.g., slow absorption, degradation of the active
agent by fluids present in the gastrointestinal tract and/or
first-pass inactivation in the liver. With regard to the
bioerodible (hydrolysable) polymeric carrier, it will be
appreciated that virtually any such carrier can be used, so long as
the desired drug release profile is not compromised, and the
carrier is compatible with the compound of Formula I or Formula II,
and any other components that may be present in the buccal dosage
unit. Generally, the polymeric carrier comprises hydrophilic
(water-soluble and water-swellable) polymers that adhere to the wet
surface of the buccal mucosa. Examples of polymeric carriers useful
herein include acrylic acid polymers and co, e.g., those known as
"carbomers" (Carbopol.RTM., which may be obtained from B.F.
Goodrich, is one such polymer). Other components may also be
incorporated into the buccal dosage forms described herein include,
but are not limited to, disintegrants, diluents, binders,
lubricants, flavoring, colorants, preservatives, and the like. For
buccal or sublingual administration, the compositions may take the
form of tablets, lozenges, or gels.
[0159] Transdermal formulations described herein may be
administered using a variety of devices that include, but are not
limited to, U.S. Pat. Nos. 3,598,122, 3,598,123, 3,710,795,
3,731,683, 3,742,951, 3,814,097, 3,921,636, 3,972,995, 3,993,072,
3,993,073, 3,996,934, 4,031,894, 4,060,084, 4,069,307, 4,077,407,
4,201,211, 4,230,105, 4,292,299, 4,292,303, 5,336,168, 5,665,378,
5,837,280, 5,869,090, 6,923,983, 6,929,801 and 6,946,144.
[0160] The transdermal dosage forms described herein may
incorporate certain pharmaceutically acceptable excipients. In one
embodiments, the transdermal formulations described herein include
at least three components: (1) a formulation of a compound of
Formula I or Formula II; (2) a penetration enhancer; and (3) an
aqueous adjuvant. In addition, transdermal formulations can include
additional components such as, but not limited to, gelling agents,
creams and ointment bases, and the like. In some embodiments, the
transdermal formulation can further include a woven or non-woven
backing material to enhance absorption and prevent the removal of
the transdermal formulation from the skin. In other embodiments,
the transdermal formulations described herein can maintain a
saturated or supersaturated state to promote diffusion into the
skin.
[0161] Formulations suitable for transdermal administration of
compounds described herein may employ transdermal delivery devices
and transdermal delivery patches and can be lipophilic emulsions or
buffered, aqueous solutions, dissolved and/or dispersed in a
polymer or an adhesive. Such patches may be constructed for
continuous, pulsatile, or on demand delivery of pharmaceutical
agents. Still further, transdermal delivery of the compounds
described herein can be accomplished by means of iontophoretic
patches and the like. Additionally, transdermal patches can provide
controlled delivery of the compounds of Formula I or Formula II.
The rate of absorption can be slowed by using rate-controlling
membranes or by trapping the compound within a polymer matrix or
gel. Conversely, absorption enhancers can be used to increase
absorption. An absorption enhancer or carrier can include
absorbable pharmaceutically acceptable solvents to assist passage
through the skin. For example, transdermal devices are in the form
of a bandage comprising a backing member, a reservoir containing
the compound optionally with carriers, optionally a rate
controlling barrier to deliver the compound to the skin of the host
at a controlled and predetermined rate over a prolonged period of
time, and means to secure the device to the skin.
[0162] Formulations that include a compound of Formula I or Formula
II, suitable for intramuscular, subcutaneous, or intravenous
injection may include physiologically acceptable sterile aqueous or
non-aqueous solutions, dispersions, suspensions or emulsions, and
sterile powders for reconstitution into sterile injectable
solutions or dispersions. Examples of suitable aqueous and
non-aqueous carriers, diluents, solvents, or vehicles including
water, ethanol, polyols (propyleneglycol, polyethylene-glycol,
glycerol, cremophor and the like), suitable mixtures thereof,
vegetable oils (such as olive oil) and injectable organic esters
such as ethyl oleate. Proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of
dispersions, and by the use of surfactants. Formulations suitable
for subcutaneous injection may also contain additives such as
preserving, wetting, emulsifying, and dispensing agents. Prevention
of the growth of microorganisms can be ensured by various
antibacterial and antifungal agents, such as parabens,
chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like. Prolonged absorption of the injectable
pharmaceutical form can be brought about by the use of agents
delaying absorption, such as aluminum monostearate and gelatin.
[0163] For intravenous injections, compounds described herein may
be formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hank's solution, Ringer's solution, or
physiological saline buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. For other parenteral injections, appropriate
formulations may include aqueous or nonaqueous solutions,
preferably with physiologically compatible buffers or
excipients.
[0164] Parenteral injections may involve bolus injection or
continuous infusion. Formulations for injection may be presented in
unit dosage form, e.g., in ampoules or in multi-dose containers,
with an added preservative. The pharmaceutical composition
described herein may be in a form suitable for parenteral injection
as a sterile suspensions, solutions or emulsions in oily or aqueous
vehicles, and may contain formulatory agents such as suspending,
stabilizing and/or dispersing agents. Pharmaceutical formulations
for parenteral administration include aqueous solutions of the
active compounds in water-soluble form. Additionally, suspensions
of the active compounds may be prepared as appropriate oily
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acid
esters, such as ethyl oleate or triglycerides, or liposomes.
Aqueous injection suspensions may contain substances which increase
the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Optionally, the suspension may
also contain suitable stabilizers or agents which increase the
solubility of the compounds to allow for the preparation of highly
concentrated solutions. Alternatively, the active ingredient may be
in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0165] In certain embodiments, delivery systems for pharmaceutical
compounds may be employed, such as, for example, liposomes and
emulsions. In certain embodiments, compositions provided herein can
also include an mucoadhesive polymer, selected from among, for
example, carboxymethylcellulose, carbomer (acrylic acid polymer),
poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic
acid/butyl acrylate copolymer, sodium alginate and dextran.
[0166] In some embodiments, the compounds described herein may be
administered topically and can be formulated into a variety of
topically administrable compositions, such as solutions,
suspensions, lotions, gels, pastes, medicated sticks, balms, creams
or ointments. Such pharmaceutical compounds can contain
solubilizers, stabilizers, tonicity enhancing agents, buffers and
preservatives.
[0167] The compounds described herein may also be formulated in
rectal compositions such as enemas, rectal gels, rectal foams,
rectal aerosols, suppositories, jelly suppositories, or retention
enemas, containing suppository bases such as cocoa butter or other
glycerides, as well as synthetic polymers such as
polyvinylpyrrolidone, PEG, and the like. In suppository forms of
the compositions, a low-melting wax such as, but not limited to, a
mixture of fatty acid glycerides, optionally in combination with
cocoa butter is first melted.
[0168] In other embodiments, the formulations described herein,
which include a compound of Formula I or Formula II, are blood
brain barrier-permeable (BBB-permeable) nanoparticle formulations.
Methods of producing such BBB-permeable nanoparticle formulations
include, but are not limited to, for example, U.S. Pat. Nos.
6,117,454 and 7,025,991.
Examples of Methods of Dosing and Treatment Regimens
[0169] The compounds described herein can be used in the
preparation of medicaments for the treatment of neurodegenerative
diseases or conditions that would benefit, at least in part, from
neuroprotection. In addition, a method for treating any of the
diseases or conditions described herein in a subject in need of
such treatment, involves administration of pharmaceutical
compositions containing at least one compound of Formula I or
Formula II described herein, or a pharmaceutically acceptable salt,
pharmaceutically acceptable N-oxide, pharmaceutically active
metabolite, pharmaceutically acceptable prodrug, or
pharmaceutically acceptable solvate thereof, in therapeutically
effective amounts to said subject.
[0170] The compositions containing the compound(s) described herein
can be administered for prophylactic and/or therapeutic treatments.
In therapeutic applications, the compositions are administered to a
patient already suffering from a disease or condition, in an amount
sufficient to cure or at least partially arrest the symptoms of the
disease or condition. Amounts effective for this use will depend on
the severity and course of the disease or condition, previous
therapy, the patient's health status, weight, and response to the
drugs, and the judgment of the treating physician.
[0171] In prophylactic applications, compositions containing the
compounds described herein are administered to a patient
susceptible to or otherwise at risk of a particular disease,
disorder or condition. Such an amount is defined to be a
"prophylactically effective amount or dose." In this use, the
precise amounts also depend on the patient's state of health,
weight, and the like. When used in a patient, effective amounts for
this use will depend on the severity and course of the disease,
disorder or condition, previous therapy, the patient's health
status and response to the drugs, and the judgment of the treating
physician.
[0172] In the case wherein the patient's condition does not
improve, upon the doctor's discretion the administration of the
compounds may be administered chronically, that is, for an extended
period of time, including throughout the duration of the patient's
life in order to ameliorate or otherwise control or limit the
symptoms of the patient's disease or condition.
[0173] In the case wherein the patient's status does improve, upon
the doctor's discretion the administration of the compounds may be
given continuously; alternatively, the dose of drug being
administered may be temporarily reduced or temporarily suspended
for a certain length of time (i.e., a "drug holiday"). The length
of the drug holiday can vary between 2 days and 1 year, including
by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7
days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50
days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days,
250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The
dose reduction during a drug holiday may be from 10%-100%,
including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
100%.
[0174] Once improvement of the patient's conditions has occurred, a
maintenance dose is administered if necessary. Subsequently, the
dosage or the frequency of administration, or both, can be reduced,
as a function of the symptoms, to a level at which the improved
disease, disorder or condition is retained. Patients can, however,
require intermittent treatment on a long-term basis upon any
recurrence of symptoms.
[0175] The amount of a given agent that will correspond to such an
amount will vary depending upon factors such as the particular
compound, disease or condition and its severity, the identity
(e.g., weight) of the subject or host in need of treatment, but can
nevertheless be determined according to the particular
circumstances surrounding the case, including, e.g., the specific
agent being administered, the route of administration, the
condition being treated, and the subject or host being treated. In
general, however, doses employed for adult human treatment will
typically be in the range of 0.02-5000 mg per day, preferably
1-1500 mg per day. The desired dose may conveniently be presented
in a single dose or as divided doses administered simultaneously
(or over a short period of time) or at appropriate intervals, for
example as two, three, four or more sub-doses per day.
[0176] The pharmaceutical composition described herein may be in
unit dosage forms suitable for single administration of precise
dosages. In unit dosage form, the formulation is divided into unit
doses containing appropriate quantities of one or more compound.
The unit dosage may be in the form of a package containing discrete
quantities of the formulation. Non-limiting examples are packaged
tablets or capsules, and powders in vials or ampoules. Aqueous
suspension compositions can be packaged in single-dose
non-reclosable containers. Alternatively, multiple-dose reclosable
containers can be used, in which case it is typical to include a
preservative in the composition. By way of example only,
formulations for parenteral injection may be presented in unit
dosage form, which include, but are not limited to ampoules, or in
multi-dose containers, with an added preservative.
[0177] The daily dosages appropriate for the compounds described
herein described herein are from about 0.01 to 2.5 mg/kg per body
weight. An indicated daily dosage in the larger mammal, including,
but not limited to, humans, is in the range from about 0.5 mg to
about 100 mg, conveniently administered in divided doses,
including, but not limited to, up to four times a day or in
extended release form. Suitable unit dosage forms for oral
administration include from about 1 to 50 mg active ingredient. The
foregoing ranges are merely suggestive, as the number of variables
in regard to an individual treatment regime is large, and
considerable excursions from these recommended values are not
uncommon. Such dosages may be altered depending on a number of
variables, not limited to the activity of the compound used, the
disease or condition to be treated, the mode of administration, the
requirements of the individual subject, the severity of the disease
or condition being treated, and the judgment of the
practitioner.
[0178] Toxicity and therapeutic efficacy of such therapeutic
regimens can be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, including, but not limited
to, the determination of the LD50 (the dose lethal to 50% of the
population) and the ED50 (the dose therapeutically effective in 50%
of the population). The dose ratio between the toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio between LD50 and ED50. Compounds exhibiting
high therapeutic indices are preferred. The data obtained from cell
culture assays and animal studies can be used in formulating a
range of dosage for use in human. The dosage of such compounds lies
preferably within a range of circulating concentrations that
include the ED50 with minimal toxicity. The dosage may vary within
this range depending upon the dosage form employed and the route of
administration utilized.
Combination Treatments
[0179] The neuroprotective compound compositions described herein
can also be used in combination with other therapeutic reagents
that are selected for their therapeutic value for the condition to
be treated. In general, the compositions described herein and, in
embodiments where combinational therapy is employed, other agents
do not have to be administered in the same pharmaceutical
composition, and may, because of different physical and chemical
characteristics, have to be administered by different routes.
[0180] In certain instances, it may be appropriate to administer at
least one neuroprotective compound described herein in combination
with another therapeutic agent. By way of example only, if one of
the side effects experienced by a patient upon receiving one of the
neuroprotective compounds described herein is nausea, then it may
be appropriate to administer an anti-nausea agent in combination
with the initial therapeutic agent. Or, by way of example only, the
therapeutic effectiveness of one of the compounds described herein
may be enhanced by administration of an adjuvant (i.e., by itself
the adjuvant may have minimal therapeutic benefit, but in
combination with another therapeutic agent, the overall therapeutic
benefit to the patient is enhanced). Or, by way of example only,
the benefit experienced by a patient may be increased by
administering one of the compounds described herein with one or
more (e.g., one, two, or three) other therapeutic agents (which
also includes a therapeutic regimen) that also have a therapeutic
benefit. In any case, regardless of the disease, disorder or
condition being treated, the overall benefit experienced by the
patient may simply be additive of the multiple therapeutic agents
or the patient may experience a synergistic (i.e., a greater than
additive) benefit due to their specific combination.
[0181] The compounds may be administered concurrently (e.g.,
simultaneously, essentially simultaneously or within the same
treatment protocol) or sequentially, depending upon the nature of
the disease, disorder, or condition, the condition of the patient,
and the actual choice of compounds used.
[0182] In any case, the multiple therapeutic agents (one of which
is a compound of Formula I or Formula II described herein) may be
administered in any order, or even simultaneously. If
simultaneously, the multiple therapeutic agents may be provided in
a single, unified form, or in multiple forms (by way of example
only, either as a single pill or as two separate pills). One of the
therapeutic agents may be given in multiple doses, or both may be
given as multiple doses. If not simultaneous, the timing between
the multiple doses may vary from more than zero weeks to less than
four weeks. In addition, the combination methods, compositions and
formulations are not to be limited to the use of only two agents;
the use of multiple therapeutic combinations is also
envisioned.
[0183] The pharmaceutical agents which make up the combination
therapy disclosed herein may be a combined dosage form or in
separate dosage forms intended for substantially simultaneous
administration. The pharmaceutical agents that make up the
combination therapy may also be administered sequentially, with
either therapeutic compound being administered by a regimen calling
for two-step administration. The two-step administration regimen
may call for sequential administration of the active agents or
spaced-apart administration of the separate active agents. The time
period between the multiple administration steps may range from, a
few minutes to several hours, depending upon the properties of each
pharmaceutical agent, such as potency, solubility, bioavailability,
plasma half-life and kinetic profile of the pharmaceutical agent.
Circadian variation of the target molecule concentration may also
determine the optimal dose interval.
[0184] In addition, the compounds described herein also may be used
in combination with procedures that may provide additional or
synergistic benefit to the patient. By way of example only,
patients are expected to find therapeutic and/or prophylactic
benefit in the methods described herein, wherein pharmaceutical
composition of a compound disclosed herein and/or combinations with
other therapeutics are combined with genetic testing to determine
whether that individual is a carrier of a mutant gene that is known
to be correlated with certain diseases or conditions.
[0185] The compounds described herein and combination therapies can
be administered before, during or after the occurrence of a disease
or condition, and the timing of administering the composition
containing a compound can vary. Thus, for example, the compounds
can be used as a prophylactic and can be administered continuously
to subjects with a propensity to develop conditions or diseases in
order to prevent the occurrence of the disease or condition. A
compound is preferably administered as soon as is practicable after
the onset of a disease or condition is detected or suspected, and
for a length of time necessary for the treatment of the disease,
such as, for example, from about 1 month to about 3 months. The
length of treatment can vary for each subject. For example, the
compound or a formulation containing the compound can be
administered for at least 2 weeks, about 1 month to about 5 years,
and from about 1 month to about 3 years.
Exemplary Therapeutic Agents for Use in Combination with a
Neuroprotective Compound
Agents for Treating Multiple Sclerosis
[0186] Where a subject is suffering from or at risk of suffering
from multiple sclerosis, a neuroprotective compound disclosed
herein can be used together with one or more of the following
exemplary multiple sclerosis therapeutic agents in any combination:
Interferon .beta.-1a, Interferon .beta.-1b, glatiramer acetate
(Copaxone.RTM.), mitoxantrone (Novantrone.RTM.), low dose
naltrexone, Natalizumab (Tysabri.RTM.), Sativex.RTM., Aimspro
(Goats Serum), Trimesta (Oral Estriol), Laquinimod, FTY720
(Fingolimod), MBP8298, NeuroVax.TM., Tovaxin.TM., Revimmune,
CHR-1103, BHT-3009, BG-12, Cladribine, daclizumab (Zenapax)
Rituximab (Rituxan), cyclophosphamide, Campath, Fampridine-SR,
MN-166, Temsirolimus, or RPI-78M.
Agents for Treating Dementia (e.g., Alzheimer's Disease or
Aids-Related Dementia)
[0187] Where a subject is suffering from or at risk of suffering
from dementia, a neuroprotective compound disclosed herein can be
used together with one or more agents or methods for treating
dementia in any combination. Examples of therapeutic
agents/treatments for treating dementia include, but are not
limited to any of the following: Flurizan.TM. (MPC-7869, r
flurbiprofen), memantine, galantamine, rivastigmine, donezipil,
tacrine, A.beta..sub.1-42 immunotherapy, resveratrol,
(-)-epigallocatechin-3-gallate, statins, vitamin C, or vitamin
E.
Agents for Treating Parkinson's Disease
[0188] Where a subject is suffering from or at risk of suffering
from Parkinson's Disease, a neuroprotective compound disclosed
herein can be used together with one or more agents or methods for
treating Parkinson's disease in any combination. Examples of
therapeutic agents/treatments for treating Parkinson's Disease
include, but are not limited to any of the following: L-dopa,
carbidopa, benserazide, tolcapone, entacapone, bromocriptine,
pergolide, pramipexole, ropinirole, cabergoline, apomorphine,
lisuride, selegiline, or rasagiline.
Agents for Treating Amyotroyhic Lateral Sclerosis
[0189] Where a subject is suffering from or at risk of suffering
from Amyotrophic Lateral Sclerosis, a neuroprotective compound
disclosed herein can be used together with one or more agents or
methods for treating Amyotrophic Lateral Sclerosis in any
combination. Examples of therapeutic agents/treatments for treating
Parkinson's Disease include, but are not limited to any of the
following: riluzole, insulin-like growth factor 1, or ketogenic
diet.
Agents for Treating Huntington's Disease
[0190] Where a subject is suffering from or at risk of suffering
from Huntington's Disease, a neuroprotective compound disclosed
herein can be used together with one or more agents or methods for
treating Huntington's Disease in any combination. Examples of
therapeutic agents/treatments for treating Huntington's Disease
include, but are not limited to any of the following: dopamine
receptor blockers, creatine, CoQ10, minocycline, exercise,
antioxidants, antidepressants (notably, but not exclusively,
selective serotonin reuptake inhibitors SSRIs, such as sertraline,
fluoxetine, and paroxetine), dopamine antagonists, (e.g.,
tetrabenazine), or RNAi-mediated silencing of mutant Huntingtin
expression.
Agents for Treating Autoimmune Inflammatory, or Allergic
conditions
[0191] Where a subject is suffering from or at risk of suffering
from an autoimmune, inflammatory disease, or allergic condition
that affects the nervous system (see, e.g., Allan et al. (2003),
Philos Trans R Soc Lond B Biol Sci, 358(1438):1669-1677), a
neuroprotective compound disclosed herein can be used together with
one or more of the following therapeutic agents in any combination:
immunosuppressants (e.g., tacrolimus, cyclosporin, rapamicin,
methotrexate, cyclophosphamide, azathioprine, mercaptopurine,
mycophenolate, or FTY720), glucocorticoids (e.g., prednisone,
cortisone acetate, prednisolone, methylprednisolone, dexamethasone,
betamethasone, triamcinolone, beclometasone, fludrocortisone
acetate, deoxycorticosterone acetate, aldosterone), non-steroidal
anti-inflammatory drugs (e.g., salicylates, arylalkanoic acids,
2-arylpropionic acids, N-arylanthranilic acids, oxicams, coxibs, or
sulphonanilides), Cox-2-specific inhibitors (e.g., valdecoxib,
celecoxib, or rofecoxib), leflunomide, gold thioglucose, gold
thiomalate, aurofin, sulfasalazine, hydroxychloroquinine,
minocycline, TNF-.alpha. binding proteins (e.g., infliximab,
etanercept, or adalimumab), abatacept, anakinra, interferon-.beta.,
interferon-.gamma., interleukin-2, allergy vaccines,
antihistamines, antileukotrienes, beta-agonists, theophylline, or
anticholinergics.
Agents for Treating Thromboembolic Disorders
[0192] Where a subject is suffering from or at risk of suffering
from a thromboembolic disorder (e.g., stroke), the subject can be
treated with a neuroprotective compound disclosed herein in any
combination with one or more other anti-thromboembolic agents.
Examples of anti-thromboembolic agents include, but are not limited
any of the following: thrombolytic agents (e.g., alteplase
anistreplase, streptokinase, urokinase, or tissue plasminogen
activator), heparin, tinzaparin, warfarin, dabigatran (e.g.,
dabigatran etexilate), factor Xa inhibitors (e.g., fondaparinux,
draparinux, rivaroxaban, DX-9065a, otamixaban, LY517717, or YM150),
ticlopidine, clopidogrel, CS-747 (prasugrel, LY640315),
ximelagatran, or BIBR 1048.
Agents for Treating an HIV Infection
[0193] Where the subject is suffering from an HIV infection (e.g.,
suffering from AIDS), any of the neuroprotective compounds
disclosed herein can be administered to the subject
prophylactically or therapeutically to treat AIDs-related dementia
in combination with one or more anti-HIV compounds administered to
treat the HIV infection. Examples of anti-HIV compounds include,
but are not limited to, AZT (zidovudine, Retrovir), ddI
(didanosine, Videx), 3TC (lamivudine, Epivir), d4T (stavudine,
Zerit), abacavir (Ziagen), and FTC (emtricitabine, Emtriva),
tenofovir (Viread), efavirenz (Sustiva), nevirapine (Viramune),
lopinavir/ritonavir (Kaletra), indinavir (Crixivan), ritonavir
(Norvir), nelfinavir (Viracept), saquinavir hard gel capsules
(Invirase), atazanavir (Reyataz), amprenavir (Agenerase),
fosamprenavir (Telzir), tipranavir (Aptivus), or T20 (enfuvirtide,
Fuzeon)
Antipsychotic Compounds
[0194] Where the subject is suffering from schizophrenia, which has
recently been found to be characterized by a progressive
neurodegenerative process (see, e.g., Perez-Neri et al. (2006),
Neurochem Res, 31(10):1279-1294), any of the neuroprotective
compounds disclosed herein can be administered to the subject
prophylactically or therapeutically in combination with one or more
antipsychotic compounds for treatment of schizophrenia. Examples of
antipsychotic compounds include, but are not limited to, clozapine,
risperidone, olanzapine, quetiapine, ziprasidone, aripiprazole,
paliperidone, sertindole, zotepine, amisulpride, bifeprunox,
melperone, chlorpromazine (largactil, thorazine), fluphenazine,
haloperidol, molindone, thiothixene, thioridazine, trifluoperazine,
loxapine, perphenazine, prochlorperazine, pimozide, thiothixene, or
zuclopenthixol.
Antiepileptic Compounds
[0195] Where the subject is suffering from epilepsy, any of the
neuroprotective compounds disclosed herein can be administered to
the subject prophylactically or therapeutically in combination with
one or more antiepileptic compounds. Examples of antiepileptic
compounds include, but are not limited to, carbamazepine, clobazam,
clonazepam, ethosuximide, felbamate, fosphenyloin, flurazepam,
gabapentin, lamotrigine, levetiracetam, oxcarbazepine, mephenyloin,
phenobarbital, phenyloin, pregabalin, primidone, sodium valproate,
tiagabine, topiramate, valproate semisodium, valproic acid,
vigabatrin, diazepam, or lorazepam.
Neuroprotective Compounds and Compositions
[0196] In some embodiments, one or more of the neuroprotective
modified terpenoid compounds disclosed herein can be used in
combination with one or more neuroprotective compounds or
compositions to treat a subject suffering from or at risk of
neurodegenerative condition. Examples of neuroprotective compounds
include, but are not limited to, any of the following: resveratrol,
GPI 1046, epigallocatechin gallate, .alpha.-lipoic acid, Omega-3
fatty acids (e.g., docosahexaenoic acid or eicosapentaenoic acid),
Vitamin E (tocopherol), carnitine, cytidine diphosphocoline
(citicholine), coenzyme Q10, curcumin, salviolonic acid B, folic
acid, Gingko biloba extract, ginsenoside Rb1, ginsenoside Rg3,
L-Glutathione, grape seed extract, lutein, zeaxanthin,
methylcobalamin, N-acetyl-L-cysteine, pycnogenol, quercetin, or
taurine.
EXAMPLES
[0197] The following specific examples are to be construed as
merely illustrative, and not limitative of the remainder of the
disclosure in any way whatsoever. Without further elaboration, it
is believed that one skilled in the art can, based on the
description herein, utilize the present invention to its fullest
extent. All publications cited herein are hereby incorporated by
reference in their entirety. Where reference is made to a URL or
other such identifier or address, it is understood that such
identifiers can change and particular information on the internet
can come and go, but equivalent information can be found by
searching the internet. Reference thereto evidences the
availability and public dissemination of such information.
Example 1
Identification of Neuroprotective Compounds
[0198] We sought to identify neuroprotective compounds using an in
vitro neuroprotection assay as described in detail below. We
screened a specific collection/library of compounds that we
identified as potentially active agents. This particular collection
contains 2000 compounds of which 50% are FDA-approved compounds,
30% are natural products, and 20% are other bioactive compounds. As
shown in FIG. 1, the vast majority of the compounds conferred
little or no neuroprotection or were even neurotoxic, with only
about 3% exhibiting neuroprotective activity. From this subset of
potential compounds (i.e., those exhibiting neuroprotective
activity), we selected a further limited group of modified
terpenoids. A larger series of modified terpenoid compounds was
then screened in the same type of assay as described below.
Exemplary neuroprotective modified terpenoids are shown in FIG.
2.
Example 2
Pharmaceutical Compositions
Example 2a
Parenteral Composition
[0199] To prepare a parenteral pharmaceutical composition suitable
for administration by injection, 100 mg of a water-soluble salt of
a compound of any of Formula I, Formula II, Table 2 or Table 3, is
dissolved in DMSO and then mixed with 10 mL of 0.9% sterile saline.
The mixture is incorporated into a dosage unit form suitable for
administration by injection.
Example 2b
Oral Composition
[0200] To prepare a pharmaceutical composition for oral delivery,
100 mg of a compound of any of Formula I, Formula II, Table 2 or
Table 3, is mixed with 750 mg of starch. The mixture is
incorporated into an oral dosage unit for, such as a hard gelatin
capsule, which is suitable for oral administration.
Example 2c
Sublingual (Hard Lozenge) Composition
[0201] To prepare a pharmaceutical composition for buccal delivery,
such as a hard lozenge, mix 100 mg of a compound of any of Formula
I, Formula II, Table 2 or Table 3, with 420 mg of powdered sugar
mixed, with 1.6 mL of light corn syrup, 2.4 mL distilled water, and
0.42 mL mint extract. The mixture is gently blended and poured into
a mold to form a lozenge suitable for buccal administration.
Example 2d
Inhalation Composition
[0202] To prepare a pharmaceutical composition for inhalation
delivery, 20 mg of a compound of any of Formula I, Formula II,
Table 2 or Table 3, is mixed with 50 mg of anhydrous citric acid
and 100 mL of 0.9% sodium chloride solution. The mixture is
incorporated into an inhalation delivery unit, such as a nebulizer,
which is suitable for inhalation administration.
Example 2e
Rectal Gel Composition
[0203] To prepare a pharmaceutical composition for rectal delivery,
100 mg of a compound of any of Formula I, Formula II, Table 2 or
Table 3, is mixed with 2.5 g of methylcelluose (1500 mPa), 100 mg
of methylparapen, 5 g of glycerin and 100 mL of purified water. The
resulting gel mixture is then incorporated into rectal delivery
units, such as syringes, which are suitable for rectal
administration.
Example 2f
Topical Gel Composition
[0204] To prepare a pharmaceutical topical gel composition, 100 mg
of a compound of any of Formula I, Formula II, Table 2 or Table 3,
is mixed with 1.75 g of hydroxypropyl celluose, 10 mL of propylene
glycol, 10 mL of isopropyl myristate and 100 mL of purified alcohol
USP. The resulting gel mixture is then incorporated into
containers, such as tubes, which are suitable for topical
administration.
Example 2g
Ophthalmic Solution Composition
[0205] To prepare a pharmaceutical ophthalmic solution composition,
100 mg of a compound of any of Formula I, Formula II, Table 2 or
Table 3, is mixed with 0.9 g of NaCl in 100 mL of purified water
and filtered using a 0.2 micron filter. The resulting isotonic
solution is then incorporated into ophthalmic delivery units, such
as eye drop containers, which are suitable for ophthalmic
administration. Example 3: Biological Assays and Analyses
Example 3A
Exemplary Modified Terpenoids are Neuroprotective against an
Oxidative Stressor and a Neurotoxic Protein
[0206] We sought to evaluate the protective efficacy of a number of
modified terpenoid compounds against the against many different
neurotoxins, ranging from the chemotoxic 6-0HDA, NMDA,
3-nitropropionic acid (3-NP), and viral proteins such as Tat and
gp120. Thus, we established an in vitro neuroprotection assay using
rat mixed hippocampal cultures, in which we evaluated the
protective efficacy of neuroprotective compounds disclosed herein.
The oxidative stressor 3-NP was used to elicit toxicity in the rat
hippocampal cultures to mimic the oxidative damage, reactive oxygen
species production and ensuing neurodegeneration resulting from HIV
infection. Another measure of neurotoxicity which results from HIV
infection was evaluated by exposure of the hippocampal cultures to
HIV-1 Tat (Li et al (2005), Neurotox Res, 8(1-2):119-134).
[0207] Rat mixed hippocamal neuronal cultures were generated from
freshly dissected rat hippocampi (embryonic day 18) in neurobasal
media containing 5% fetal bovine serum and 2% B27 supplement. The
cells were plated into 96 well plates at a density of
4.times.10.sup.5 cells/mL and routinely used on days 11-14
following culturing. Cell viability was assessed with MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay. The MTT assay is based on the ability of a mitochondrial
dehydrogenase enzyme from viable cells to cleave the tetrazolium
rings of the pale yellow MTT and form dark blue formazan crystals.
See Mosmann (1983), J Immunol Methods, 65(1-2):55-63. These
crystals are largely impermeable to cell membranes, and thus
accumulate within healthy cells. The resultant formazan
precipitates are solubilized with DMSO and read on a multiwell
scanning spectrophotometer (ELISA reader). The number of surviving
cells is directly proportional to the level of the formazan product
created.
[0208] Mixed hippocampal cultures were incubated with 3-NP (0.5-10
mM) for 18 hours and then assessed for viability using an MTT
assay. As shown in FIG. 3, titration of 3-NP levels for neurotoxic
effects demonstrated that 3 mM 3-NP treatment consistently induced
25-35% cytotoxicity in rat mixed hippocampal cultures.
[0209] The assay system was validated using two neuroprotective
agents, GPI 1046 and Resveratrol. Both of these compounds has
demonstrated antioxidant and/or neuroprotective activities in
numerous in vitro and in vivo assays (for review, see Poulter et
al. (2004), Neuroscience, 128(1):1-6; Caporello, et al. (2006), J
Neurochem, 98(1):146-155; Zamin et al. (2006), Neurobiol Dis,
24(1):176-182). Cultures were preincubated with GPI 1046 or
Resveratrol for one hour prior to an 18 hour exposure to 3 mM 3-NP.
These "positive control" neuroprotective compounds significantly
protected rat neurons from oxidative damage elicited by 3-NP (FIG.
4A) in the rat mixed hippocampal culture assay system described
above. The same neuroprotective compounds were evaluated for
efficacy against HIV-1 Tat protein toxicity using the same 1 hour
preincubation protocol. As with the 3-NP neurotoxicity assay, these
compounds protected hippocampal neurons from Tat toxicity as well
(FIG. 4B). These data indicated that the measurement of
neuroprotection against 3-NP toxicity likely serves as a good
indicator of protective activity against HIV-1 Tat toxicity.
[0210] Using the validated 3 mM 3-NP neurotoxicity assay described
above, we evaluated the neuroprotective efficacy of approximately
2000 compounds from the Spectrum Collection (MicroSource Discovery)
as described in Example 1. Several of the neuroprotective compounds
identified in this collection were modified terpenoids. Thus, we
tested an expanded collection of modified terpenoids in the in
vitro neuroprotection assay. As shown in Tables 2 and 3, a number
of modified terpenoid compounds were identified as having
neuroprotective activity against 3-NP. NP assay data for four of
these compounds are shown in FIG. 5. These compounds protected the
mixed hippocampal cultures significantly, with nearly complete
protection provided by 10 .mu.M of Khivorin, about 60-70%
protection resulting from odoratone, and about 50% protection from
gedunin and angolensic acid, methyl ester treatment. The 3-NP
protection dose-response characteristics of the modified terpenoids
Khivorin, Gedunin, Nomilin, and Limonin are shown in FIGS. 6A-6D,
respectively. Gedunin and Limonin also dose dependently protected
hippocampal cultures from HIV-1 Tat toxicity (FIG. 7), with nearly
complete neuroprotection provided by 1-10 .mu.M Gedunin. Thus, some
modified terpenoids also protect hippocampal neurons from HIV-1
neurotoxic protein degeneration.
[0211] We also tested the ability of Limonin to protect against
N-methyl-D-Aspartic Acid (NMDA) excitotoxicity, and
6-Hydroxydopamine (6-0HDA), a dopaminergic neurotoxin. As shown in
FIG. 8, Limonin dose-dependently blocked the neurotoxicity of both
NMDA (FIG. 8A) and 6-OHDA (FIG. 8B) conferring complete protection
at the highest Limonin dose tested (10 .mu.M).
[0212] Finally, we sought to determine if the compounds tested in
rat neuronal cultures would also be effective on cultured human
fetal neurons. Indeed, as shown in FIG. 9, Limonin dose-dependently
protected human fetal neuronal cultures against both 3-NP (FIG. 9A)
and 6-OHDA.
[0213] Based on these data, we concluded that various modified
terpenoid compounds are neuroprotective. TABLE-US-00002 TABLE 2
Modified Terpenoids having Significant 3-NP Neuroprotective
Activity Compound Structure % Protection 3alpha-
ACETOXYDIHYDRODEOXY- GEDUNIN ##STR24## 151 1,3-DIDEACETYLKHIVORIN
##STR25## 129 DEOXODIHYDROGEDUNIN ##STR26## 101 3beta-
ACETOXYDEOXYANGOLENSIC ACID, METHYL ESTER ##STR27## 128
TRIDESACETOXYKHIVORIN ##STR28## 97 7beta-HYDROXY-7-
DESACETOXYKHIVORINIC ACID, METHYL ESTER ##STR29## 84 3beta-
HYDROXYDEOXODIHYDRO- GEDUNIN ##STR30## 82 DEOXODEOXYDIHYDROGED-
UNIN ##STR31## 77 KHIVORIN ##STR32## 70 EPOXYGEDUNIN ##STR33## 69
7-EPIKHIVORIN ##STR34## 65 3beta,7beta- DIACETOXYDEOXODEACE-
TOXYDEOXYDIHYDROGEDUNIN ##STR35## 61 DESACETYL (7)KHIVORINIC ACID,
METHYL ESTER ##STR36## 58 3-DEOXO-3beta- ACETOXYDEOXYDIHYDRO-
GEDUNIN ##STR37## 56 3beta- HYDROXYDEOXODIHYDRO- DEOXYGEDUNIN
##STR38## 56 DEOXYGEDUNOL ACETATE ##STR39## 54 ISOGEDUNIN ##STR40##
50 GEDUNOL ##STR41## 45 2,3-DIHYDROLSOGEDUNIN ##STR42## 44
7-DEACETOXY-7-OXO- KHIVORINIC ACID, METHYL ESTER ##STR43## 42
TRIDESACETOXYKHIVORIN ##STR44## 39 3beta- ACETOXYDEOXODIHYDRO-
GEDUNIN ##STR45## 39 DEACETOXY-7-OXOGEDUNIN ##STR46## 38
DEOXYKHIVORIN ##STR47## 36 7-DEACETOXY-7- OXOKHIVORIN ##STR48## 36
3alpha-HYDROXY-3- DEOXYANGOLENSIC ACID METHYL ESTER ##STR49## 35
ANGOLENSIC ACID, METHYL ESTER ##STR50## 33 7-DEAGETYLKHIVORIN
##STR51## 32 3beta- HYDROXYDEOXYDESACE- TOXY-7-OXOGEDUNIN ##STR52##
30 3-alpha- HYDROXYDEOXYGEDININ ##STR53## 28 DIHYDROGEDUNIN
##STR54## 28 6-HYDROXYANGOLENSIC ACID METHYL ESTER ##STR55## 26
1,2alpha- EPOXYDEACETOXYDIHYDRO- GEDUNIN ##STR56## 25
7-DEACETOXY-7- OXODEOXYGEDUNIN ##STR57## 24 DEOXYGEDUNIN ##STR58##
23 GEDUNIN ##STR59## 40 DEACETYLGEDUNIN ##STR60## 19 DIHYDROGEDUNIN
ETHANEDITHIOKETAL ##STR61## 19 1,7-DIDEACETOXY-1,7- DIOXO-3-
DEACETYLKHIVORIN ##STR62## 15 1,3-DIDEACETYL-7- DEACETOXY-7-
OXOKHIVORIN ##STR63## 15 1 (2)alpha- EPOXYDEOXYDIHYDROG- EDUNIN
##STR64## 14
[0214] TABLE-US-00003 TABLE 3 Modified Terpenoids Exhibiting
Neuroprotective Activity against 3-NP % Protection Structure vs
3-NP 3beta-ACETOXYDEOXODIHYDROGEDUNIN 39 CARAPIN-8(9)-ENE 38
DEACETOXY-7-OXOGEDUNIN 38 KHAYANTHONE 36 DEOXYKHIVORIN 36
7-DEACETOXY-7-OXOKHIVORIN 36 ANGOLENSIC ACID, METHYL ESTER 33
7-DEACETYLKHIVORIN 32 FISSINOLIDE 28 6-HYDROXYANGOLENSIC ACID
METHYL ESTER 26 DEOXYGEDUNIN 23 DEACETYLGEDUNIN 19 BUSSEIN 18
CARAPIN 18 ENTANDROPHRAGMIN 18 UTILIN 16
1,3-DIDEACETYL-7-DEACETOXY-7- 15 OXOKHIVORIN
1,7-DIDEACETOXY-1,7-DIOXO-3- 15 DEACETYLKHIVORIN KHAYASIN C 13
DIHYDROFISSINOLIDE 12 8beta-HYDROXYCARAPIN, 3,8-HEMIACETAL 9
MEXICANOLIDE 9 QUASSIN 8 3-DEACETYLKHIVORIN 7 PRIEURIANIN 6
Example 3B
In Vitro Modeling of Blood Brain Barrier Permeability to Test
Compounds
[0215] In vitro models of the Blood Brain Barrier from human brain
microvascular endothelial cells (HBMEC) that were isolated and
characterized have been published previously (see, e.g., Stins et
al. (1997), J Neuroimmunol, 76(1-2):81-90; Cucullo et al. (2007),
Epilepsia, 48(3):505-516). These HBMEC possess gamma glutamyl
transpeptidase (GGTP) and drug transporter P-glycoprotein, and
junctional proteins as seen by ZO1 immuno-staining, thereby
demonstrating their brain endothelial cell characteristics. In
vitro BBB models were constructed by growing HBMEC on microporous
membranes (0.4 .mu.m pore size) in the upper compartment of
semipermeable Transwell.TM. tissue culture inserts (24 wells,
Corning-Costar). The upper compartment compares to blood side and
bottom compartment to brain side. Transmission electron microscopy
revealed a smooth endothelial cell monolayer, typical rod shaped
Weibel-Palade bodies and tight junctions. Polarity was shown after
treatment with TNF-.alpha., which resulted in an apical expression
of ICAM-1, which is in agreement with data of Wong et al. (1992), J
Neuroimmunol, 39(1-2): 11-21. The presence of junctional proteins
is seen by ZO1 immuno-staining and Western blotting for ZO-1,
beta-catenin and occludin-1, showing that HBMEC possess endothelial
and brain characteristics and functions.
[0216] Propidium iodide (PI) (MW=600) at 0.5 mg/ml, an indicator of
in vitro blood brain barrier ("BBB") integrity, is applied to the
upper compartment along with a vehicle solution or a solution
containing test compound at a concentration of 1 .mu.M. At 2 and 4
hours post drug treatment, levels of compound are measured in top
and bottom compartments by mass spectrometery as described in Tian
et al. (2004), Rapid Comm Mass Spec, 18:3099-3104. The level of
test compound detected in the bottom compartment is then normalized
for differences in PI permeability between the test compound
solution and the vehicle control solution.
Example 3C
In Vivo Assessment of Neuroprotective Modified Terpenoid Compounds
in a 3-NP-Induced Neurotoxicity Animal Model
[0217] In order to determine the in vivo efficacy of compounds
identified as neuroprotective against 3-NP in vitro, as described
herein, we employ a 3-NP-induced neurotoxicity model in rats. See,
e.g., Kumar et al. (2006), Behav Pharmacol, 17(5-6):485-492.
[0218] To induce 3-NP neurotoxicity in vivo, 12-week-old male Lewis
rats weighing 340-370 gm are administered intraperitoneal
injections of 3-NP (20 mg/kg) for 4 days. The animals are divided
into three treatment groups (n=12 per group) as follows:
[0219] Group 1 is administered Khivorin, Gedunin, Odoratone,
Angolensic acid, or another modified terpenoid compound once daily
(10 mg/kg; oral gavage) beginning four days prior to and continuing
for four days subsequent to the beginning of the 3-NP
injections.
[0220] Group 2 is administered saline vehicle (negative control),
by oral gavage, beginning four days prior to and continuing for
four days subsequent to the beginning of the 3-NP injections.
[0221] Group 3 is administered resveratrol (positive control) (10
mg/kg; oral gavage) by oral gavage, beginning four days prior to
and continuing for four days subsequent to the beginning of the
3-NP injections.
[0222] Subsequent to the beginning of the 3-NP injections, animals
are assessed for significant loss of body weight, a decline in
motor function (locomotor activity, movement pattern, and vacuous
chewing movements) and cognitive deficits (e.g., impairment in
learning or memory). Differences of performance between groups are
analyzed at each time point by two-tailed t test.
[0223] Twenty-four hours after the last 3-NP injection animals are
anesthetized, perfused transcardially with saline, followed by
ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4.
Brains are immediately removed and postfixed overnight in the same
fixative and then cryoprotected in 30% sucrose in 0.1 M phosphate
buffer, pH 7.4. Sequential coronal sections (30 .mu.m) are made on
a freezing microtome, starting from the anterior aspect of the
corpus callosum throughout the entire striatum. For histological
assessment, every sixth section (210 .mu.m interval) is processed
for cresyl violet staining to assess cell loss and neuronal
degeneration. Cresyl violet staining is performed with standard
protocols.
[0224] Stereological analysis of lesion volumes are performed by
digitally acquiring cresyl violet-stained sections through the
striatum at 4.times. objective using a computerized image analysis
system. Lesion volumes for each group of animals are calculated by
summing the cross-sectional areas of the lesion in each section and
multiplying this value by the distance between sections.
[0225] Compounds found to confer a significant reduction in
3-NP-induced behavioral deficits or neuroanatomical lesion volume
are considered to be neuroprotective in vivo.
Example 3D
In Vivo Assessment of Identified Neuroprotective Compounds in a
Focal Ischemia Animal Model
[0226] A problem with ongoing methodology used to test the efficacy
of neuroprotective compounds in vivo in the classic Experimental
Autoimmune Encephalomyelitis (EAE) model is that
inflammation-mediated lesions are randomly distributed within the
central nervous system, thereby making uniform quantification of
axonal damage and neuronal cell death difficult. In contrast, a
focal lesion model induces a localized inflammatory response and
subsequent lesion only within a specific region of the spinal cord.
The focal lesion model therefore enables a more accurate assessment
of the efficacy of neuroprotective agents because a specific region
of the spinal cord and brain are compared between animals.
Furthermore, the focal lesion model decreases variability found
within the classic EAE models and therefore decreases the number of
animals required.
[0227] Lewis rats (12-15 rats per treatment group) are treated with
a test compound at a dose of 0.1, 1, and 10 mg/kg s.c or p.o. or
with vehicle once daily for 3 days prior to immunization with MOG
and incomplete Freunds Adjuvant as described below. Eighteen days
later, the animals are subjected to laminectomy of the dorsal
column at T8 and injected with TNF-.alpha. and .gamma.-IFN to
induce a focal EAE lesion as described below. Three weeks later,
the animals are sacrificed. Outcome measures include behavioral
studies (Basso-Beattie-Bresnahan scale), evoked potentials (nerve
conduction velocity), radiological outcomes (Diffusion Tensor
Imaging, Magnetic Resonance Imaging), and histology measurements
(Luxol Fast Blue, Toluidine Blue, Myelin Basic Protein,
phosphoNeurofilament and Axonal degeneration). A second cohort
includes another set of animals where the same drug regimen listed
above is initiated following the laminectomy.
Animals
[0228] For these experiments, Lewis rats (10-12 weeks old) are
allowed free access to food and water to acclimate 7-10 days before
the initiation of experiments. At the time of the study, the
animals weigh 200-250 g.
Induction of Myelin Oligodendrocyte Glycoprotein (MOG)
Sensitivity
[0229] Animals are injected subcutaneously at the base of the tail
with 100 .mu.l of recombinant MOG.sub.1-125 (250-500 .mu.g/mL)
emulsified in incomplete Freund's adjuvant. No manipulations are
performed for 18-30 days after immunization to allow for the immune
system to develop a sensitivity to MOG. This procedure does not
induce clinical symptoms of EAE.
Induction of Focal EAE Lesion
[0230] To induce an EAE lesion within a specific region of the
spinal cord, a steriotaxic injection of TNF-.alpha. and IFN-.gamma.
is administered within the spinal cord after laminectomy. The
animals are shaved prior to initiation of the procedure.
Anesthetized animals are placed in the prone position, and a
midline incision is made. Following the dissection of fascia and
muscle, a laminectomy is performed at T8 with Rongeur forceps. A
focal EAE lesion within the cortical spinal tract is induced by
administering 2 .mu.L of 250 ng of TNF-.alpha. and 150 Units of
IFN-.gamma. dissolved in phosphate buffered saline with trace
amounts of Monastral Blue using a capillary glass tube. Following
surgery the wound is sutured first through the fascia, and then the
skin with a 4.0 vicryl thread or with wound clips. Animals are
warmed and allowed to recover.
[0231] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *