U.S. patent application number 16/003517 was filed with the patent office on 2019-01-17 for compositions and methods for treating beta-amyloid related diseases.
The applicant listed for this patent is AMERICAN LIFE SCIENCE PHARMACEUTICALS, INC.. Invention is credited to Amy ALLAN, Gregory HOOK, Robert J. TERNANSKY.
Application Number | 20190015472 16/003517 |
Document ID | / |
Family ID | 43926081 |
Filed Date | 2019-01-17 |
View All Diagrams
United States Patent
Application |
20190015472 |
Kind Code |
A1 |
TERNANSKY; Robert J. ; et
al. |
January 17, 2019 |
COMPOSITIONS AND METHODS FOR TREATING BETA-AMYLOID RELATED
DISEASES
Abstract
In alternative embodiments the invention provides compositions
and methods for ameliorating diseases and conditions having a
beta-amyloid component, including Alzheimer's disease (AD),
Vascular Dementia (VD), dementia, pre-dementia, Cognitive
Dysfunction Syndrome (CDS) and loss of cognition in humans and in
non-human animal. In alternative embodiment the invention provides
analogs of AB-007 and its acid form E64c (loxistatin), their
preparation, and pharmaceutical compositions thereof and methods of
making and using same. In alternative embodiments compositions of
the invention are deuterated analogs of AB-007 (or E64d) and E64c
(or loxistatin). In alternative embodiments compositions of the
invention are metabolically blocked forms as compared to AB-007 and
loxistatin. In alternative embodiments compositions of the
invention are used to ameliorate (including treat, slow, reverse or
prevent) a disease or condition which can be ameliorated by partial
or complete inhibition of a cysteine protease, e.g., AD, VD, CDS.
The invention also provides alternative dosage forms and
formulations for AB-007 and loxistatin, and for compounds of this
invention, which can be used e.g., to treat AD, VD and CDS, in
humans and in non-human animals.
Inventors: |
TERNANSKY; Robert J.; (San
Diego, CA) ; ALLAN; Amy; (San Diego, CA) ;
HOOK; Gregory; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMERICAN LIFE SCIENCE PHARMACEUTICALS, INC. |
San Diego |
CA |
US |
|
|
Family ID: |
43926081 |
Appl. No.: |
16/003517 |
Filed: |
June 8, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14734999 |
Jun 9, 2015 |
10022418 |
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16003517 |
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12910747 |
Oct 22, 2010 |
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14734999 |
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PCT/US2010/044683 |
Aug 6, 2010 |
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12910747 |
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61293783 |
Jan 11, 2010 |
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61232383 |
Aug 7, 2009 |
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61232388 |
Aug 7, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61J 1/035 20130101;
C07K 5/06139 20130101; A61P 25/28 20180101; A61K 45/06 20130101;
A61K 38/05 20130101; A61K 31/336 20130101 |
International
Class: |
A61K 38/05 20060101
A61K038/05; C07K 5/078 20060101 C07K005/078; A61K 45/06 20060101
A61K045/06; A61K 31/336 20060101 A61K031/336; A61J 1/03 20060101
A61J001/03 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with government support under grant
number 4R44AG032784 awarded by the National Institutes of Health
(NIH). The government has certain rights in the invention.
Claims
1-41. (canceled)
42: A method for treating or ameliorating, or treating the symptoms
of, or being palliative for: a cognitive dysfunction or a loss of
cognition, or a traumatic brain injury, comprising: administering
or having administered to an individual in need thereof a
pharmaceutical composition comprising a compound of Formula I:
##STR00081## wherein for both Formula I and Formula II, X.sup.1
through X.sup.11 are -D (deuterium) (positions X.sup.1 through
X.sup.11 are deuterated), and X.sup.12 through X.sup.20 (Formula I)
or X.sup.12 through X.sup.26 (Formula II) are --H (hydrogen).
43: The method of claim 42, wherein for both Formula I and Formula
II, X.sup.1 through X.sup.7 are -D (deuterium) (positions X.sup.1
through X.sup.7 are deuterated), and X.sup.8 through X.sup.30
(Formula I) or X.sup.8 through X.sup.26 (Formula II) are --H
(hydrogen).
44: The method of claim 42, wherein the compound of Formula I has a
stereospecificity as set forth in Formula III: ##STR00082##
45: The method of claim 44, wherein the compound of Formula I or
Formula II is conjugated to a chemical delivery system (CDS), or
further comprises a chemical delivery system (CDS) selected from
the group comprising a pyridinium, a 1,4 dihydrotrigonelline
esters, or a dihydroquinoline or a dihydroisoquinoline, and
optionally the pyridinium is selected from the group consisting of
a 3-methyl-1-propylpyridinium, a 1-butyl-3-methylpyridinium and a
1-butyl-4-methylpyridinium.
46: The method of claim 42, wherein compound of Formula I or
Formula II is formulated in combination with, or together with, a
selegiline, selegiline hydrochloride, or a deprenyl.
47: The method of claim 42, wherein the compound of Formula I or
Formula II is formulated in unit dosage form, and optionally a unit
dosage is between about 1 mg and about 400 mg; or is between about
1 mg and about 250 mg; or is about 5 mg and about 150 mg; or is
between about 1 mg and about 75 mg; or is about 5 mg, about 10 mg,
about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg,
about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg,
about 65 mg, about 70 mg, or about 75 mg.
48: The method of claim 42, wherein the compound of Formula I or
Formula II is formulated for administration as a liquid dosage form
or a solid dosage form, or for parenteral administration or enteral
administration.
49: The method of claim 42, wherein the compound of Formula I or
Formula II is formulated for administration as, or in the form of,
an injectable sterile formulation, a gel, a lotion, a spray, an
aerosol, a powder, a patch, an adhesive tape, a gel, a liquid, an
elixir, a syrup, a suspension, a lyophilate, a lozenge, a pill, a
geltab, a lozenge, a tablet, a capsule and/or an implant.
50: The method of claim 42, wherein the compound of Formula I or
Formula II is formulated as a chow, or a feed or a feed
supplement.
51: The method of claim 42, wherein the compound of Formula I or
Formula II is formulated in combination with or together with a
nutritional supplement or a vitamin.
52: The method of claim 42, wherein the compound of Formula I or
Formula II is formulated in combination with or together with a
non-steroidal anti-inflammatory drug.
53: The method of claim 42, wherein the compound of Formula I or
Formula II is formulated in combination with or together with an
antioxidant.
54: The method of claim 42, wherein the compound of Formula I or
Formula II is formulated for administration as an implantable
infusion system, and the compound is administered by an implantable
infusion system.
55: The method of claim 42, wherein the compound of Formula I or
Formula II is formulated with microencapsulation to delay
disintegration and adsorption in the gastrointestinal tract to
provide a sustained action over a longer period.
56: The method of claim 42, wherein t wherein the compound of
Formula I or Formula II is formulated with a time delay material,
and optionally the time delay material comprises a glyceryl
monostearate or a glyceryl distearate. wherein the compound of
Formula I or Formula II is the composition is formulated with a
solid or a liquid carrier.
58: The method of claim 42, wherein the compound of Formula I or
Formula II is formulated as or with: an aqueous or a non-aqueous
solvent, an aqueous or a non-aqueous solution, a suspension, an
emulsion or a solid, and optionally the non-aqueous solvent
comprises a propylene glycol, a polyethylene glycol, a vegetable
oil, an injectable organic ester, and optionally the aqueous
carrier comprises water, an ethanol, an alcoholic/aqueous solution,
a glycerol, a saline or a buffered media.
59: The method of claim 42, wherein the compound of Formula I or
Formula II is: (a) formulated in a liposome; (b) administered using
an implant, an osmotic pump, an implantable infusion system or an
intrathecal catheter; or (c) formulated as a pharmaceutical
composition packaged in a blister pack, blister packettes, blister
package, lidded blister, blister card, clamshell, tray or
shrinkwrap.
60: A method for treating or ameliorating, or treating the symptoms
of, or being palliative for: a cognitive dysfunction or a loss of
cognition, or a traumatic brain injury, comprising: administering
or having administered to an individual in need thereof a
pharmaceutical composition comprising a compound of Formula IV:
##STR00083## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, and
R.sup.5 are -D (deuterium), and R.sup.6, R.sup.7, R.sup.8, R.sup.9,
R.sup.10 and R are --H (hydrogen).
61: The method of claim 62, wherein the compound of Formula IV
R.sup.1 through R.sup.3 are -D (deuterium) and R.sup.4 through
R.sup.10 are --H (hydrogen) and R is ethyl; or, the compound of
Formula IV R.sup.1 through R.sup.3 are -D (deuterium) and R.sup.4
through R.sup.10 and R are --H (hydrogen).
Description
RELATED APPLICATIONS
[0001] This application is a continuation of International Patent
Application Serial No. PCT/US2010/044683, filed Aug. 6, 2010, which
claims the benefit of priority under 35 U.S.C. .sctn. 119(e) of
U.S. Provisional Patent Application Ser. No. ("USSN") 61/293,783,
filed Jan. 11, 2010; U.S. Ser. No. 61/232,383, filed Aug. 7, 2009;
and U.S. Ser. No. 61/232,388, filed Aug. 7, 2009. Each of the
aforementioned applications are expressly incorporated herein by
reference in their entirety and for all purposes.
FIELD OF THE TECHNOLOGY
[0003] The invention generally relates to medicinal chemistry,
veterinary medicine and cell biology. In alternative embodiments
the invention provides compositions and methods for ameliorating
diseases and conditions having a beta-amyloid component, including
Alzheimer's disease (AD), Vascular Dementia (VD), dementia,
pre-dementia, Cognitive Dysfunction Syndrome (CDS) and loss of
cognition, in humans and in non-human animals. In alternative
embodiments the invention provides analogs of AB-007 (E64d, or
loxistatin) and its acid form E64c (loxistatin acid), their
preparation, and pharmaceutical compositions thereof and methods of
making and using same. In alternative embodiments compositions of
the invention are deuterated analogs of AB-007 (or E64d or
loxistatin) and E64c (or loxistatin acid). In alternative
embodiments compositions of the invention are metabolically blocked
forms as compared to AB-007 and loxistatin. In alternative
embodiments compositions of the invention are used to ameliorate
(including treat, slow, reverse or prevent) a disease or condition
which can be ameliorated by partial or complete inhibition of a
cysteine protease, e.g., Alzheimer's disease (AD), Vascular
Dementia (VD), dementia, pre-dementia, Cognitive Dysfunction
Syndrome (CDS) and loss of cognition in humans and in non-human
animal. The invention also provides alternative dosage forms and
formulations for AB-007 (E64d, loxistatin) and loxistatin acid
(E64c), and for compounds of this invention.
BACKGROUND
[0004] AB-007 (also called loxistatin, E64d, EST or
((2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl
ester) is an ethyl ester prodrug, 342.4 mol wt (MW), which is
completely converted in vivo to its acid form E64c (also called
loxistatin acid or Ep 475, 314.4 mol wt, which irreversibly
inhibits proteases belonging to the cysteine protcase class by
covalently binding to sulfhydryl groups in the proteases' active
sites.
##STR00001##
[0005] Two hydroxylated metabolites of loxistatin (or E64d) have
been observed:
##STR00002##
This metabolism, or in vivo hydroxylation, can result in lowering
the effective concentration of the drug and shortens its half life
in vivo.
[0006] Cathepsin B is co-localized with beta amyloid (A.beta.) in
plaques of AD brains and is elevated in cerebrospinal fluid (CSF)
of Alzheimer Disease (AD) patients. Also, age-related changes in
cathepsin B expression are consistent with the late age of onset of
Alzheimer's.
[0007] These findings, among others, indicate a role for cathepsin
B in AD.
SUMMARY
[0008] In alternative embodiments the invention provides
compositions and methods for treating, preventing, reversing,
slowing the progression of and/or ameliorating diseases and
conditions having a beta-amyloid (.beta.-amyloid, or A.beta.)
component, including Alzheimer's disease (AD), Vascular Dementia
(VD), dementia, pre-dementia, Cognitive Dysfunction Syndrome (CDS)
and loss of cognition.
[0009] In alternative embodiments, the invention provides a
compound of Formula I:
##STR00003##
wherein
[0010] R is --H or alkyl;
[0011] each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, and R.sup.10 is independently
selected from the group consisting of --H, -D, --F, --OH, and
--CH.sub.3;
or a pharmaceutically acceptable salt or solvate thereof; with the
proviso that at least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, and R.sup.10 is not
--H.
[0012] In alternative embodiments, the compounds have the
stereochemistry of Formula I':
##STR00004##
[0013] In alternative embodiments, each of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, and
R.sup.10 is independently selected from the group consisting of --H
and -D. In alternative embodiments, each of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, and R.sup.5, is --H and each of R.sup.6, R.sup.7,
R.sup.8, R.sup.9, and R.sup.10 is independently selected from the
group consisting of --H and -D. In alternative embodiments, each of
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.10 is H and
each of R.sup.6, R.sup.7, R.sup.8, and R.sup.9 is independently
selected from the group consisting of --H and -D. In alternative
embodiments, each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, and R.sup.10 is independently
selected from the group consisting of --H and --F. In alternative
embodiments, each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
and R.sup.10 is --H and each of R.sup.6, R.sup.7, R.sup.8, and
R.sup.9 is independently selected from the group consisting of --H
and --F. In alternative embodiments, each of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, and
R.sup.10 is independently selected from the group consisting of --H
and --OH. In alternative embodiments, each of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.5, and R.sup.10 is --H and each of R.sup.6,
R.sup.7, R.sup.8, and R.sup.9 is independently selected from the
group consisting of --H and --OH. In alternative embodiments, each
of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, R.sup.9, and R.sup.10 is independently selected from the
group consisting of --H and --CH.sub.3. In alternative embodiments,
each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, and R.sup.10
is --H and each of R.sup.6, R.sup.7, R.sup.8, and R.sup.9 is
independently selected from the group consisting of --H and
--CH.sub.3.
[0014] In alternative embodiments, the invention provides a
compound selected from the group consisting of:
##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010## ##STR00011## ##STR00012##
or a pharmaceutically acceptable salts, hydrates, stereoisomers or
solvates thereof.
[0015] In alternative embodiments, the invention provides a
compound selected from the group consisting of:
##STR00013##
or a pharmaceutically acceptable salt, hydrate, stereoisomer or
solvate thereof.
[0016] In alternative embodiments, the invention provides
pharmaceutical compositions, dosage form or formulation comprising
a therapeutically effective amount of a compound of the invention,
or a pharmaceutically acceptable salt, hydrate, stereoisomer or
solvate thereof, with a pharmaceutically acceptable carrier or
excipient,
[0017] and optionally the pharmaceutical composition, dosage form
or formulation further comprises at least one other pharmaceutical
composition, dosage form or formulation used to treat or
ameliorate, or treat the symptoms of, or be palliative for, a
cognitive dysfunction or a loss of cognition, a dementia or a
pre-dementia, Alzheimer's disease (AD), Vascular Dementia (VD),
and/or a Cognitive Dysfunction Syndrome (CDS) in humans or in a
non-human animal, wherein optionally the at least one other
pharmaceutical composition, dosage form or formulation comprises a
selegiline (e.g., selegiline hydrochloride) or deprenyl, or
ANIPRYL.TM.; a donepezil (ARICEPT.TM.); a carbamate; edrophonium or
comparable reversible acetylcholinesterase inhibitor (e.g.,
TENSILON.TM., ENLON.TM., REVERSOL.TM.); a neostigmine (e.g.,
PROSTIGMIN.TM., VAGOSTIGMIN.TM.); a galantamine (e.g., NIVALIN.TM.,
RAZADYNE.TM., RAZADYNE ER.TM., REMINYL.TM.); a rivastigmine (e.g.,
EXELON); a tarenflurbil or R-flurbiprofen (e.g., FLURIZAN.TM.);
and, any combination or equivalent thereof.
[0018] In alternative embodiments, of the pharmaceutical
compositions, dosage forms or formulations, the composition is
suitable for (or formulated for) topical, oral, parenteral,
intrathecal or intravenous infusion administration, wherein
optionally said composition is suitable for (or formulated for)
administration as a (or in the form of a) patch, adhesive tape,
gel, liquid or suspension, powder, spray, aerosol, lyophilate,
lozenge, pill, geltab, tablet, capsule and/or implant. The
pharmaceutical composition, dosage form or formulation can be
suitable for (or formulated for) human or veterinary
administration, wherein optionally said composition is suitable for
(or formulated for) administration to a domestic, zoo, laboratory
or farm animal, and optionally the animal is a dog or a cat.
[0019] In alternative embodiments, the invention provides methods
of inhibiting a cysteine protease, or a cathepsin, a caspase or a
calpain, or a cathepsin L or a cathepsin B, or a cathepsin F, H, K,
L1, L2, O, S, W, X or Z, in an individual, a tissue, an organ or a
cell, comprising contacting said cell, tissue, organ or individual
with a compound of the invention, or a pharmaceutical composition,
dosage form or formulation of the invention, or a compound of
Formula I,
[0020] wherein optionally the contacting is in vitro, ex vivo or in
vivo, and optionally the individual is a human or a non-human
animal, and optionally the individual or non-human animal is a
domestic, zoo, laboratory or farm animal, and optionally the animal
is a dog or a cat,
[0021] and optionally the tissue, organ or cell comprises a muscle
cell, a nerve cell, muscle tissue, peripheral nervous system (PNS)
and/or central nervous system (CNS) or brain,
[0022] and optionally by administering the compound or
pharmaceutical composition, dosage form or formulation to the
individual, a tissue, an organ or a cell a dementia or
pre-dementia, a vascular dementia (VD) and/or an Alzheimer's
disease (AD), is prevented, treated or ameliorated,
[0023] and optionally the compound or pharmaceutical composition,
dosage form or formulation is administered to prevent, treat or
ameliorate pre-clinical Alzheimer's, a mild cognitive impairment
and/or Alzheimer's dementia or pre-dementia.
[0024] In alternative embodiments, the invention provides methods
of inhibiting a cysteine protease, or a cathepsin, a caspase or a
calpain, or a cathepsin L or a cathepsin B, or a cathepsin F, H, K,
L1, L2, O, S, W, X or Z, in an individual or a patient, comprising
administering to said individual or patient in need thereof an
effective amount of a compound of the invention, or a
pharmaceutical composition, dosage form or formulation of the
invention, or a compound of Formula I,
[0025] wherein optionally the administering is ex vivo or in vivo,
and optionally the individual is a human or a non-human animal, and
optionally the individual or non-human animal is a domestic, zoo,
laboratory or farm animal, and optionally the animal is a dog or a
cat,
[0026] and optionally the compound or pharmaceutical composition,
dosage form or formulation is targeted to, or directly administered
to (or into) a tissue, organ or cell, wherein optionally the
tissue, organ or cell comprises a muscle cell, a nerve cell, muscle
tissue, peripheral nervous system (PNS) and/or central nervous
system (CNS) or brain,
[0027] and optionally by administering the compound or
pharmaceutical composition, dosage form or formulation the
individual or patient is treated for (as a therapeutic or
prophylactic treatment) a dementia or pre-dementia, a vascular
dementia (VD) and/or an Alzheimer's disease (AD),
[0028] and optionally the compound or pharmaceutical composition,
dosage form or formulation is administered to prevent, treat or
ameliorate pre-clinical Alzheimer's, a mild cognitive impairment
and/or Alzheimer's dementia or pre-dementia.
[0029] In alternative embodiments, the invention provides methods
for preventing, slowing the progression of, treating or
ameliorating a cognitive impairment, a dementia or pre-dementia, a
vascular dementia (VD) or an Alzheimer's disease (AD), comprising
administering to an individual or a patient, comprising
administering to said individual or patient in need thereof an
effective amount of a compound of the invention, or a
pharmaceutical composition, dosage form or formulation of the
invention, or a compound of Formula I, wherein optionally the
administering is ex vivo or in vivo, and optionally the individual
is a human or a non-human animal, and optionally the individual or
non-human animal is a domestic, zoo, laboratory or farm animal, and
optionally the animal is a dog or a cat), and optionally the
compound or pharmaceutical composition, dosage form or formulation
is administered to prevent, treat or ameliorate pre-clinical
Alzheimer's, a mild cognitive impairment and/or Alzheimer's
dementia or pre-dementia.
[0030] In alternative embodiments, the invention provides methods
of reversing, slowing, reducing or preventing the effects of
.beta.-amyloid in an individual or patient comprising administering
to the individual or patient in need thereof an effective amount of
a compound of the invention, or a pharmaceutical composition,
dosage form or formulation of the invention, or a compound of
Formula I, and optionally by reversing, slowing, reducing or
preventing the effects of .beta.-amyloid in the individual or
patient, a dementia or pre-dementia, a vascular dementia (VD)
and/or an Alzheimer's disease (AD), is treated or ameliorated,
wherein optionally the administering is ex vivo or in vivo, and
optionally the individual is a human or a non-human animal, and
optionally the individual or non-human animal is a domestic, zoo,
laboratory or farm animal, and optionally the animal is a dog or a
cat, and optionally the compound or pharmaceutical composition,
dosage form or formulation is administered to prevent, treat or
ameliorate pre-clinical Alzheimer's, a mild cognitive impairment
and/or Alzheimer's dementia or pre-dementia.
[0031] In alternative embodiments, the invention provides methods
of reversing, slowing, reducing or preventing neuronal cell death
or apoptosis in a cell, an organ, a tissue, an individual or a
patient comprising administering to the cell or tissue, or the
individual or patient in need thereof, an effective amount of a
compound of the invention, or a pharmaceutical composition, dosage
form or formulation of the invention, or a compound of Formula I,
and optionally by reversing, slowing, reducing or preventing
neuronal cell death or apoptosis in the cell, organ, tissue,
individual or patient, a dementia or pre-dementia, a vascular
dementia (VD) and/or an Alzheimer's disease (AD) is treated or
ameliorated, wherein optionally the administering is ex vivo or in
vivo, and optionally the individual is a human or a non-human
animal, and optionally the individual or non-human animal is a
domestic, zoo, laboratory or farm animal, and optionally the animal
is a dog or a cat, and optionally the compound or pharmaceutical
composition, dosage form or formulation is administered to prevent,
treat or ameliorate pre-clinical Alzheimer's, a mild cognitive
impairment and/or Alzheimer's dementia or pre-dementia.
[0032] In alternative embodiments, the invention provides methods
of slowing, reversing, reducing or preventing neuronal cell death
in an individual, subject or patient comprising (a) administering
to said individual, subject or patient an effective amount of a
compound comprising:
[0033] (i) a compound of the invention, or a pharmaceutical
composition, dosage form or formulation of the invention, or a
compound of Formula I; or
[0034] (ii) a loxistatin (also called E64d or AB-007) (also called
(2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl
ester), or loxistatin acid (also called E64c) conjugated to (or
comprising) a chemical delivery system (CDS); or a composition as
described in U.S. Patent Application Nos. 20080227806, 20080176841
and/or 20100048717;
[0035] such that neuronal cell death is slowed, reversed, reduced
and/or prevented, wherein said effective amount is between about 1
mg and about 400 mg; or is between about 1 mg and about 250 mg; or
is about 5 mg and about 150 mg; or is between about 1 mg and about
75 mg; or is about 5 mg, about 10 mg, about 15 mg, about 20 mg,
about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg,
about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, or
about 75 mg;
[0036] (b) the method of (a), wherein the compound is formulated in
a gel, spray, aerosol, powder, liquid or solid dosage form, or is
formulated for administration as a (or in the form of a) patch,
adhesive tape, gel, liquid or suspension, powder, spray, aerosol,
lyophilate, lozenge, pill, geltab, tablet, capsule and/or
implant;
[0037] (c) the method of (b), wherein the solid dosage form
comprises an implant, a pill, a capsule, a geltab, a tablet or a
lozenge; or
[0038] (d) the method of (a), (b) or (c), wherein the compound is
administered as a once a day, or twice a day (bid), or three times
a day (tid) formulation, optionally as an oral dosage,
[0039] and optionally the compound or pharmaceutical composition,
dosage form or formulation is administered to prevent, treat or
ameliorate a cognitive impairment, a dementia or pre-dementia, a
vascular dementia (VD), an Alzheimer's disease (AD), or a
pre-clinical Alzheimer's disease (AD), an Alzheimer's dementia or
pre-dementia and/or a mild cognitive impairment;
[0040] and optionally the individual is a human or a non-human
animal, and optionally the individual or non-human animal is a
domestic, zoo, laboratory or farm animal, and optionally the animal
is a dog or a cat.
[0041] In alternative embodiments, the invention provides methods
of slowing, reversing, reducing or preventing the effects or
formation of .beta.-amyloid, or .beta.-amyloid accumulation, or
.beta.-amyloid plaque formation in an individual, subject or
patient comprising
[0042] (a) administering to said individual, subject or patient an
effective amount of a compound comprising:
[0043] (i) a compound of the invention, or a pharmaceutical
composition, dosage form or formulation of the invention, or a
compound of Formula I; or
[0044] (ii) a loxistatin (also called E64d or AB-007) (also called
(2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl
ester), or loxistatin acid (also called E64c) conjugated to (or
comprising) a chemical delivery system (CDS); or a composition as
described in U.S. Patent Application Nos. 20080227806, 20080176841
and/or 20100048717;
[0045] such that the effects or formation of .beta.-amyloid, or
.beta.-amyloid accumulation, or .beta.-amyloid plaque formation is
slowed, reversed, reduced and/or prevented, wherein said effective
amount is between about 1 mg and about 400 mg; or is between about
1 mg and about 250 mg; or is about 5 mg and about 150 mg; or is
between about 1 mg and about 75 mg; or is about 5 mg, about 10 mg,
about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg,
about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg,
about 65 mg, about 70 mg, or about 75 mg;
[0046] (b) the method of (a), wherein the compound is formulated in
a gel, spray, aerosol, powder, liquid or solid dosage form, or is
formulated for administration as a (or in the form of a) patch,
adhesive tape, gel, liquid or suspension, powder, spray, aerosol,
lyophilate, lozenge, pill, geltab, tablet, capsule and/or
implant;
[0047] (c) the method of (b), wherein the solid dosage form
comprises an implant, a pill, a capsule, a geltab, a tablet or a
lozenge; or
[0048] (d) the method of (a), (b) or (c), wherein the compound is
administered as a once a day, or a twice a day (bid), or a three
times a day (tid) formulation, optionally as an oral dosage,
[0049] and optionally the compound or pharmaceutical composition,
dosage form or formulation is administered to prevent, treat or
ameliorate a cognitive impairment, a dementia or pre-dementia, a
vascular dementia (VD), an Alzheimer's disease (AD) or a
pre-clinical Alzheimer's disease (AD), a mild cognitive impairment
and/or Alzheimer's dementia or pre-dementia, and optionally the
individual is a human or a non-human animal, and optionally the
individual or non-human animal is a domestic, zoo, laboratory or
farm animal, and optionally the animal is a dog or a cat.
[0050] In alternative embodiments, the invention provides methods
of slowing, reversing, reducing or preventing a cognitive
impairment, a dementia, an Alzheimer's Disease (AD), a vascular
dementia (VD) and/or an Alzheimer's dementia or pre-dementia, in an
individual, subject or patient comprising
[0051] (a) administering to said individual, subject or patient an
effective amount of a compound comprising:
[0052] (i) a compound of the invention, or a pharmaceutical
composition, dosage form or formulation of the invention, or a
compound of Formula I; or
[0053] (ii) a loxistatin (also called E64d or AB-007) (also called
(2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl
ester), or loxistatin acid (also called E64c) conjugated to (or
comprising) a chemical delivery system (CDS); or a composition as
described in U.S. Patent Application Nos. 20080227806, 20080176841
and/or 20100048717;
[0054] such that the cognitive impairment, AD, VD, dementia or
pre-dementia, is slowed, reversed, reduced and/or prevented,
wherein said effective amount is between about 1 mg and about 400
mg; or is between about 1 mg and about 250 mg; or is about 5 mg and
about 150 mg; or is between about 1 mg and about 75 mg; or is about
5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30
mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55
mg, about 60 mg, about 65 mg, about 70 mg, or about 75 mg;
[0055] (b) the method of (a), wherein the compound is formulated in
a gel, spray, aerosol, powder, liquid or solid dosage form, or is
formulated for administration as a (or in the form of a) patch,
adhesive tape, gel, liquid or suspension, powder, spray, aerosol,
lyophilate, lozenge, pill, geltab, tablet, capsule and/or
implant;
[0056] (c) the method of (b), wherein the solid dosage form
comprises an implant, a pill, a capsule, a geltab, a tablet or a
lozenge; or
[0057] (d) the method of (a), (b) or (c), wherein the compound is
administered as a once a day, or a twice a day (bid), or a three
times a day (tid) formulation, optionally as an oral dosage,
[0058] and optionally the compound or pharmaceutical composition,
dosage form or formulation is administered to prevent, treat or
ameliorate a cognitive impairment, a dementia or pre-dementia, a
vascular dementia (VD), Alzheimer's disease (AD), or a pre-clinical
Alzheimer's, a mild cognitive impairment and/or Alzheimer's
dementia or pre-dementia, and optionally the individual is a human
or a non-human animal, and optionally the individual or non-human
animal is a domestic, zoo, laboratory or farm animal, and
optionally the animal is a dog or a cat.
[0059] In alternative embodiments, the invention provides methods
of slowing, reversing, reducing or preventing a Cognitive
Dysfunction Syndrome (CDS) in a non-human animal, e.g., dogs and
cats, including Canine or Cat (feline) Cognitive Dysfunction (CCD),
comprising
[0060] (a) administering to said individual, subject or patient an
effective amount of a compound comprising an inhibitor of a
cysteine protease, or a cathepsin, a caspase or a calpain, or a
cathepsin L or a cathepsin B, or a cathepsin F, H, K, L1, L2, O, S,
W, X or Z, in an individual, a tissue, an organ or a cell,
[0061] wherein optionally the inhibitor comprises: [0062] (i) a
compound of the invention, or a pharmaceutical composition, dosage
form or formulation of the invention, or a compound of Formula I;
[0063] (ii) a loxistatin (also called E64d or AB-007) (also called
(2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl
ester), or loxistatin acid (also called E64c) conjugated to (or
comprising) a chemical delivery system (CDS); or a composition as
described in U.S. Patent Application Nos. 20080227806, 20080176841
and/or 20100048717; [0064] (iii) an odanacatib or MK-0674, or
equivalents thereof; [0065] (iv) a diazomethyl ketone, a
fluoromethyl ketone, an acyloxymethyl ketone, an
O-acylhydroxylamine or a vinyl sulfone, or equivalents thereof;
and/or [0066] (v) a reversible hydrazide inhibitor of cathepsin B
comprising a ZLIII115A and ZLIII43A, or equivalents thereof; [0067]
(vi) a cystatin, or a cystatin A, cystatin B, cystatin C (or
cystatin 3) or cystatin D, or a type 1 cystatin (a stefin), a type
2 cystatin or a kininogen;
[0068] wherein optionally the Cognitive Dysfunction Syndrome (CDS)
is slowed, reversed, reduced and/or prevented, wherein said
effective amount is between about 1 mg and about 400 mg; or is
between about 1 mg and about 250 mg; or is about 5 mg and about 150
mg; or is between about 1 mg and about 75 mg; or is about 5 mg,
about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg,
about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg,
about 60 mg, about 65 mg, about 70 mg, or about 75 mg;
[0069] (b) the method of (a), wherein the compound is formulated in
a gel, spray, aerosol, powder, liquid or solid dosage form, or is
formulated for administration as a (or in the form of a) patch,
adhesive tape, gel, liquid or suspension, powder, spray, aerosol,
lyophilate, lozenge, pill, geltab, tablet, capsule and/or
implant;
[0070] (c) the method of (b), wherein the solid dosage form
comprises an implant, a pill, a capsule, a geltab, a tablet or a
lozenge; or
[0071] (d) the method of (a), (b) or (c), wherein the compound is
administered as a once a day, or a twice a day (bid), or a three
times a day (tid) formulation, optionally as an oral dosage,
[0072] and optionally the individual is a human or a non-human
animal, and optionally the individual or non-human animal is a
domestic, zoo, laboratory or farm animal, and optionally the animal
is a dog or a cat, and optionally CDS encompasses a Canine or Cat
(feline) Cognitive Dysfunction (CCD).
[0073] In alternative embodiments, the invention provides products
of manufacture, e.g., provides a blister pack or a plurality of
blister packettes, a blister package, a lidded blister or a blister
card or packet, a clamshell, a tray or a shrink wrap, or a paper,
plastic or cellophane package or a plurality of packettes,
comprising:
[0074] (a) a compound of the invention, or a pharmaceutical
composition, dosage form or formulation of the invention, and/or a
compound of Formula I; and
[0075] (b) at least one other (e.g., at least one additional,
different) compound, pharmaceutical composition, dosage form or
formulation of (a), wherein the at least one other (e.g., at least
one additional, different) compound, pharmaceutical composition,
dosage form or formulation is used to treat or ameliorate, or treat
the symptoms of, or be palliative for: a cognitive dysfunction or a
loss of cognition, a dementia or a pre-dementia, Alzheimer's
disease (AD), Vascular Dementia (VD), and/or a Cognitive
Dysfunction Syndrome (CDS), in humans or in a non-human animal,
[0076] wherein optionally the at least one other pharmaceutical
composition, dosage form or formulation comprises a selegiline
(e.g., selegiline hydrochloride) or deprenyl, or ANIPRYL.TM.; a
donepezil (ARICEPT.TM.); a carbamate; edrophonium or comparable
reversible acetylcholinesterase inhibitor (e.g., TENSILON.TM.,
ENLON.TM., REVERSOL.TM.); a neostigmine (e.g., PROSTIGMIN.TM.,
VAGOSTIGMIN.TM.); a galantamine (e.g., NIVALIN.TM., RAZADYNE.TM.,
RAZADYNE ER.TM., REMINYL.TM.); a rivastigmine (e.g., EXELON); a
tarenflurbil or R-flurbiprofen (e.g., FLURIZAN.TM.); and, any
combination or equivalent thereof, or a nutritional supplement or a
vitamin, e.g., vitamin E, vitamin B12 or a folic acid supplement,
or any equivalent or combination thereof, or a pain treatment or
pain palliative or an anti-inflammatory drug, e.g., an ibuprofen
(e.g., ADVIL.TM., MOTRIN.TM.), naproxen sodium (ALEVE.TM.),
indomethacin (INDOCIN.TM.), or any equivalent or combination
thereof, or a non-steroidal anti-inflammatory drug (a NSAID), e.g.,
a cyclooxygenase (COX) (or prostaglandin synthase) inhibitor, e.g.,
an etodolac (e.g., LODINE.TM., LODINE SR.TM. or ECCOXOLAC.TM.),
naproxen, celecoxib, rofecoxib, etoricoxib, valdecoxib, parecoxib,
nabumetone, diclofenac, lumiracoxib, or equivalent, or a
neuropathic pain analgesic such as gabapentin or pregabalin.
[0077] In alternative embodiments, the invention provides methods
for lowering body or blood homocysteine or cystathionine in an
individual, subject or patient, comprising
[0078] (a) administering to said individual, subject or patient an
effective amount of a compound comprising an inhibitor of a
cysteine protease, or a cathepsin, a caspase or a calpain, or a
cathepsin L or a cathepsin B, or a cathepsin F, H, K, L1, L2, O, S,
W, X or Z, in an individual, a tissue, an organ or a cell, wherein
optionally the inhibitor comprises: [0079] (i) a compound of the
invention, or a pharmaceutical composition, dosage form or
formulation of the invention, or a compound of Formula I; [0080]
(ii) a loxistatin (also called E64d or AB-007) (also called
(2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl
ester), or loxistatin acid (also called E64c) conjugated to (or
comprising) a chemical delivery system (CDS); or a composition as
described in U.S. Patent Application Nos. 20080227806, 20080176841
and/or 20100048717; [0081] (iii) an odanacatib or MK-0674, or
equivalents thereof; [0082] (iv) a diazomethyl ketone, a
fluoromethyl ketone, an acyloxymethyl ketone, an
O-acylhydroxylamine or a vinyl sulfone, or equivalents thereof;
and/or [0083] (v) a reversible hydrazide inhibitor of cathepsin B
comprising a ZLIII115A and ZLIII43A, or equivalents thereof; [0084]
(vi) a cystatin, or a cystatin A, cystatin B, cystatin C (or
cystatin 3) or cystatin D, or a type 1 cystatin (a stefin), a type
2 cystatin or a kininogen;
[0085] wherein optionally said effective amount is between about 1
mg and about 400 mg; or is between about 1 mg and about 250 mg; or
is about 5 mg and about 150 mg; or is between about 1 mg and about
75 mg; or is about 5 mg, about 10 mg, about 15 mg, about 20 mg,
about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg,
about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, or
about 75 mg;
[0086] (b) the method of (a), wherein the compound is formulated in
a gel, spray, aerosol, powder, liquid or solid dosage form, or is
formulated for administration as a (or in the form of a) patch,
adhesive tape, gel, liquid or suspension, powder, spray, aerosol,
lyophilate, lozenge, pill, geltab, tablet, capsule and/or
implant;
[0087] (c) the method of (b), wherein the solid dosage form
comprises an implant, a pill, a capsule, a geltab, a tablet or a
lozenge; or
[0088] (d) the method of (a), (b) or (c), wherein the compound is
administered as a once a day, or a twice a day (bid), or a three
times a day (tid) formulation, optionally as an oral dosage;
[0089] and optionally the individual is a human or a non-human
animal, and optionally the individual or non-human animal is a
domestic, zoo, laboratory or farm animal, and optionally the animal
is a dog or a cat;
[0090] and optionally the compound is administered to lower
homocysteine levels to below 10 micromoles/liter.
[0091] In alternative embodiments, the invention provides methods
for lowering or decreasing the risk of heart attacks, strokes,
blood clot formation in an individual, subject or patient,
comprising
[0092] (a) administering to an individual, subject or patient an
effective amount of a compound comprising an inhibitor of a
cysteine protcase, or a cathepsin, a caspase or a calpain, or a
cathepsin L or a cathepsin B, or a cathepsin F, H, K, L1, L2, O, S,
W, X or Z, in an individual, a tissue, an organ or a cell,
[0093] wherein optionally the inhibitor comprises: [0094] (i) a
compound of any of the invention, or a pharmaceutical composition,
dosage form or formulation of the invention, or a compound of
Formula I; [0095] (ii) a loxistatin (also called E64d or AB-007)
(also called
(2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl
ester), or loxistatin acid (also called E64c) conjugated to (or
comprising) a chemical delivery system (CDS); or a composition as
described in U.S. Patent Application Nos. 20080227806, 20080176841
and/or 20100048717; [0096] (iii) an odanacatib or MK-0674, or
equivalents thereof; [0097] (iv) a diazomethyl ketone, a
fluoromethyl ketone, an acyloxymethyl ketone, an
O-acylhydroxylamine or a vinyl sulfone, or equivalents thereof;
[0098] and/or [0099] (v) a reversible hydrazide inhibitor of
cathepsin B comprising a ZLIII115A and ZLIII43A, or equivalents
thereof; [0100] (vi) a cystatin, or a cystatin A, cystatin B,
cystatin C (or cystatin 3) or cystatin D, or a type 1 cystatin (a
stefin), a type 2 cystatin or a kininogen;
[0101] wherein optionally said effective amount is between about 1
mg and about 400 mg; or is between about 1 mg and about 250 mg; or
is about 5 mg and about 150 mg; or is between about 1 mg and about
75 mg; or is about 5 mg, about 10 mg, about 15 mg, about 20 mg,
about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg,
about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, or
about 75 mg;
[0102] (b) the method of (a), wherein the compound is formulated in
a gel, spray, aerosol, powder, liquid or solid dosage form, or is
formulated for administration as a (or in the form of a) patch,
adhesive tape, gel, liquid or suspension, powder, spray, aerosol,
lyophilate, lozenge, pill, geltab, tablet, capsule and/or
implant;
[0103] (c) the method of (b), wherein the solid dosage form
comprises an implant, a pill, a capsule, a geltab, a tablet or a
lozenge; or
[0104] (d) the method of (a), (b) or (c), wherein the compound is
administered as a once a day, or a twice a day (bid), or a three
times a day (tid) formulation, optionally as an oral dosage;
[0105] and optionally the individual is a human or a non-human
animal, and optionally the individual or non-human animal is a
domestic, zoo, laboratory or farm animal, and optionally the animal
is a dog or a cat;
[0106] and optionally the compound is administered to lower
homocysteine levels to below 10 micromoles/liter.
[0107] In alternative embodiments, the invention provides methods
for treating, preventing, slowing the progression of, reversing or
ameliorating traumatic central nervous system (CNS) (e.g., brain)
injury (due to, e.g., trauma, infection, surgery, and the like) and
related (e.g., as sequclae or side effects) post-traumatic
injuries, including traumatic war neurosis, post traumatic stress
disorder (PTSD) or post-traumatic stress syndrome (PTSS), and
cognitive, learning or memory impairments resulting therefrom, in
an individual, subject or patient, comprising:
[0108] (a) administering to an individual, subject or patient an
effective amount of a compound comprising an inhibitor of a
cysteine protease, or a cathepsin, a caspase or a calpain, or a
cathepsin L or a cathepsin B, or a cathepsin F, H, K, L1, L2, O, S,
W, X or Z, in an individual, a tissue, an organ or a cell,
[0109] wherein optionally the inhibitor comprises: [0110] (i) a
compound of the invention, or a pharmaceutical composition, a
dosage form or a formulation of the invention, or a compound of
Formula I; [0111] (ii) a loxistatin (also called E64d or AB-007)
(also called
(2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl
ester), or loxistatin acid (also called E64c) conjugated to (or
comprising) a chemical delivery system (CDS); or a composition as
described in U.S. Patent Application Nos. 20080227806, 20080176841
and/or 20100048717; [0112] (iii) an odanacatib or MK-0674, or
equivalents thereof; [0113] (iv) a diazomethyl ketone, a
fluoromethyl ketone, an acyloxymethyl ketone, an
O-acylhydroxylamine or a vinyl sulfone, or equivalents thereof;
and/or [0114] (v) a reversible hydrazide inhibitor of cathepsin B
comprising a ZLIII115A and ZLIII43A, or equivalents thereof; [0115]
(vi) a cystatin, or a cystatin A, cystatin B, cystatin C (or
cystatin 3) or cystatin D, or a type 1 cystatin (a stefin), a type
2 cystatin or a kininogen;
[0116] wherein optionally said effective amount is between about 1
mg and about 400 mg; or is between about 1 mg and about 250 mg; or
is about 5 mg and about 150 mg; or is between about 1 mg and about
75 mg; or is about 5 mg, about 10 mg, about 15 mg, about 20 mg,
about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg,
about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, or
about 75 mg;
[0117] (b) the method of (a), wherein the compound is formulated in
a gel, spray, aerosol, powder, liquid or solid dosage form, or is
formulated for administration as a (or in the form of a) patch,
adhesive tape, gel, liquid or suspension, powder, spray, aerosol,
lyophilate, lozenge, pill, geltab, tablet, capsule and/or
implant;
[0118] (c) the method of (b), wherein the solid dosage form
comprises an implant, a pill, a capsule, a geltab, a tablet or a
lozenge; or
[0119] (d) the method of (a), (b) or (c), wherein the compound is
administered as a once a day, or a twice a day (bid), or a three
times a day (tid) formulation, optionally as an oral dosage;
[0120] and optionally the individual is a human or a non-human
animal, and optionally the individual or non-human animal is a
domestic, zoo, laboratory or farm animal, and optionally the animal
is a dog or a cat.
[0121] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0122] FIG. 1 graphically illustrates data showing the measurement
of brain A.beta..sub.40 and A.beta..sub.42 for 10 mg/kg/day dosing
after 7 days compared to vehicle treated controls, as discussed in
detail in Example 8, below.
[0123] FIG. 2 graphically illustrates data showing the measurements
of brain A.beta..sub.1-40 in guinea pigs administered between 0 and
50 mg/kg/day AB-007 via oral gavage for 7 days, as discussed in
detail in Example 8, below.
[0124] FIG. 3 graphically illustrates data showing the measurements
of brain A.beta..sub.1-42 in guinea pigs administered between 0 and
50 mg/kg/day AB-007 via oral gavage for 7 days, as discussed in
detail in Example 8, below.
[0125] FIG. 4 graphically illustrates data showing the measurements
of Cathepsin B activity after administration of between 0 and 50
mg/kg/day AB-007 to guinea pigs via oral gavage for 7 days, as
discussed in detail in Example 8, below.
[0126] FIG. 5 graphically illustrates data showing the measurements
of BACE-1 activity after administration of between 0 and 50
mg/kg/day AB-007 to guinea pigs via oral gavage for 7 days, as
discussed in detail in Example 8, below.
[0127] FIG. 6 graphically illustrates data showing that once-a-day
oral administration of AB-007 (E64d, loxistatin) to guinea pigs
results in a dose response reduction in FIG. 6(A) brain
A.beta.(40), FIG. 6(B) brain A.beta.(42), FIG. 6(C) CSF A.beta.,
FIG. 6(D) CSF A.beta.(42), FIG. 6(E) plasma A.beta.(40) and FIG.
6(F) plasma A.beta.(42), as discussed in detail in Example 9,
below.
[0128] FIG. 7A graphically illustrates data showing that once-a-day
for one week oral administration of AB-007 (E64d, loxistatin) to
guinea pigs results in a dose response reduction brain cathepsin B
activity; FIG. 7(B) (lower graph) illustrates that the same
treatment results in an increase in brain BACE1 activity, as
discussed in detail in Example 9, below.
[0129] FIG. 8 graphically illustrates data showing paired guinea
pig data for brain A.beta.(40) or A.beta.(42) versus (vs) brain
cathepsin B or BACE1 activity, respectively for the combined AB-007
(E64d, loxistatin) dose groups: FIG. 8(A) Brain A.beta.(40) vs
brain cathepsin B activity shows a significant positive
correlation, showing that brain cathepsin B inhibition reduces
brain A.beta.(40); FIG. 8(B) Brain A.beta.(42) vs brain cathepsin B
activity also has a significant positive correlation, showing brain
cathepsin B inhibition reduces brain A.beta.(42); FIG. 8(C): Brain
A.beta.(40) vs brain BACE1 activity demonstrates a small negative
correlation; and, FIG. 8(D) Brain A.beta.(42) vs brain BACE1
activity has a negative correlation, as discussed in detail in
Example 9, below.
[0130] FIG. 9 graphically illustrates data showing paired guinea
pig data for brain A.beta.(40) vs A.beta.(42) and brain cathepsin B
vs BACE1 activity for all AB-007 (E64d, loxistatin) dose groups
combined: FIG. 9(A) Brain A.beta.(40) vs A.beta.(42) shows a
significant positive correlation; and, FIG. 9(B) Brain cathepsin B
vs BACE1 activity shows a slight negative correlation, as discussed
in detail in Example 9, below.
[0131] FIG. 10 graphically illustrates data showing that once-a-day
for a week oral administration of the invention's exemplary
composition of the invention, the hepta-deuterated "E64d7", to
guinea pigs results in a dose response reduction in FIG. 10(A)
brain A.beta.(40), FIG. 10(B) brain A.beta.(42), FIG. 10(C) CSF
A.beta., FIG. 10(D) CSF A.beta.(42), FIG. 10(E) plasma A.beta.(40)
and FIG. 10(F) plasma A.beta.(42), as discussed in detail in
Example 9, below.
[0132] FIG. 11A (upper graph) graphically illustrates data showing
that once-a-day oral administration of E64d7 to guinea pigs results
in a dose response reduction of brain cathepsin B activity, which
is similar to the biphasic lowering of A.beta.; FIG. 11B (lower
graph) graphically illustrates data showing that same treatment
results in an increase in brain BACE1 activity, as discussed in
detail in Example 9, below.
[0133] FIG. 12 graphically illustrates data showing paired guinea
pig data for brain A.beta.(40) or A.beta.(42) vs brain cathepsin B
or BACE1 activity, respectively for the combined "E64d7" dose
groups: FIG. 12(A) Brain A.beta.(40) vs brain cathepsin B activity
shows a significant positive correlation; FIG. 12(B) Brain
A.beta.(42) vs brain cathepsin B activity also has a significant
positive correlation; FIG. 12(C) Brain A.beta.(40) vs brain BACE1
activity demonstrates a small negative correlation; FIG. 12(D)
Brain A.beta.(42) vs brain BACE1 activity has a negative
correlation, as discussed in detail in Example 9, below.
[0134] FIG. 13 graphically illustrates data showing paired guinea
pig data for brain A.beta.(40) vs A.beta.(42) and brain cathepsin B
vs BACE1 activity for all "E64d7" dose groups combined: FIG. 13(A)
(upper graph) Brain A.beta.(40) vs A.beta.(42) shows a significant
positive correlation; FIG. 13(B) Brain cathepsin B vs BACE1
activity shows a slight negative correlation, as discussed in
detail in Example 9, below.
[0135] FIG. 14 graphically illustrates data showing the brain
A.beta.(40) and A.beta.(42) data from the AB-007 (E64d,
loxistatin)-doped mouse chow experiments, results from two
experimental groups are shown: FIG. 14(A) Feeding the E64d doped
chow for 1 or 3 months caused a significant reduction in brain
A.beta.(40) in both the young and old mice relative to age-matched
controls; FIG. 14(B) the E64d doped chow fed for 1 or 3 months also
caused a significant reduction in brain A.beta.(42) in both the
young and old mice relative age-matched controls, as discussed in
detail in Example 9, below.
[0136] FIG. 15 graphically compares the data shown in FIG. 14, for
brain A.beta.(40) and A.beta.(42) levels in young and old animals
feed E64d doped chow for 1 or 3 months: FIG. 15(A) The brain
A.beta.(40) data from 1 month feeding; FIG. 15(B) the brain
A.beta.(40) data from 3 month feeding; FIG. 15(C) the brain
A.beta.(42) data from 1 month feeding; FIG. 15(D) the brain
A.beta.(42) data from 3 month feeding are shown, as discussed in
detail in Example 9, below.
[0137] FIG. 16 graphically illustrates data showing the effects of
AB-007 (E64d, loxistatin)-doped chow on the spatial memory deficit
that develops in transgenic APPlon mice, as discussed in detail in
Example 9, below.
[0138] FIG. 17 graphically illustrates data showing a paired data
analysis from the mouse experiments between brain A.beta.(40) or
A.beta.(42) peptides and latency period for young and old mice
either treated or not with AB-007 (E64d, loxistatin): FIG. 17(A)
graphically illustrates the effect of feeding young mice either the
control or E64d-doped chow for 1 month on brain A.beta.(40) peptide
and latency period is shown; FIG. 17(B) graphically illustrates the
effect of the E64d-doped chow feeding on young animals on brain
A.beta.(42) peptide vs. latency period is shown; FIG. 17(C)
graphically illustrates the effect of the E64d-doped chow feeding
on old animals on brain A.beta.(40) peptide and latency period is
shown; FIG. 17(D) graphically illustrates the effect of the
E64d-doped chow feeding on old animals on brain A.beta.(42) and
latency period is shown; as discussed in detail in Example 9,
below.
[0139] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0140] In alternative embodiments the invention provides
compositions and methods for preventing, treating, slowing the
progress of, reversing or ameliorating diseases and conditions
having a beta-amyloid component, including cognitive dysfunctions
and loss of cognition, dementias and pre-dementias, Alzheimer's
disease (AD), Vascular Dementia (VD), and Cognitive Dysfunction
Syndrome (CDS) in humans or in a non-human animal.
[0141] In alternative embodiments the invention provides analogs of
AB-007 (also called loxistatin or E64d) and its acid form E64c
(also called loxistatin acid), their preparation, and
pharmaceutical compositions thereof and methods of making and using
same. In alternative embodiments, AB-007 (loxistatin) and/or E64c
(loxistatin acid) are derivatized, e.g. deuterated, in one or more
sites, e.g., a site relevant to a metabolic site, e.g., a site on
the molecule involved and/or altered (e.g., hydroxylated) in its
metabolism or breakdown in vivo. In alternative embodiments, the
derivatization, e.g., a deuteration, results in altered metabolism,
e.g., slowed or blocked metabolism, of a composition of the
invention. Thus, in alternative embodiment compositions of the
invention have "better" pharmacokinetic properties than the
"parent" AB-007 (loxistatin, E64d) or E64c (loxistatin acid), e.g.,
because (noting the invention is not limited by any particular
mechanism of action) the derivatization slows or inhibits the
hydroxylation of AB-007 or E64c, a lower dosage is effective and/or
more potent and an individual can be given a lower dosage to
produce the same effect from a comparative dosage and/or
formulation of the "parent" AB-007 or E64c.
[0142] In one embodiment, to block or slow the drug's (a
composition of the invention) metabolism, e.g., hydroxylation, one
or more hydrogen atoms extracted in the in vivo metabolic process
and/or otherwise involved in the metabolic process is substituted
by a moiety which is more difficult to (e.g., enzymatically)
remove, e.g., deuterium. In alternative embodiments, one or more
selected hydrogens are substituted (replaced) with a hydroxyl,
deuterium, fluorine or a methyl group or a combination thereof.
[0143] For example, in alternative embodiments, compositions of the
invention comprise a hepta-deuterated (or 7-position deuterated)
AB-007 (loxistatin, E64d) and a hepta-deuterated (or 7-position
deuterated) loxistatin acid (E64c):
##STR00014##
(the above illustrated isoform of hepta-deuterated (or 7-position
deuterated) AB-007 (loxistatin, E64d) is designated "E64d7" (see,
e.g., Example 9, below), and the above illustrated isoform of
hepta-deuterated (or 7-position deuterated) E64c (or loxistatin
acid) is designated "E64c7").
[0144] In alternative embodiments, compositions of the invention
also comprise a 1-, 2-, 3-, 4-, 5-, 6- and/or 7-position
substitutions with e.g., a deuterium and/or a fluorine, as
alternative embodiments to the hepta-deuterated (or 7-position
deuterated) species of the invention. While the invention is not
limited by any particular mechanism of action, these alternative
embodiments are based on the observed two hydroxylated metabolites
of AB-007 (loxistatin, E64d), as illustrated above e.g., in the
hepta-deuterated isoform designated "E64d7" and "E64c7".
[0145] In alternative embodiments the invention provides
compositions comprising Formula I, which illustrates compounds of
the invention resulting from these described substitutions:
##STR00015##
wherein
[0146] R is --H or alkyl;
[0147] each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.8, R.sup.9, and R.sup.10 is independently
selected from the group consisting of --H, -D, --F, --OH,
--CH.sub.3 and/or a pharmaceutically acceptable salt, hydrate,
stereoisomer, or solvate thereof; with the proviso that at least
one of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6,
R.sup.7, R.sup.8, R.sup.9, and R.sub.10 is not hydrogen (--H).
[0148] For example, alternative embodiments of the invention
comprise both ester and acid forms (e.g., E64c and E64c (loxistatin
acid) forms, respectively) comprising or consisting of, in addition
to the illustrated deuterated embodiments, including the
hepta-deuterated embodiments (where e.g. all R.sup.1, R.sup.2,
R.sup.3 positions can be deuterated or otherwise substituted),
alternative exemplary species can have only R.sup.1 and R.sup.3
positions completely or partially deuterated or otherwise
substituted, or only R.sup.1 positions completely or partially
deuterated or otherwise substituted, or only R.sup.3 positions
completely or partially deuterated or otherwise substituted, or
only the R.sup.2 position deuterated or otherwise substituted, or a
combination of the R.sup.1 and R.sup.2 or R.sup.2 and R.sup.3
positions completely or partially deuterated or otherwise
substituted, and the like.
[0149] Alternative embodiments of the invention comprise
derivatized analogs, e.g., metabolically blocked or otherwise
altered derivatives, of AB-007 (loxistatin, E64d) and loxistatin
acid (E64c), including deuterated, hydroxylated, fluorinated or
methylated analogs or derivatives, or any combination thereof.
[0150] With regard to deuterated compounds of the invention,
including those of Formula I, it will be recognized that some
variation of natural isotopic abundance occurs in a synthesized
compound depending upon the origin of chemical materials used in
the synthesis. Thus, a preparation of AB-007 (loxistatin, E64d) or
loxistatin acid (E64c) will inherently contain small amounts of
deuterated isotopologues. The concentration of naturally abundant
stable hydrogen and carbon isotopes, notwithstanding this
variation, is small and immaterial as compared to the degree of
stable isotopic substitution of compounds of this disclosure. See,
for instance, Wada E et al., Seikagaku 1994, 66:15; Ganes L Z et
al., Comp Biochem Physiol Mol Integr Physiol 1998, 119:725.
Accordingly, for compounds and compositions of this invention,
which include pharmaceutical preparations and formulations, when a
particular position is designated as having deuterium ("-D"), it is
understood that the abundance of deuterium at that position is
greater than, or substantially greater than, the natural abundance
of deuterium, which is 0.015%. For example, alternative embodiments
of the invention comprise analogs of AB-007 (loxistatin, E64d)
and/or loxistatin acid (E64c) having greater than 0.02%, or greater
than about 0.1% deuterium. In one embodiment, the deuterium
substitution, or "enrichment", occurs at a specific position or
positions. In one embodiment, the deuterium enrichment is no less
than about 1%. In a further embodiment, the deuterium enrichment is
no less than about 10%. In a further embodiment, the deuterium
enrichment is no less than about 20%. In a further embodiment, the
deuterium enrichment is no less than about 50%. In a further
embodiment, the deuterium enrichment is no less than about 70%. In
a further embodiment, the deuterium enrichment is no less than
about 80%. In a further embodiment, the deuterium enrichment is no
less than about 90%. In a further embodiment, the deuterium
enrichment is no less than about 95%. In one embodiment, the
deuterated (or otherwise substituted) compound of the invention has
a slower rate of metabolism, e.g., slower rate of hydroxylation,
than a corresponding protonated (non-deuterated, non-substituted)
compound.
[0151] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which this application pertains.
The following definitions are provided to assist the reader in the
practice of the application.
[0152] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the application pertains.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice for testing of the
disclosure of the present application, materials and methods
typically employed are described herein.
[0153] In alternative embodiments, the terms "alkyl" and
"substituted alkyl" are interchangeable and include substituted,
optionally substituted and unsubstituted C.sub.1 to C.sub.12, or
longer, straight chain (a linear C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10,
C.sub.11 or C.sub.1-12 or longer carbon chain), or branched
saturated aliphatic hydrocarbon groups, substituted, optionally
substituted and unsubstituted C.sub.1 to C.sub.12 straight chain
(or longer) or branched unsaturated aliphatic hydrocarbon groups.
The invention also comprises compound comprising substituted,
optionally substituted and unsubstituted C.sub.3 to C.sub.8, or
C.sub.4 to C.sub.8 or C.sub.5 to C.sub.8 cyclic saturated aliphatic
hydrocarbon groups, substituted, optionally substituted and
unsubstituted C.sub.3 to C.sub.8, or C.sub.4 to C.sub.8 or C.sub.5
to C.sub.8 cyclic unsaturated aliphatic hydrocarbon groups having
the specified number of carbon atoms. In alternative embodiments,
the definition of "alkyl" includes but is not limited to: methyl
(Me), trideuteromethyl (--CD.sub.3), ethyl (Et), propyl (Pr), butyl
(Bu), pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, ethenyl,
propenyl, butenyl, penentyl, hexenyl, heptenyl, octenyl, nonenyl,
decenyl, undecenyl, isopropyl (i-Pr), isobutyl (i-Bu), tert-butyl
(t-Bu), sec-butyl (s-Bu), isopentyl, neopentyl, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,
cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl,
methylcyclopropyl, ethylcyclohexenyl, butenylcyclopentyl,
adamantyl, norbomyl and the like. Alkyl or hydrogen substituents
can be independently selected from the group consisting of
deuterium, halogen, --OH, --SH, --NH.sub.2, --CN, --NO.sub.2,
.dbd.O, .dbd.CH.sub.2, trihalomethyl, carbamoyl,
arylC.sub.0-10alkyl, heteroarylC.sub.0-10alkyl, C.sub.1-10alkyloxy,
arylC.sub.0-10alkyloxy, C1-10alkylthio, arylC.sub.0-10alkylthio,
C.sub.1-10alkylamino, arylC.sub.0-10alkylamino,
N-aryl-N--C.sub.0-10alkylamino, C.sub.1-10alkylcarbonyl,
arylC.sub.0-10alkylcarbonyl, C.sub.1-10 alkylcarboxy,
arylC.sub.0-10alkylcarbooalkylcarboxy,
C.sub.1-10alkylcarbonylamino, arylC.sub.0-10alkylcarbonylamino,
tetrahydrofuryl, morpholinyl, piperazinyl, hydroxypyronyl,
--C.sub.0-10alkylCOOR.sup.A and --C.sub.0-10alkylCONR.sup.BR.sup.C
wherein R.sup.A, R.sup.B and R.sup.C are independently selected
from the group consisting of hydrogen, deuterium, alkyl, aryl.
[0154] In alternative embodiments, a composition of the invention,
including a pharmaceutical composition of the invention, can be
administered to any patient, individual or "subject", including any
mammal, such as human patients and non-human primates, as well as
experimental animals such as rabbits, rats, and mice, and other
animals. Animals include all vertebrates, e.g., mammals and
non-mammals, such as dogs, cats, sheep, cows, pigs, rabbits,
chickens, including any domestic, farm or zoo animals, and etc.
Subjects for practicing the therapeutic methods of this invention
can be human patients. In alternative embodiments compositions of
the invention are administered to individuals or subjects, e.g.,
patients, to ameliorate (including treat, slow, reverse or prevent)
a disease or condition which can be ameliorated by partial or
complete inhibition of a cysteine protcase, e.g., a dementia or
pre-dementia, a cognitive loss, Alzheimer's disease (AD) or a
vascular dementia (VD) or Cognitive Dysfunction Syndrome (CDS) in a
non-human animal.
[0155] In alternative embodiments, patients, individuals or
subjects treated using compositions and methods of the invention
include those already suffering from a dementia or pre-dementia, or
suspected from suffering from a dementia or pre-dementia, or a
partial loss of cognition, as well as those prone to developing a
dementia or pre-dementia, e.g., Alzheimer's disease (AD), a
vascular dementia (VD) or Cognitive Dysfunction Syndrome (CDS) in a
non-human animal. For example, in one embodiment, patients,
individuals or subjects (including humans and animals, e.g., dogs
and cats) treated using compositions and methods of the invention
are asymptomatic but have been diagnosed as pre-conditional or
predisposed to AD or a dementia or pre-dementia because of a
genetic test, family history and/or brain or body scan (e.g., PET
or CAT) detection of central nervous system (CNS, e.g., brain)
plaques, e.g., plaques comprising beta amyloid.
[0156] In alternative embodiments, "treating" or "ameliorating"
includes e.g. one or more of (i) preventing any cognitive loss or
dementia or pre-dementia (e.g., AD or pre-dementia or vascular
dementia, or VD) from occurring (e.g. prophylaxis); (ii) inhibiting
any cognitive loss or dementia or pre-dementia or arresting or
slowing or reversing its development or progress; and (iii)
relieving one or more symptoms, e.g., loss of cognition or memory,
associated with a dementia or pre-dementia or AD. In alternative
embodiments, "treatment" includes the administration of the
compositions or dosage forms of this invention, and practicing the
methods of this invention, to prevent or delay the onset of the
symptoms, complications, or biochemical indicia of a dementia or
pre-dementia, such as AD, VD and Cognitive Dysfunction Syndrome
(CDS) in a non-human animal, alleviating or ameliorating the
symptoms of a dementia or pre-dementia, AD, VD and/or CDS, or
reversing or arresting or inhibiting further development of the
disease. In alternative embodiments, "treatment" further refers to
any indicia of success in the treatment or amelioration or
prevention or reversal of a dementia or pre-dementia, AD, VD and/or
CDS, or a related pathological process (e.g., neuronal or muscle
death), including any objective or subjective parameter such as
abatement; remission; diminishing of symptoms or making the disease
condition more tolerable to the patient; slowing in the rate of
degeneration or decline; or making the final point of degeneration
less debilitating. Detailed procedures for the treatment or
amelioration of a dementia or pre-dementia, AD, VD and/or CDS, or
symptoms thereof can be based on objective or subjective
parameters, including the results of an examination by a physician,
a cognitive or psychological test, a biopsy, a radiograph, a CAT or
PET scan and/or an MRI and the like.
[0157] In alternative embodiments, compounds of the invention can
also comprise crystal forms, salts, solvates and hydrates. Thus,
these compounds may crystallize with, for example, waters of
hydration, or one, a number of, or any fraction thereof of
molecules of the mother liquor solvent. The solvates and hydrates
of such compounds are included within the scope of this
disclosure.
[0158] In alternative embodiments, compounds of the invention
(e.g., compounds of Formula I), comprise asymmetric carbon atoms.
For example, compounds of Formula I can have the stereochemistry
denoted in Formula I':
##STR00016##
wherein in alternative embodiment: R is --H or alkyl; each of
R.sub.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, R.sup.9, and R.sup.10 is independently selected from the
group consisting of --H, -D, --F, --OH, --CH.sub.3, or a
pharmaceutically acceptable salt or solvate thereof; with the
proviso that at least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, and R.sup.10 is not
--H.
[0159] In alternative embodiments, compounds of the invention exist
as (comprise) individual respective stereoisomers that are
substantially free from another possible stereoisomer. In
alternative embodiments, the term "substantially free of other
stereoisomers" as used herein means less than about 15%, 20%, 25%,
30%, 35%, 40%, 50% or 55% of other stereoisomers, or less than
about 10% of other stereoisomers, or less than about 5% of other
stereoisomers, or less than about 2% of other stereoisomers, or
less than about 1% or less of other stereoisomers, or less than
"X"% of other stereoisomers (wherein X is a number between 0 and
100, inclusive) are present. Methods of obtaining or synthesizing
an individual enantiomer for a given compound are known in the art
and may be applied as practicable to final compounds or to starting
material or intermediates.
[0160] In alternative embodiments, compounds of the invention
comprise or are formulated as pharmaceutically acceptable salts.
Pharmaceutically acceptable salts of the compounds disclosed herein
include suitable acid addition or base salts thereof. In
alternative embodiments, compounds of the invention are formulated
as described in Berge et al, J Pharm Sci, 66, 1-19 (1977).
[0161] In alternative embodiments, compounds of the invention
formulated as salts that are formed, for example, with strong
inorganic acids such as mineral acids, e.g. hydrohalic acids such
as hydrochloride, hydrobromide and hydroiodide, sulphuric acid,
phosphoric acid sulphate, bisulphate, hemisulphate, thiocyanate,
persulphate and sulphonic acids; with strong organic carboxylic
acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which
are unsubstituted or substituted (e.g., by halogen), such as acetic
acid; with saturated or unsaturated dicarboxylic acids, for example
oxalic, malonic, succinic, maleic, fumaric, phthalic or
tetraphthalic; with hydroxycarboxylic acids, for example ascorbic,
glycolic, lactic, malic, tartaric or citric acid; with amino acids,
for example aspartic or glutamic acid; with benzoic acid; or with
organic sulfonic acids, such as (C.sub.1-C.sub.4)-alkyl- or
aryl-sulfonic acids which are unsubstituted or substituted (for
example, by a halogen) such as methane- or p-toluene sulfonic acid.
Compounds of the invention also encompass salts which are not
pharmaceutically acceptable, for example, a salt may still be
valuable as an intermediate in a synthetic or analytical process or
protocol.
[0162] In alternative embodiments, compounds of the invention
comprise any acceptable salt for example, acetate,
trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate,
malate, pantothenate, adipate, alginate, aspartate, benzoate,
butyrate, digluconate, cyclopentanate, glucoheptanate,
glycerophosphate, oxalate, heptanoate, hexanoate, fumarate,
nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate,
pivalate, proprionate, tartrate, lactobionate, pivolate,
camphorate, undecanoate and succinate, organic sulphonic acids such
as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate,
camphorsulphonate, 2-naphthalenesulphonate, benzenesulphonate,
p-chlorobenzenesulphonate and p-toluenesulphonate; and inorganic
acids such as hydrochloride, hydrobromide, hydroiodide, sulphate,
bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and
sulphonic acids. Pharmaceutical compositions as disclosed herein
can be prepared in accordance with methods well known and routinely
practiced in the art. See, e.g., Remington: The Science and
Practice of Pharmacy, Mack Publishing Co., 20.sup.th ed., 2000; and
Sustained and Controlled Release Drug Delivery Systems, J. R.
Robinson, ed., Marcel Dekker, Inc., New York, 1978.
[0163] In some embodiments, the compounds of the invention are
provided in the form of pharmaceutically acceptable salts
comprising an amine that is basic in nature and can react with an
inorganic or organic acid to form a pharmaceutically acceptable
acid addition salt; e.g., such salts comprise inorganic acids such
as hydrochloric, hydrobromic, hydriodic, sulfuric and phosphoric
acid, as well as organic acids such as para-toluenesulfonic,
methanesulfonic, oxalic, para-bromophenylsulfonic, carbonic,
succinic, citric, benzoic and acetic acid, and related inorganic
and organic acids; or optionally such pharmaceutically acceptable
salts comprise sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,
phosphate, mono-hydrogenphosphate, dihydrogenphosphate,
metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate,
propionate, decanoate, caprylate, acrylate, formate, isobutyrate,
caprate, heptanoate, propiolate, oxalate, malonate, succinate,
suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,
hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,
dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate,
terephthalate, sulfonate, xylenesulfonate, phenylacetate,
phenylpropionate, phenylbutyrate, citrate, lactate,
.beta.-hydroxybutyrate, glycollate, maleate, tartrate,
methanesulfonate, propanesulfonates, naphthalene-1-sulfonate,
naphthalene-2-sulfonate, mandelate, hippurate, gluconate,
lactobionate, and the like salts.
[0164] In alternative embodiments, compounds of the invention,
including pharmaceutical compositions and formulations of the
invention, and compounds used to practice the methods of this
invention, comprise compositions manufactured under "Good
manufacturing practice" or GMP, or "current good manufacturing
practices" (cGMP), conditions.
[0165] In alternative embodiments, formulations and pharmaceutical
compositions of the invention comprise a therapeutically effective
dose or efficacious dose of the active ingredient, e.g., one or
more compounds of this invention, e.g., comprising a derivatized
form, e.g., a deuterated form, e.g., which can be a metabolically
blocked derivative, of AB-007 (loxistatin, E64d) and/or loxistatin
acid (E64c), or a stereoisomer, salt, hydrate and/or solvate
thereof, which in alternative embodiments is mixed with a
pharmaceutically acceptable solvent, carrier or excipient. In
alternative embodiments, formulations and pharmaceutical
compositions of the invention comprise a therapeutically effective
dose or efficacious dose of an active ingredient (e.g., an
inhibitor of a cysteine protease, or a cathepsin, a caspase or a
calpain, or a cathepsin L or a cathepsin B, or a cathepsin F, H, K,
L1, L2, O, S, W, X or Z) for the amelioration, prevention or
treatment of Alzheimer's disease (AD).
Conjugates of Compositions of the invention
[0166] In alternative embodiments a composition of this invention,
or a composition used to practice the methods of this invention
(e.g., an inhibitor of a cysteine protease, or a cathepsin, a
caspase or a calpain, or a cathepsin L or a cathepsin B, or a
cathepsin F, H, K, L1, L2, O, S, W, X or Z), is linked to (e.g.,
conjugated or otherwise joined to) a chemical delivery system
(CDS); e.g., a CDS such that the ratio of brain to periphery drug
(a composition of this invention) levels can be increased. This
embodiment can offer significant advantages in the treatments
because the drug (e.g., a composition of this invention) because of
the CDS linkage is concentrated in the brain, thereby reducing the
dose and toxicity. For example, CDS prodrugs of the invention
(e.g., comprising a composition of this invention) can be made
using blood-brain barrier (BBB) penetrating moieties as described
in U.S. Patent Application No. 20080227806.
[0167] In alternative embodiments, a composition of this invention,
or a composition used to practice the methods of this invention
(e.g., an inhibitor of a cysteine protease, or a cathepsin, a
caspase or a calpain, or a cathepsin L or a cathepsin B, or a
cathepsin F, H, K, L1, L2, O, S, W, X or Z), is linked to a CDS
prodrug which exploits the bidirectional properties of the
blood-brain barrier to lock-in the active compound (e.g., a
composition of this invention) in the brain and allow sustained
release of active compound, e.g., a cysteine protease inhibitor
composition of this invention. In alternative embodiments, CDS
moieties that can be linked to a composition of this invention, or
a composition used to practice the methods of this invention (e.g.,
an inhibitor of a cysteine protease, or a cathepsin, a caspase or a
calpain, or a cathepsin L or a cathepsin B, or a cathepsin F, H, K,
L1, L2, O, S, W, X or Z), comprise pyridimium and pyridimium
derivatives (e.g., 3-methyl-1-propylpyridinium,
1-butyl-3-methylpyridinium, and 1-butyl-4-methylpyridinium),
1,4-dihydrotrigoneline esters and/or amides, including
dihydroquinoline- and dihydroisoquinoline-based targetor moieties
for brain-specific chemical delivery. In alternative embodiments a
1,4 dihydrotrigoneline esters or amides are used; they are
hydrophobic and allow the CDS prodrug (comprising a composition of
this invention) to readily cross the blood-brain barrier. A
hydrophobic 1,4 dihydrotrigoneline can be converted in vivo to a
hydrophilic quaternary form. This conversion takes place throughout
the body via oxidation but without generating active or reactive
radical intermediates. In the periphery the charged quaternary form
is rapidly eliminated from the body but in the brain the charged
form (e.g., of a composition of this invention) is locked behind
the blood-brain barrier. This results in the brain being the only
site containing the CDS prodrug and drug (e.g., a composition of
this invention) after a relatively short period of time. There, the
CDS prodrug continues to be metabolized and release the cysteine
protease inhibitor composition of this invention. As a result,
there is a sustained release of the drug composition in the brain.
Thus, the CDS prodrug not only delivers the cysteine protease
inhibitor moiety composition to the brain, it also provides
preferential delivery there, thus targeting the brain.
[0168] Compositions of this invention, or a composition used to
practice the methods of this invention (e.g., an inhibitor of a
cysteine protease, or a cathepsin, a caspase or a calpain, or a
cathepsin L or a cathepsin B, or a cathepsin F, H, K, L1, L2, O, S,
W, X or Z), also can be linked or conjugated to a polypeptide,
peptide or liposome to target the composition to the brain
vasculature or otherwise expedite penetration of the BBB. For
example, in one embodiment, a composition of this invention is
linked or conjugated to a short peptide motif to create a
penetration composition for specific transport across a biological
barrier sealed by a tight junction, e.g., the BBB, e.g., as
described in U.S. pat. App. Pub. no. 20060251713.
Formulations and Pharmaceutical Compositions
[0169] In alternative embodiments a composition of this invention
(which include a derivatized form, e.g., a deuterated form, e.g.,
which can be a metabolically blocked derivative, of AB-007
(loxistatin, E64d) and/or loxistatin acid (E64c), or a
stereoisomer, salt, hydrate and/or solvate thereof), or a
composition used to practice the methods of this invention (e.g.,
an inhibitor of a cysteine protease, or a cathepsin, a caspase or a
calpain, or a cathepsin L or a cathepsin B, or a cathepsin F, H, K,
L1, L2, O, S, W, X or Z), is formulated for administration by any
or a variety of means including orally, parenterally, by inhalation
spray, nasally, topically, intrathecally, intrathecally,
intracerebrally, epidurally, intracranially or rectally.
Formulations of the invention can comprise pharmaceutically
acceptable carriers, adjuvants and vehicles. In alternative
embodiments, composition of this invention, or a composition used
to practice the methods of this invention, are formulated for
parenteral administration, including administration intrathecally,
intracerebrally or epidurally (into a intrathecal, intracerebral,
epidural space), subcutaneously, intravenously, intramuscularly
and/or intraarterially; e.g., by injection routes but also
including a variety of infusion techniques. Intraarterial,
intrathecal, intracranial, epidural, intravenous and other
injections as used in some embodiments can include administration
through catheters or pumps, e.g., an intrathecal pump, or an
implantable medical device (which can be an intrathecal pump or
catheter).
[0170] In alternative embodiments a compound of the invention, or a
composition used to practice the methods of this invention, can be
formulated in accordance with a routine procedure(s) adapted for a
desired administration route. Accordingly, in alternative
embodiments compounds used to practice the invention are formulated
or manufactured as lyophilates, powders, lozenges, liposomes,
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0171] In alternative embodiments a compound of the invention, or a
composition used to practice the methods of this invention, can be
formulated as a preparation for implantation or injection. Thus,
for example, the compounds can be formulated with suitable
polymeric or hydrophobic materials (e.g., as an emulsion in an
acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives (e.g., as a sparingly soluble salt). Alternatively, the
active ingredient (e.g., a composition of the invention) can be in
powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use. Suitable alternative and exemplary
formulations for each of these methods of administration can be
found, for example, in Remington: The Science and Practice of
Pharmacy, A. Gennaro, ed., 20th edition, Lippincott, Williams &
Wilkins, Philadelphia, Pa.
[0172] In alternative embodiments a compound of the invention, or a
composition used to practice the methods of this invention, can be
formulations for parenteral administration comprising any common
excipient, e.g., sterile water or saline, a polyalkylene glycol
such as a polyethylene glycol, an oil of synthetic or vegetable
origin, a hydrogenated naphthalene and the like. In alternative
embodiments, a compound used to practice the invention can be a
biocompatible, biodegradable lactide polymer, a lactide/glycolide
copolymer, or polyoxyethylene-polyoxypropylene copolymers can be
useful excipients to control the release of active compounds.
[0173] In alternative embodiments, a composition of the invention,
or a composition used to practice the methods of this invention, is
administered using parenteral delivery systems such as
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, intrathecal catheters, pumps and
implants, and/or use of liposomes. Formulations for parenteral
administration can also include glycocholate for buccal
administration, methoxysalicylate for rectal administration, or
citric acid for vaginal administration. Formulations for inhalation
administration can contain as excipients, for example, lactose, or
can be aqueous solutions containing, for example,
polyoxyethylene-9-auryl ether, glycocholate and deoxycholate, or
oily solutions for administration in the form of nasal drops, or as
a gel to be applied intranasally.
[0174] In alternative embodiments, formulations of the invention,
or a composition used to practice the methods of this invention,
are administered intranasally. When given by this route, examples
of appropriate dosage forms are a nasal spray or dry powder, as is
known to those skilled in the art. For example, a nasal formulation
can comprise a conventional surfactant, generally a non-ionic
surfactant. When a surfactant is employed in a nasal formulation,
the amount present will vary depending on the particular surfactant
chosen, the particular mode of administration (e.g. drop or spray)
and the effect desired.
[0175] In alternative embodiments, a pharmaceutical composition of
the invention, or a composition used to practice the methods of
this invention, is in the form of a sterile injectable preparation,
such as a sterile injectable aqueous or oleaginous suspension. This
suspension may be formulated according to the known art using those
suitable dispersing or wetting agents and suspending agents. The
sterile injectable preparation may also be a sterile injectable
solution or suspension in a non-toxic parenterally acceptable
diluent or solvent, such as a solution in 1,3-butane-diol or
prepared as a lyophilized powder. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In alternative embodiments,
sterile fixed oils are conventionally be employed as a solvent or
suspending medium. For this purpose any bland fixed oil may be
employed including synthetic mono- or diglycerides. In alternative
embodiments, fatty acids such as oleic acid may likewise be used in
the preparation of injectables. Formulations for intravenous
administration can comprise solutions in sterile isotonic aqueous
buffer. Where necessary, the formulations can also include a
solubilizing agent and a local anesthetic to ease pain at the site
of the injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampule (ampoule) or sachet indicating
the quantity of active agent. Where the compound is to be
administered by infusion, it can be dispensed in a formulation with
an infusion bottle containing sterile pharmaceutical grade water,
saline or dextrose/water. Where the compound is administered by
injection, an ampule of sterile water for injection or saline can
be provided so that the ingredients can be mixed prior to
administration.
[0176] In alternative embodiments, formulations of the invention,
or a composition used to practice the methods of this invention,
further comprise aqueous and non-aqueous sterile injection
solutions that can contain (comprise) antioxidants, buffers,
bacteriostats, bactericidal antibiotics and solutes that render the
formulation isotonic with the bodily fluids of the intended
recipient; and/or aqueous and non-aqueous sterile suspensions,
which can include suspending agents and thickening agents.
[0177] In alternative embodiments, compounds of the invention, or a
composition used to practice the methods of this invention, are
formulated for topical administration, e.g., in the form of a
liquid, lotion, cream or gel. Topical administration can be
accomplished by application directly on the treatment area. For
example, such application can be accomplished by rubbing the
formulation (such as a lotion or gel) onto the skin of the
treatment area, or by a spray application of a liquid formulation
onto the application or treatment area.
[0178] In alternative embodiments, formulations of the invention,
or a composition used to practice the methods of this invention,
comprise a bioimplant or a bioimplant material, and also can be
coated with a compound of the invention or another compounds so as
to improve interaction between cells and the implant.
[0179] In alternative embodiments, formulations of the invention,
or a composition used to practice the methods of this invention,
comprise minor amounts of wetting or emulsifying agents, or pH
buffering agents.
[0180] In alternative embodiments, formulations of the invention,
or a composition used to practice the methods of this invention,
are formulated as a suppository, with traditional binders and
carriers such as triglycerides.
[0181] In alternative embodiments, formulations of the invention,
or a composition used to practice the methods of this invention,
comprise oral formulations such as tablets, pills, troches,
lozenges (see, e.g., as described in U.S. Pat. No. 5,780,055),
aqueous or oil suspensions, dispersible powders or granules,
emulsions, hard or soft capsules or geltabs, gels, jellies, syrups
and/or elixirs. Compositions intended for oral use may be prepared
according to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions may contain one
or more agents including sweetening agents, taste-masking agents,
flavoring agents, coloring agents and preserving agents, in order
to provide a palatable preparation. Oral formulations can include
standard carriers such as pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium
saccharine, cellulose, magnesium carbonate, etc. Tablets containing
the active ingredient in admixture with non-toxic pharmaceutically
acceptable excipient which are suitable for manufacture of tablets
are acceptable. These excipients may be, for example, inert
diluents, such as calcium or sodium carbonate, lactose, calcium or
sodium phosphate; granulating and disintegrating agents, such as
maize starch, or alginic acid; binding agents, such as starch,
gelatin or acacia; and lubricating agents, such as magnesium
stearate, stearic acid or talc. Tablets may be uncoated or may be
coated by known techniques including microencapsulation to delay
disintegration and adsorption in the gastrointestinal tract and
thereby provide a sustained action over a longer period. For
example, a time delay material such as glyceryl monostearate or
glyceryl distearate alone or with a wax may be employed.
[0182] In alternative embodiments, formulations for oral use are
hard gelatin capsules where the active ingredient is mixed with an
inert solid diluent, for example calcium phosphate or kaolin, or as
soft gelatin capsules wherein the active ingredient is mixed with
water or an oil medium, such as peanut oil, liquid paraffin or
olive oil.
[0183] In alternative embodiments, formulations of the invention,
or a composition used to practice the methods of this invention,
comprise aqueous suspensions comprising the active material (e.g.,
a compound of this invention) in admixture with excipients suitable
for the manufacture of aqueous suspensions. Exemplary excipients
include a suspending agent, such as sodium carboxymethylcellulose,
methylcellulose, hydroxypropyl methylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing
or wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethyleneoxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous
suspension may also contain one or more preservatives such as ethyl
or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or
more flavoring agents and one or more sweetening agents, such as
sucrose or saccharin.
[0184] In alternative embodiments, formulations of the invention,
or a composition used to practice the methods of this invention,
comprise oil suspensions that can be formulated by suspending the
active ingredient (e.g., a compound of this invention) in a
vegetable oil, such as arachis oil, olive oil, sesame oil or
coconut oil, or in a mineral oil such as liquid paraffin. The oral
suspensions may contain a thickening agent, such as beeswax, hard
paraffin or ectyl alcohol. Sweetening agents, such as those set
forth above, and flavoring agents may be added to provide a
palatable oral preparation. These compositions may be preserved by
the addition of an antioxidant such as ascorbic acid.
[0185] In alternative embodiments, pharmaceutical formulations
comprising the compounds of the invention, or a composition used to
practice the methods of this invention, include an agent which
controls release of the compound, thereby providing a timed or
sustained release compound.
[0186] Carriers
[0187] In alternative embodiments, pharmaceutically acceptable
carriers for manufacturing or formulating compounds of this
invention, or a composition used to practice the methods of this
invention, comprise aqueous or non-aqueous solutions, suspensions,
emulsions and solids. Examples of non-aqueous solvents suitable for
use as disclosed herein include, but are not limited to, propylene
glycol, polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. In alternative
embodiments, aqueous carriers can comprise water, ethanol,
alcoholic/aqueous solutions, glycerol, emulsions and/or
suspensions, including saline and buffered media. Oral carriers can
be elixirs, syrups, capsules, tablets and the like.
[0188] In alternative embodiments, liquid carriers are used to
manufacture or formulate compounds of this invention, or a
composition used to practice the methods of this invention,
including carriers for preparing solutions, suspensions, emulsions,
syrups, elixirs and pressurized compounds. The active ingredient
(e.g., a composition of this invention) can be dissolved or
suspended in a pharmaceutically acceptable liquid carrier such as
water, an organic solvent, a mixture of both or pharmaceutically
acceptable oils or fats. The liquid carrier can comprise other
suitable pharmaceutical additives such as solubilizers,
emulsifiers, buffers, preservatives, sweeteners, flavoring agents,
suspending agents, thickening agents, colors, viscosity regulators,
stabilizers or osmo-regulators.
[0189] In alternative embodiments, liquid carriers used to
manufacture or formulate compounds of this invention comprise water
(partially containing additives as above, e.g. cellulose
derivatives, preferably sodium carboxymethyl cellulose solution),
alcohols (including monohydric alcohols and polyhydric alcohols,
e.g. glycols) and their derivatives, and oils (e.g. fractionated
coconut oil and arachis oil). For parenteral administration, the
carrier can also include an oily ester such as ethyl oleate and
isopropyl myristate. Sterile liquid carriers are useful in sterile
liquid form comprising compounds for parenteral administration. The
liquid carrier for pressurized compounds disclosed herein can be
halogenated hydrocarbon or other pharmaceutically acceptable
propellant.
[0190] In alternative embodiments, solid carriers are used to
manufacture or formulate compounds of this invention, or a
composition used to practice the methods of this invention,
including solid carriers comprising substances such as lactose,
starch, glucose, methylcellulose, magnesium stearate, dicalcium
phosphate, mannitol and the like. A solid carrier can further
include one or more substances acting as flavoring agents,
lubricants, solubilizers, suspending agents, fillers, glidants,
compression aids, binders or tablet-disintegrating agents; it can
also be an encapsulating material. In powders, the carrier can be a
finely divided solid which is in admixture with the finely divided
active compound. In tablets, the active compound is mixed with a
carrier having the necessary compression properties in suitable
proportions and compacted in the shape and size desired. Suitable
solid carriers include, for example, calcium phosphate, magnesium
stearate, talc, sugars, lactose, dextrin, starch, gelatin,
cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange
resins. A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active ingredient
in a free flowing form such as a powder or granules, optionally
mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(e.g., sodium starch glycolate, cross-linked povidone, cross-linked
sodium carboxymethyl cellulose) surface active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent. The tablets may optionally be coated or scored and may be
formulated so as to provide slow or controlled release of the
active ingredient therein using, for example, hydroxypropyl
methylcellulose in varying proportions to provide the desired
release profile. Tablets may optionally be provided with an enteric
coating, to provide release in parts of the gut other than the
stomach.
[0191] In alternative embodiments, parenteral carriers are used to
manufacture or formulate compounds of this invention, or a
composition used to practice the methods of this invention,
including parenteral carriers suitable for use as disclosed herein
include, but are not limited to, sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's and fixed
oils. Intravenous carriers can comprise fluid and nutrient
replenishers, electrolyte replenishers such as those based on
Ringer's dextrose and the like. Preservatives and other additives
can also comprise, for example, antimicrobials, antioxidants,
chelating agents, inert gases and the like.
[0192] In alternative embodiments, carriers used to manufacture or
formulate compounds of this invention, or a composition used to
practice the methods of this invention, can be mixed as needed with
disintegrants, diluents, granulating agents, lubricants, binders
and the like using conventional techniques known in the art. The
carriers can also be sterilized using methods that do not
deleteriously react with the compounds, as is generally known in
the art.
[0193] The invention also provides articles of manufacture and kits
containing (comprising) compounds of this invention, or a
composition used to practice the methods of this invention,
including pharmaceutical compositions and formulations. By way of
example only a kit or article of manufacture can include a
container (such as a bottle) with a desired amount of a compound
(or pharmaceutical composition of a compound) described herein.
Such a kit or article of manufacture can further include
instructions for using the compound (or pharmaceutical composition
of a compound) described herein. The instructions can be attached
to the container, or can be included in a package (such as a box or
a plastic or foil bag) holding the container.
[0194] The compounds of the invention, or a composition used to
practice the methods of this invention, can be delivered to the
body or targeted to a specific tissue or organ (e.g., a muscle or a
brain) by any method or protocol, e.g., including ex vivo "loading
of cells" with a composition of the invention; where the "loaded
cell" is the administered intramuscularly, or intrathecally,
intracerebrally, or epidurally into the central nervous system
(CNS), e.g., as described in U.S. Pat. App. Pub. No.
20050048002.
[0195] In alternative embodiments, compounds of the invention, or a
composition used to practice the methods of this invention, are
first lyophilized and then suspended in a hydrophobic medium, e.g.,
comprising aliphatic, cyclic or aromatic molecules, e.g., as
described in U.S. Pat. App. Pub. No. 20080159984.
Methods of Use
[0196] In alternative embodiments, a composition of this invention,
which includes a derivatized form, e.g., a deuterated form, e.g.,
which can be a metabolically blocked derivative, of AB-007
(loxistatin, E64d) and/or loxistatin acid (E64c), or a
stereoisomer, salt, hydrate or solvate thereof, or a composition
used to practice the methods of this invention, is used as a
cysteine protease inhibitor. In alternative embodiments
compositions of the invention, or a composition used to practice
the methods of this invention, are used to ameliorate (including
treat, slow, reverse or prevent) a disease or condition which can
be ameliorated by partial or complete inhibition of a cysteine
protease, e.g., Alzheimer's disease (AD) or vascular dementia (VD),
or Cognitive Dysfunction Syndrome (CDS) in a non-human animal.
[0197] While the invention is not limited by any particular
mechanism of action, because cysteine proteases, including
cathepsins (e.g., cathepsin B, or a cathepsin F, H, K, L1, L2, O,
S, W, X or Z), caspases and calpains, are involved in a dementia or
pre-dementia and/or a neurological pathology, and as these enzymes
can be important in the development of cognitive loss or a dementia
or pre-dementia, including cognitive loss or a dementia or
pre-dementia due to AD or vascular dementia (VD), in alternative
embodiments the compositions of the invention are used to
ameliorate dementia or pre-dementia, neurological pathology,
Alzheimer's disease (AD), Cognitive Dysfunction Syndrome (CDS) in a
non-human animal, and/or vascular dementia (VD). The invention
provides compositions (e.g., pharmaceutical compositions or
formulations) and methods for preventing, slowing the progression
of, reversing or ameliorating a cognitive loss, a dementia or
pre-dementia, a neurological pathology, AD, VD and/or Cognitive
Dysfunction Syndrome (CDS) in a non-human animal, and/or any
disease of condition that can be ameliorated by complete or partial
inhibition of a cysteine protease such as a cathepsin (e.g., a
cathepsin B, or a cathepsin F, H, K, L1, L2, O, S, W, X or Z), a
caspase and/or a calpain.
[0198] While the invention is not limited by any particular
mechanism of action, in alternative embodiments, a composition of
this invention is used to ameliorate a neurological disease, a
dementia or pre-dementia and/or AD, by slowing the accumulation of,
reversing or preventing .beta.-amyloid (A.beta.) and plaque
formation, abnormal phosphorylation and/or aggregation of the
microtubule-associated protein tau, and/or massive neuronal loss.
In alternative embodiments, a composition of this invention is used
to ameliorate hereditary forms of AD (e.g., familial AD, or FAD);
and while the invention is not limited by any particular mechanism
of action, mutations identified in hereditary forms of AD as well
as abundant animal models and in vitro data strongly implicate AP
and the polypeptide from which it is derived, the amyloid precursor
protein (APP), as the principal factor driving the development of
AD.
[0199] In alternative embodiments, the present application provides
a method for inhibiting or slowing the activity of a cysteine
protease enzyme, e.g., a cathepsin (e.g., a cathepsin B, or a
cathepsin F, H, K, L1, L2, O, S, W, X or Z), a caspase and/or a
calpain, comprising contacting cells containing the cysteine
protease enzyme (e.g., the cathepsin B, etc.) with an effective
amount of a compound of this invention, e.g. a compound of Formula
I, which can be a metabolically blocked analog (e.g., deuterated
analog) of AB-007 (loxistatin, E64d) and/or loxistatin acid (E64c),
or a stereoisomer, salt, hydrate or solvate of a composition of
this invention, e.g. a compound encompassed by Formula I.
[0200] In one aspect the present application provides a method for
preventing, slowing the progression of, reversing, or treating
Alzheimer's disease (AD), VD or Cognitive Dysfunction Syndrome
(CDS) in a non-human animal, comprising administering to a patient
in need thereof an effective amount a compound of this invention,
e.g. a compound of Formula I, which can be a metabolically blocked
analog of AB-007 (loxistatin, E64d) and/or loxistatin acid (E64c),
or a stereoisomer, salt, hydrate or solvate of a composition of
this invention, e.g. a compound encompassed by Formula I.
[0201] The present application also provides a method of reducing
or preventing the effects of .beta.-amyloid in an individual or
subject (e.g., a patient or a non-human animal) comprising
administering to said individual or subject (e.g., patient or a
non-human animal) an effective amount of a compound of this
invention, e.g. a compound of Formula I, which can be a
metabolically blocked analog of AB-007 (loxistatin, E64d) and/or
loxistatin acid (E64c), or a stereoisomer, salt, hydrate or solvate
of a composition of this invention, e.g. a compound encompassed by
Formula I, such that the effects of .beta.-amyloid are reduced or
prevented.
[0202] The present application also provides a method of
preventing, slowing or reversing neuronal cell apoptosis or death
in an individual or subject (e.g., a patient or a non-human animal)
comprising administering to said individual or subject (e.g.,
patient or a non-human animal) an effective amount of a compound of
this invention, e.g. a compound of Formula I, which can be a
metabolically blocked analog of AB-007 (loxistatin, E64d) and/or
loxistatin acid (E64c), or a stereoisomer, salt, hydrate or solvate
of a composition of this invention, e.g. a compound encompassed by
Formula I, such that neuronal cell death is prevented.
Dosages
[0203] In alternative embodiments, compositions of the invention,
or compositions used to practice the methods of the invention, are
formulated and administered in a variety of different dosages and
treatment regimens, depending on the disease or condition to be
ameliorated, the condition of the individual to be treated, the
goal of the treatment, and the like, as to be routinely determined
by the clinician, see e.g., the latest edition of Remington: The
Science and Practice of Pharmacy, Mack Publishing Co., supra.
[0204] In alternative embodiments, the effective amount of a
compound of this invention, e.g. a compound of Formula I, which can
be a metabolically blocked analog of AB-007 (loxistatin, E64d)
and/or loxistatin acid (E64c), or a stereoisomer, salt, hydrate or
solvate of this invention, e.g. a compound encompassed by Formula
I, or a composition used to practice the methods of the invention,
is between about 0.1 mg and about 20.0 mg per kg of body weight of
the individual or subject (e.g., patient). In another variation,
the effective amount is between about 0.1 mg and about 10.0 mg per
kg of body weight of the individual or subject (e.g., patient) or
between about 0.1 mg and about 5.0 mg per kg of body weight of the
patient. Alternately, the effective amount is between about 0.2 mg
and about 2 mg per kg of body weight of the individual or subject
(e.g., patient).
[0205] In a further variation of any of the disclosed aspects or as
an alternative embodiment, the administration of a therapeutically
effective amount of a compound of this invention, e.g. a compound
of Formula I, which can be a metabolically blocked analog of AB-007
(loxistatin, E64d) and/or loxistatin acid (E64c), or a
stereoisomer, salt, hydrate or solvate of a composition of this
invention, e.g. a compound encompassed by Formula I, elicits an
"improved" clinical effect during the treatment in said individual,
subject or patient per dosage unit thereof as compared to the
"parent" AB-007 (loxistatin, E64d) and/or loxistatin acid (E64c);
e.g., by administering a composition of this invention instead of
AB-007 (loxistatin, E64d) and/or loxistatin acid (E64c), a lower
dosage can be used.
[0206] While the invention is not limited by any particular
mechanism of action, in one embodiment, it is predicated in part on
the discovery that AB-007, at dosages significantly lower than that
suggested in the art, are effective in treating a loss in cognitive
abilities, a dementia or pre-dementia (e.g. as in AD and VD) and
Cognitive Dysfunction Syndrome (CDS) in a non-human animal. AB-007
(loxistatin, E64d) has been shown to be a neuroprotectant and thus
may not only reduce the likely cause of AD, abnormal A.beta.
accumulation, but also protect against A.beta. toxicity and thus be
effective in treating a loss in cognitive abilities, a dementia or
pre-dementia (e.g. as in AD and VD) and Cognitive Dysfunction
Syndrome (CDS) in a non-human animal.
[0207] Cathepsin B inhibitors administered
intracerebroventricularly (icv) (0.15 mg/kg/day for 30 days) to
guinea pigs, which express A.beta. identical to human and have APP
containing the human wt (wild type) .beta.-secretase site sequence,
have been found to reduce brain A.beta. and CTF.beta. (C-terminal
.beta.-secretase fragment (CTF.beta.) derived from APP) by up to
70%. Moreover, this treatment also reduces the A.beta. and
CTF.beta. in synaptosome fractions, which are a measure of brain
regulated secretion.
[0208] It has been reported that deleting the cathepsin B gene in
transgenic mice expressing wt hAPP (the allele present in the
majority of humans) resulted in 67% less brain A.beta.340 and
A.beta.42 and 41% less CTF.beta. than in corresponding transgenic
controls expressing cathepsin B.
[0209] SiRNA silencing cathepsin B reduces secretion of A.beta. in
primary rat hippocampal cells, which express APP containing the
human wild-type .beta.-secretase site. Moreover, it has been shown
that a cathepsin B inhibitor reduces regulated secretion of A.beta.
in the hippocampal cells.
[0210] Cathepsin B does not efficiently cleave the Swedish (Swc)
mutant .beta.-secretase site in APP, and neither inhibiting
cathepsin B nor deleting the cathepsin B gene reduces A.beta. or
CTF.beta. in animals expressing that site. Most AD patients express
APP having the wt .beta.-secretase site whereas only a small number
express the Swe .beta.-secretase site.
[0211] In contrast, cathepsin B inhibitors are efficacious at
lowering A.beta. in models expressing the wt APP secretase cleavage
site; thus, it is anticipated that they also do so in most
Alzheimer's patients (most humans have the wt APP secretase
cleavage site). Thus, the compositions and methods of this
invention are effective in most Alzheimer's patients.
[0212] Oral AB-007 (loxistatin, E64d) is rapidly and completely
converted in the gut to E64c (loxistatin acid) as shown above. In
humans, the only metabolites are E64c and two hydroxylated forms of
E64c. No drug accumulation occurs in humans. The serum half-life
(t.sub.1/2) in humans for E64c and the hydroxylated E64c forms is
about 1.3 and 2.5 hours, respectively. The Area Under the Curve
(AUC) in humans for E64c and the hydroxylated E64c forms is about
4.6 and 5.08 .mu.g/hr/mL, respectively. The total urinary excretion
rate in humans is about 30%.
[0213] Radioactive AB-007 (loxistatin, E64d) studies in animals
have shown that the primary absorption site is the small intestine,
where about 60% of an oral dose is absorbed. The maximum tissue
concentration occurs 0.5 hours after administration and, excluding
the gastrointestinal track, the liver and kidneys have the highest
concentrations at about 25 and 12 times that of the plasma
concentration, respectively. All other organs, including the brain,
have a concentration corresponding to the plasma concentration or
concentrations lower than plasma.
[0214] The LD.sub.50 for oral AB-007 (loxistatin, E64d) is over
10,000 mg/kg and 5,000 mg/kg for rats and dogs, respectively. The
subacute no-effect dose for oral AB-007 is under 80 mg/kg/day and
40 mg/kg/day in rats and dogs, respectively. Chronic toxicity data
indicate that the liver and kidney are the target organs for
toxicity and that no-effect dose level is below 2 mg/kg/day and 5
mg/kg/day in rats and dogs, respectively.
[0215] Rats appear to be a particularly sensitive species to AB-007
(loxistatin, E64d) toxicity as the hepatic injury seen in rats does
not occur in other species. High oral doses of AB-007 (500 mg/kg
and 1 g/kg) depress the central nervous system, causing sedation
and mild inhibition of pentetrazol-induced seizure, but have no
effect on the cardiovascular system. The maximal intravenous or
oral AB-007 doses do not influence vision or hearing in rats.
[0216] In healthy human volunteers, single or continuous 1 week
oral administration of AB-007 (loxistatin, E64d) at about 5
mg/kg/day causes no change in pulse, blood pressure, body
temperature, grip force, electrocardiogram, or physician
observations. Moreover, there are no changes in hematological
tests, including leukocyte count, erythrocyte count, hemoglobin,
hematocrit, platelet count, reticulocyte count, differential
leukocyte count, blood chemistry, including GOT, GPT, LDH, A1-P,
LAP, .gamma.-GTP, CPK, BUN, creatinine, uric acid, total
cholesterol, triglycerides, serum total proteins, protein fraction,
Cl, Na, K, Ca, P, and urinalyses including pH, proteins, sugar,
urobilinogen, occult blood and sediment.
[0217] Oral radioactive AB-007 (loxistatin, E64d) data in animals
show that the resulting brain drug dose, although low, is
nonetheless ample for reducing brain A.beta. via inhibition of
cathepsin B. A 5 mg/kg oral AB-007 dose results in E64c brain
concentrations in the rat and hamster that are 100 fold and 300
fold, respectively, greater than that needed to inhibit by 50%
cathepsin B cleavage of the wt .beta.-secretase cleavage site
(IC.sub.50).
[0218] As detailed in the Examples below, a daily 10 mg/kg guinea
pig oral dose of AB-007 (loxistatin, E64d) has been shown to reduce
brain A.beta..sub.40 and A.beta..sub.42 by 42% relative to vehicle
treated controls. A 10 mg/kg guinea pig oral dose allometrically
scales to a 2 mg/kg human oral dose, which is well below the 5
mg/kg dose of AB-007 previously administered to humans.
[0219] In alternative embodiments, the present invention provides
dosage forms and formulations of AB-007 (loxistatin, E64d) and/or
loxistatin acid (E64c), and compositions of this invention, and
compositions used to practice this invention, comprising less than
about 1, 2, 3, 4 or 5 mg per kg body weight of a subject or
individual. In alternative embodiments, the present invention
provides dosage forms and formulations of AB-007 (loxistatin, E64d)
and/or loxistatin acid (E64c), compositions of this invention), and
compositions of this invention, and compositions used to practice
this invention, comprising more than about 5, 6, 7, 8, 9 or 10 or
more mg per kg body weight of a subject or individual. In
alternative embodiments, for an adult human subject with a body
weight of around 75 kg, the daily dosage of about 0.1 mg/kg to
about 10 mg/kg to be administered is from about 7.5 mg daily to
about 750 mg daily.
[0220] In some embodiments, human subjects with similar body
weights are treated by administered dosage of e.g., AB-007
(loxistatin, E64d), and/or loxistatin acid (E64c) and/or
compositions of this invention, and/or compositions used to
practice this invention, from about 0.1 mg/kg to about 2 mg/kg, or
from about 7.5 mg to about 150 mg daily. In other embodiments,
human subjects are administered a dosage of e.g., AB-007
(loxistatin, E64d) and/or loxistatin acid (E64c) and/or
compositions of this invention, and/or compositions used to
practice this invention, from about 0.1 mg/kg to about 0.5 mg/kg or
from about 7.5 mg to about 38 mg AB-007.
[0221] For other human subject with different body weight or non
human subject, the amount of drug, e.g., AB-007 (loxistatin, E64d)
and/or loxistatin acid (E64c) and/or compositions of this invention
or compositions used to practice this invention, to be administered
can be readily calculated based on their exact body. In alternative
embodiments, these exemplary dosage forms are administered to
patients suffering from a cognitive loss, a dementia or
pre-dementia, AD (including preclinical, pre-symptomatic forms), VD
and/or Cognitive Dysfunction Syndrome (CDS) in a non-human animal.
While the suitable drug (e.g., AB-007) dosages employed in the
present invention can be described by the amount of drug (e.g.,
AB-007) per kg of body weight of the subject, the exact amount of a
compound of this invention, or loxistatin acid or AB-007, or
compositions used to practice this invention, to be administered
can be readily (routinely) determined by the skilled artisan
(clinician). In alternative embodiments, less (derivatized)
compositions of the invention can be used in a dosage or
formulation, because use of the (derivatized) compositions of the
invention (versus the "parent" AB-007 (E64d) and/or loxistatin acid
(E64c)) can result in increasing the effective concentration of a
composition of the invention as a drug, and can also increase its
half life in vivo.
[0222] In alternative embodiments, the invention provides kits
comprising a solid dosage form of a compound of this invention, a
composition used to practice this invention, or loxistatin acid or
AB-007; and also can comprise an instruction for administering said
dosage once daily for treatment of the intended disease or
condition (or pre-condition), including e.g., a cognitive loss, a
dementia or pre-dementia, AD, VD and/or Cognitive Dysfunction
Syndrome (CDS) in a non-human animal, wherein said solid dosage
form comprises between about 1 mg and about 400 mg of the drug,
e.g., loxistatin acid or AB-007.
[0223] In alternative embodiments, the solid dosage forms of the
invention (e.g., as a pill, tablet or lozenge), including those in
the kits or pharmaceutical compositions of the invention, comprise
between about 1 mg and about 250 mg of a compound of this
invention, a composition used to practice this invention, or AB-007
(loxistatin, E64d) and/or loxistatin acid (E64c); or comprise
between about 5 mg and about 150 mg of a compound of this
invention, a composition used to practice this invention, or AB-007
(loxistatin, E64d) and/or loxistatin acid (E64c); or comprise
between about 1 mg and about 75 mg of a compound of this invention,
a composition used to practice this invention, or AB-007
(loxistatin, E64d) and/or loxistatin acid (E64c).
[0224] In alternative embodiments, the kits pharmaceutical
compositions of the invention can comprise a solid dosage form
(e.g., as a pill, tablet or lozenge) comprising about 100 mg, about
150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, or
about 400 mg of a compound of this invention, a composition used to
practice this invention, or AB-007 (loxistatin, E64d) and/or
loxistatin acid (E64c). The solid dosage form (e.g., as a pill,
tablet or lozenge) also can comprise about 5 mg, about 10 mg, about
15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40
mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65
mg, about 70 mg, or about 75 mg of a compound of this invention, a
composition used to practice this invention, or AB-007 (loxistatin,
E64d) and/or loxistatin acid (E64c). The solid dosage form (e.g.,
as a pill, tablet or lozenge) can comprise about 5 mg, about 10 mg,
about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg,
about 40 mg, about 45 mg, or about 50 mg of a compound of this
invention, a composition used to practice this invention, or AB-007
(loxistatin, E64d) and/or loxistatin acid (E64c).
[0225] The invention provides methods for treating a cognitive
loss, a dementia or pre-dementia, AD, VD and/or Cognitive
Dysfunction Syndrome (CDS) in a non-human animal, comprising
administering to an individual, subject or patient in need thereof
an effective amount of a compound of this invention, a composition
used to practice this invention, or AB-007 (loxistatin, E64d)
and/or loxistatin acid (E64c), wherein said effective amount (e.g.,
as a solid dosage, such as a pill, tablet or lozenge) is between
about 0.1 mg and about 5.0 mg per kg of body weight of said
individual, subject or patient.
[0226] In alternative embodiments, an effective amount of a
compound of this invention, a composition used to practice this
invention, or AB-007 (loxistatin, E64d) and/or loxistatin acid
(E64c) (e.g., as a solid dosage, such as a pill, tablet or lozenge)
is between about 0.1 mg and about 2.0 mg per kg of body weight of
said individual, subject or patient; or is between about 0.1 mg and
about 1.0 mg per kg of body weight; or is about 0.1 mg, about 0.15
mg, about 0.2 mg, about 0.25 mg, about 0.3 mg, about 0.35 mg, about
0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg,
about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about
0.85 mg, about 0.9 mg, about 0.95 mg, or about 1.0 mg of a compound
of this invention, or AB-007 (loxistatin, E64d) and/or loxistatin
acid (E64c) per kg of body weight; or an effective amount of a
compound of this invention, a composition used to practice this
invention, or AB-007 (loxistatin, E64d) and/or loxistatin acid
(E64c) is about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg
or about 0.3 mg per kg of body weight; or an effective amount of a
compound of this invention, a composition used to practice this
invention, or AB-007 (loxistatin, E64d) and/or loxistatin acid
(E64c) is about 0.2 mg AB-007 per kg of body weight.
[0227] The invention provides methods for treating a cognitive
loss, a dementia or pre-dementia, AD, VD and/or Cognitive
Dysfunction Syndrome (CDS) in a non-human animal, comprising
administering to an individual, subject or patient in need thereof
an effective amount of a compound of this invention, a composition
used to practice this invention, or AB-007 (loxistatin, E64d)
and/or loxistatin acid (E64c), wherein said effective amount (e.g.,
as a solid dosage, such as a pill, tablet or lozenge) is between
about 1 mg and about 400 mg; or is a solid dosage form comprising
between about 1 mg and about 250 mg; or the solid dosage form
comprises between about 5 mg and about 150; or the solid dosage
form (e.g., as a pill, tablet or lozenge) comprises between about 1
mg and about 75; or the solid dosage form comprises about 5 mg,
about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg,
about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg,
about 60 mg, about 65 mg, about 70 mg, or about 75 mg.
[0228] The invention provides methods for treating a cognitive
loss, a dementia or pre-dementia, AD, VD and/or Cognitive
Dysfunction Syndrome (CDS) in a non-human animal, comprising
administering to an individual, subject or patient in need thereof
a compound of this invention, a composition used to practice this
invention, or AB-007 (loxistatin, E64d) and/or loxistatin acid
(E64c) administered as a daily oral dosage form (e.g., as a solid
dosage, such as a pill, tablet or lozenge).
[0229] In one embodiment the invention provides methods of slowing,
reversing, reducing or preventing the effects of .beta.-amyloid in
an individual, subject or patient comprising administering to an
individual, subject or patient in need thereof an effective amount
of a compound of this invention, a composition used to practice
this invention, or AB-007 (loxistatin, E64d) and/or loxistatin acid
(E64c), wherein said effective amount (e.g., as a solid dosage,
such as a pill, tablet or lozenge) is between about 0.1 mg and
about 5.0 mg per kg of body weight of said patient; or is between
about 0.1 mg and about 2.0 mg per kg of body weight; or is between
about 0.1 mg and about 1 mg per kg of body weight; or is about 0.1
mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, about 0.3 mg, about
0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg,
about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8
mg, about 0.85 mg, about 0.9 mg, about 0.95 mg, or about 1.0 mg per
kg of body weight; or is about 0.1 mg, about 0.15 mg, about 0.2 mg,
about 0.25 mg or about 0.3 mg per kg of body weight; or is about
0.2 mg per kg of body weight.
[0230] In one embodiment the invention provides methods of slowing,
reversing, reducing or preventing the effects of .beta.-amyloid, or
.beta.-amyloid accumulation, or .beta.-amyloid plaque formation, in
an individual, subject or patient comprising administering to said
individual, subject or patient an effective amount of a compound of
this invention, a composition used to practice this invention, or
AB-007 (loxistatin, E64d) and/or loxistatin acid (E64c) such that
the effects of .beta.-amyloid are reduced or prevented, wherein
said effective amount (e.g., as a solid dosage, such as a pill,
tablet or lozenge) is between about 1 mg and about 400 mg; or is
between about 1 mg and about 250 mg; or between about 5 mg and
about 150 mg; or between about 1 mg and about 75 mg; or about 5 mg,
about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg,
about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg,
about 60 mg, about 65 mg, about 70 mg, or about 75 mg. In one
embodiment the compound of this invention, a composition used to
practice this invention, or AB-007 (loxistatin, E64d) and/or
loxistatin acid (E64c) is administered as a daily oral dosage
(e.g., solid dosage, such as a pill, tablet or lozenge) form.
[0231] In one embodiment the invention provides methods of slowing,
reversing, reducing or preventing neuronal cell death in an
individual, subject or patient comprising administering to the
individual, subject or patient in need thereof an effective amount
of a compound of this invention, a composition used to practice
this invention, or AB-007 (loxistatin, E64d) and/or loxistatin acid
(E64c), wherein said effective amount is between about 0.1 mg and
about 5.0 mg per kg of body weight; or is between about 0.1 mg and
about 2.0 mg per kg of body weight; or is between about 0.1 mg and
about 1 mg per ckg of body weight of said patient; or is about 0.1
mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, about 0.3 mg, about
0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg,
about 0.6 mg, about 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8
mg, about 0.85 mg, about 0.9 mg, about 0.95 mg, or about 1.0 mg per
kg of body weight; or is about 0.1 mg, about 0.15 mg, about 0.2 mg,
about 0.25 mg or about 0.3 mg per kg of body weight; or is about
0.2 mg per kg of body weight.
[0232] In one embodiment the invention provides methods of slowing,
reversing, reducing or preventing neuronal cell death in an
individual, subject or patient comprising administering to said
individual, subject or patient an effective amount of a compound of
this invention, a composition used to practice this invention, or
AB-007 (loxistatin, E64d) and/or loxistatin acid (E64c) such that
neuronal cell death is slowed, reversed, reduced and/or prevented,
wherein said effective amount is between about 1 mg and about 400
mg; or is (e.g., as a solid dosage form) between about 1 mg and
about 250 mg; or is about 5 mg and about 150 mg; or is between
about 1 mg and about 75 mg; or is about 5 mg, about 10 mg, about 15
mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40
mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65
mg, about 70 mg, or about 75 mg. In one embodiment, the compound of
this invention, a composition used to practice this invention, or
AB-007 (loxistatin, E64d) and/or loxistatin acid (E64c) is
administered as a daily oral dosage (e.g., solid dosage, such as a
pill, tablet or lozenge) form.
Diagnosis and Assessment of Dementia and AD
[0233] In alternative embodiments, any method, procedure or
protocol, including those based on one or more psychological,
cognitive, imaging, biomarker and/or genetic parameters and/or
measurements, are used to predict, detect, monitor the progression
of and/or monitor the effectiveness of a prophylactic or clinical
treatment (e.g., using a composition or dosage formulation of this
invention) for VD, AD and/or any other neuro-dementing disease.
This monitoring and assessment allows the skilled artisan to
determine if a modification of dosage and/or treatment regimen is
needed or desired to maximize effectiveness of a composition of
this invention for a particular individual or patient.
[0234] For example, in alternative embodiments the following
diagnostics can be used alone or in combination: determinations for
genetic risk factors or mutations (mainly for FAD cases, but also
for sporadic AD diagnostics); neuroimaging methods; diagnostics
based on biochemical/biological markers. For example, in humans a
gene variant APOE3 is associated with a neutral risk of developing
the disease, while the APOE4 variant is associated with a high
risk. Thus, an individual having the APOE4 variant is a candidate
individual to have administered a composition of this invention
and/or a method of this invention.
[0235] Risk of developing Alzheimer's appears to be slightly higher
if a first-degree relative such as a parent, sister or brother has
the disease. Thus, in alternative embodiments, an individual having
a first-degree relative with AD, VD, or a dementia or pre-dementia
is a candidate individual to have administered a composition of
this invention and/or a method of this invention.
[0236] Researchers have found that an increase of 5 micromoles of
homocysteine per liter of blood increases the risk of AD by 40%.
People with higher homocysteine levels in blood had nearly double
the risk of disease AD and other dementias in relation to people
with low blood homocysteine. Thus, in alternative embodiments, an
individual having higher homocysteine levels in blood is a
candidate individual to have administered a composition of this
invention and/or a method of this invention.
[0237] Additionally, noting the invention is not limited by any
particular mechanism of action, because the compositions and dosage
formulations of the invention can bind to homocysteine and its
precursor cystathionine, they also are effective for lowering blood
homocysteine levels. Thus, in alternative embodiments, the
invention provides methods for lowering body or blood homocysteine
or cystathionine levels by administration of a composition and/or
dosage formulation of the invention to treat, e.g., any conditions
initiated or exacerbated or associated with an elevated
homocysteine level, e.g., AD and other dementias. For example,
elevated levels of homocysteine (>10 micromoles/liter) in the
blood may be associated with atherosclerosis (e.g., or
pre-sclerotic plaques) as well as an increased risk of heart
attacks, strokes, blood clot formation, in addition to Alzheimer's
disease. Thus, in alternative embodiments, the invention provides
methods for decreasing the risk of heart attacks, strokes, blood
clot formation, AD and dementia by administration of a composition
and/or dosage formulation of the invention.
[0238] In alternative embodiments, criteria to clinically diagnose
AD, VD, or a dementia or pre-dementia and/or to monitor (assess)
the effectiveness of a composition of the invention or a dosage
formulation of the invention comprises the DSM-IIIR criteria and
the NINCDS-ADRDA criteria (National Institute of Neurological and
Communicative Disorders and Stroke (NINCDS) and the Alzheimer's
Disease and Related Disorders Association (ADRDA); see McKhann et
al., Neurology 34:939-944, 1984). The criteria for diagnosis of AD
under DSM-IIIR can include (1) dementia, (2) insidious onset with a
generally progressive deteriorating course, and (3) exclusion of
all other specific causes of dementia by history, physical
examination, and laboratory tests. Within the context of the
DSM-IIIR criteria, dementia is understood to involve "a
multifaceted loss of intellectual abilities, such as memory,
judgment, abstract thought, and other higher cortical functions,
and changes in personality and behavior." (DSM-IIR, 1987).
[0239] In alternative embodiments, NINCDS-ADRDA criteria are used;
the NINCDS-ADRDA sets forth three categories of AD, including
"probable," "possible," and "definite" AD. Clinical diagnosis of
"possible" AD may be made on the basis of a dementia or
pre-dementia syndrome, in the absence of other ncurologic,
psychiatric or systemic disorders sufficient to cause dementia.
Criteria for the clinical diagnosis of "probable" AD can include
(a) dementia established by clinical examination and documented by
a test such as the Mini-Mental test (Foldstein et al., J. Psych.
Res. 12:189-198, 1975); (b) deficits in two or more areas of
cognition; (c) progressive worsening of memory and other cognitive
functions; (d) no disturbance of consciousness; (e) onset between
ages 40 and 90, most often after age 65; and (f) absence of
systemic orders or other brain diseases that could account for the
dementia.
[0240] In alternative embodiments, criteria for definite diagnosis
of AD include histopathologic evidence obtained from a biopsy, or
after autopsy. Since confirmation of definite AD requires
histological examination from a brain biopsy specimen (which is
often difficult to obtain), it is rarely used for early diagnosis
of AD.
[0241] In alternative embodiments, neuropathologic diagnosis of AD
is used. Presence and numbers of plaques and tangles in the
neurocortex (frontal, temporal, and parietal lobes), hippocampus
and amygdala are analyzed (Khachaturian, Arch. Neurol.
42:1097-1105; Esiri, "Anatomical Criteria for the Biopsy diagnosis
of Alzheimer's Disease," Alzheimer's Disease, Current Research in
Early Diagnosis, Becker and Giacobini (eds.), pp. 239-252,
1990).
[0242] In alternative embodiments, quantitative
electroencephalographic analysis (EEG) is used to diagnose AD. This
method employs Fourier analysis of the beta, alpha, theta, and
delta bands (Riekkinen et al., "EEG in the Diagnosis of Early
Alzheimer's Disease," Alzheimer's Disease, Current Research in
Early Diagnosis, Becker and Giacobini (eds.), pp. 159-167, 1990)
for diagnosis of AD.
[0243] In alternative embodiments, quantifying the degree of neural
atrophy is used. Such atrophy can be a consequence of AD. Examples
of these methods include computed tomographic scanning (CT), and
magnetic resonance imaging (MRI) (Leedom and Miller, "CT, MRI, and
NMR Spectroscopy in Alzheimer's Disease," Alzheimer's Disease,
Current Research in Early Diagnosis, Becker and Giacobini (eds.),
pp. 297-313, 1990).
[0244] In alternative embodiments, decreased cerebral blood flow or
metabolism in the posterior temporoparietal cerebral cortex is used
to diagnose VD or AD. Decreased blood flow or metabolism by
positron emission tomography (PET) is measured, e.g., see Parks and
Becker, "Positron Emission Tomography and Neuropsychological
Studies in Dementia," Alzheimer's Disease's, Current Research in
Early Diagnosis, Becker and Giacobini (eds.), pp. 315-327, 1990.
Single photon emission computed tomography (SPECT) also can be
used, see e.g., Mena et al., "SPECT Studies in Alzheimer's Type
Dementia Patients," Alzheimer's Disease, Current Research in Early
Diagnosis, Becker and Giacobini (eds.), pp. 339-355, 1990). Xenon
inhalation methods also can be used, see, e.g., Jagust et al.,
Neurology 38:909-912; Prohovnik et al., Neurology 38:931-937; and
Waldemar et al., Senile Dementias: II International Symposium, pp.
399407, 1988.
[0245] In alternative embodiments, AD can be immunologically
diagnosed, see e.g., Wolozin, "Immunochemical Approaches to the
Diagnosis of Alzheimer's Disease," Alzheimer's Disease, Current
Research in Early Diagnosis, Becker and Giacobini (eds.), pp.
217-235, 1990. Wolozin and coworkers (Wolozin et al., Science
232:648-650, 1986) produced a monoclonal antibody "Alz50," that
reacts with a 68-kDa protein "A68," which is expressed in the
plaques and neuron tangles of patients with Alzheimer's disease.
Using the antibody Alz50 and Western blot analysis, A68 was
detected in the cerebral spinal fluid (CSF) of some Alzheimer's
patients and not in the CSF of normal elderly patients (Wolozin and
Davies, Ann. Neurol. 22:521-526, 1987). In another embodiment,
antibodies against beta-amyloid (e.g., as described in U.S. Pat.
Appl. No. 20090028869) can be used to diagnose or monitor (assess)
the effectiveness of a prophylactic or clinical treatment using a
composition of this invention.
[0246] In alternative embodiments, neurochemical markers of AD are
used, including neurochemical markers comprising reduced levels of
acetylcholinesterase (Giacobini and Sugaya, "Markers of Cholinergic
Dysfunction in Alzheimer's Disease," Alzheimer's Disease, Current
Research in Early Diagnosis, Becker and Giacobini (eds.), pp.
137-156, 1990), reduced somatostatin (Tamming a et al., Neurology
37:161-165, 1987), a negative relation between serotonin and
5-hydroxyindoleacetic acid (Volicer et al., Arch Neurol.
42:127-129, 1985), greater probenecid-induced rise in homovanyllic
acid (Gibson et al., Arch. Neurol. 42:489-492, 1985) and/or reduced
neuron-specific enolase (Cutler et al., Arch. Neurol. 43:153-154,
1986).
[0247] In alternative embodiments, various types of dementia can be
diagnosed and monitored, and the progression of the disease over an
extended period of time assessed, using (i) a memory assessment,
(ii) an extensive neuropyschological exam, (iii) an examination by
a geriatric neurologist and/or (iv) MRI imaging of the brain.
Disease progression can be documented by changes in these
parameters over time. In some embodiments, changes in the
parameters of at least one of these assessments can be used to
assess the efficacy of a composition of this invention in an
individual, patient or subject over time.
[0248] In alternative embodiments, a memory assessment is used,
e.g., adult patients with complaint of short term memory and/or
cognitive decline, whether self-referred or directed from a
clinician on the suspicion of a possible or probable memory
disorder or dementia, are evaluated (i) memory assessment (ii) an
extensive neuropsychological exam, (iii) an examination by a
geriatric neurologist and/or (iv) MRI imaging of the brain are
performed. A neuropsychology assessment captures a broad inventory
of cognitive function which aids in determining the array and
severity of deficits. These include assessments of judgment,
insight, behavior, orientation, executive Control, general
intellectual functioning, visual-spatial function, memory and/or
new learning ability. Depression, if present, is identified. A
neurological evaluation can captures the history of cognitive
alteration as well as the general medical history, and typically a
complete neurological exam is performed. A neurological examination
can also comprise laboratory studies to exclude reversible causes
of dementia including Vitamin B12, Folate, Basic Metabolic Profile,
CBC, TSH, ALT, AST, C-reactive protein, serum homocysteine, and
RPR.
[0249] In alternative embodiments, compositions of the invention
are used to treat "pre-dementia" patients, where the first symptoms
are often mistaken as related to aging or stress. Detailed
neuropsychological testing can reveal mild cognitive difficulties
up to eight years before a person fulfills the clinical criteria
for diagnosis of AD. These early symptoms can affect the most
complex daily living activities. The most noticeable deficit is
memory loss, which shows up as difficulty in remembering recently
learned facts and inability to acquire new information. Subtle
problems with the executive functions of attentiveness, planning,
flexibility, and abstract thinking, or impairments in semantic
memory (memory of meanings, and concept relationships), can also be
symptomatic of the early stages of AD. Apathy can be observed at
this stage, and remains the most persistent neuropsychiatric
symptom throughout the course of the disease. The preclinical stage
of the disease has also been termed mild cognitive impairment, but
whether this term corresponds to a different diagnostic stage or
identifies the first step of AD is a matter of dispute. In
alternative embodiments, compositions of the invention are used to
treat individuals with mild cognitive impairment and/or
pre-dementia.
Animal Cognitive Dysfunction Syndrome (CDS)
[0250] In alternative embodiments, the invention provides
pharmaceutical compositions comprising dosage forms and
formulations of the invention for administration to human and
non-human subjects, e.g., including e.g. domestic, farm,
experimental and zoo animals, including e.g., dogs and cats. In
alternative embodiments, compositions and methods of the invention
are used to treat, prevent, slow the progress of or ameliorate a
Cognitive Dysfunction Syndrome (CDS) in a non-human animal, e.g.,
dogs and cats. In one embodiment, CDS includes Canine or Cat
(feline) Cognitive Dysfunction (CCD), which is sometimes referred
to as "old dog syndrome", "brain aging", "doggie dementia" or
"senility".
[0251] Animal CDS can have a pathology mimicking Alzheimer's
disease (AD) pathology. Dogs with CDS accumulate the toxic beta
amyloid protein seen in the brains of patients with AD. The
deposits in dogs occur in similar brain regions as humans, and the
extent of deposition correlates with the level of cognitive
impairment, as in patients with AD. In dogs, disturbances in four
main behavioral categories emerge: disorientation seen in daily
walks or at home; impaired interaction with owners; sleep/wake
cycle disturbances with barking and pacing at nights and loss of
continence. These areas are often impaired in AD patients, although
of course, the manifestations are different.
[0252] Much like in people, the risk for cognitive dysfunction in
dogs and cats increases with age. In dogs as in people, there is a
progression from mild cognitive impairment to frank symptoms of
dementia. Dogs nine years and older and cats over 12 are at risk
for CDS. Over a quarter of 11 to 12 year old dogs (28 percent) and
nearly three quarters (68 percent) of 15 to 16 year old dogs are
impaired. Female gender places both dogs and women at higher risk
for cognitive impairment. Spayed female dogs are more at risk for
CDS just as women who undergo early menopause or hysterectomies
have a higher risk for AD. Accordingly, compositions and methods of
the invention are designed for and formulated for administration to
dogs starting at between about ages 9 to 11.
[0253] The evaluation for animal Cognitive Dysfunction Syndrome
(CDS), as for AD, involves screening for reversible causes of
cognitive impairment such as infections, endocrine problems and
other ailments. Brain MRIs in CDS reveal brain shrinkage or atrophy
and an increase in the size of the brain's ventricular cavities,
parallel to findings in people with AD. Brain MRIs performed for
behavioral changes may reveal large or small strokes. Other common
conditions that exacerbate CDS and predispose to strokes include
untreated hypertension, high cholesterol levels and poorly
controlled diabetes.
[0254] In alternative embodiments, the invention provides
formulations and dosages for slowing, reversing, reducing or
preventing animal Cognitive Dysfunction Syndrome (CDS).
Formulations and dosages for administering an effective amount of a
compound comprising an inhibitor of a cysteine protease, or a
cathepsin, a caspase or a calpain, or a cathepsin L or a cathepsin
B, or a cathepsin F, H, K, L1, L2, O, S, W, X or Z, which includes
but is not limited to compositions of this invention, for animal
Cognitive Dysfunction Syndrome (CDS) are discussed above.
[0255] In alternative embodiments, the invention provides
formulations and dosages comprising a composition of the invention,
or a dosage formulation of the invention, in combination with
selegiline (e.g., selegiline hydrochloride) or deprenyl, or
ANIPRYL.TM., which is one of the top medications for canine
dementia. Selegiline (or deprenyl) also is used to treat
Alzheimer's and Parkinson's in humans. Anipryl works by increasing
the level of an essential neurotransmitter called dopamine. Anipryl
has shown to reverse some of the changes associated with canine
dementia and it improves the behavior in about 75% of affected dogs
in one month. In some cases, it may take up to 60 days.
Combination Formulations
[0256] In alternative embodiments, the invention provides
pharmaceutical compositions comprising compositions, dosage forms
and formulations of the invention further comprising at least one
other (e.g., at least one additional, different) pharmaceutical
composition, dosage form or formulation used to treat or
ameliorate, or treat the symptoms of, or be palliative for: a
cognitive dysfunction or a loss of cognition, a dementia or a
pre-dementia, Alzheimer's disease (AD), Vascular Dementia (VD),
and/or a Cognitive Dysfunction Syndrome (CDS), in humans or in a
non-human animal. The at least one additional, different
pharmaceutical composition, dosage form or formulation can be any
known treatment or palliative for a dementia, AD, VD or CDS.
[0257] For example, in alternative embodiments, the at least one
other (e.g., at least one additional, different) pharmaceutical
composition, dosage form or formulation comprises a selegiline
(e.g., sclegiline hydrochloride) or deprenyl, or ANIPRYL.TM..
[0258] In alternative embodiments, the at least one other (e.g., at
least one additional, different) pharmaceutical composition, dosage
form or formulation comprises an acetylcholinesterase inhibitor
(AChEI) or anti-cholinesterase, e.g., a donepezil (ARICEPT.TM.); a
carbamate; edrophonium or comparable reversible
acetylcholinesterase inhibitor (e.g., TENSILON.TM., ENLON.TM.,
REVERSOL.TM.); a neostigmine (e.g., PROSTIGMIN.TM.,
VAGOSTIGMIN.TM.); a galantamine (e.g., NIVALIN.TM., RAZADYNE.TM.,
RAZADYNE ER.TM., REMINYL.TM.); a rivastigmine (e.g., EXELON), and
any equivalent or combination thereof.
[0259] In alternative embodiments, the at least one other (e.g., at
least one additional, different) pharmaceutical composition, dosage
form or formulation comprises a memantine or comparable blocker of
NMDA glutamate receptors (including e.g., AXURA.TM., AKATINOL.TM.,
NAMENDA.TM., EBIXA.TM., ABIXA.TM., MEMOX.TM.), any equivalent or
combination thereof.
[0260] In alternative embodiments, the at least one other (e.g., at
least one additional, different) pharmaceutical composition, dosage
form or formulation comprises a nutritional supplement or a
vitamin, e.g., vitamin E, vitamin B12 or a folic acid supplement,
or any equivalent or combination thereof.
[0261] In alternative embodiments, the at least one other (e.g., at
least one additional, different) pharmaceutical composition, dosage
form or formulation comprises a pain treatment or pain palliative
or an anti-inflammatory drug, e.g., an ibuprofen (e.g., ADVIL.TM.,
MOTRIN.TM.), naproxen sodium (ALEVE.TM.), indomethacin
(INDOCIN.TM.), and any equivalent or combination thereof. The
anti-inflammatory drug can comprise a non-steroidal
anti-inflammatory drug (a NSAID), e.g., a cyclooxygenase (COX) (or
prostaglandin synthase) inhibitor, e.g., an etodolac (e.g.,
LODINE.TM., LODINE SR.TM. or ECCOXOLAC.TM.), naproxen, celecoxib,
rofecoxib, etoricoxib, valdecoxib, parecoxib, nabumetone,
diclofenac, lumiracoxib, or equivalent. The pain palliative can
comprise a neuropathic pain analgesic such as gabapentin or
pregabalin.
[0262] In alternative embodiments, the at least one other (e.g., at
least one additional, different) pharmaceutical composition, dosage
form or formulation comprises a tarenflurbil, or R-flurbiprofen
(the single enantiomer of the racemate NSAID flurbiprofen), e.g.,
FLURIZAN.TM..
[0263] In alternative embodiments, the at least one other (e.g., at
least one additional, different) pharmaceutical composition, dosage
form or formulation comprises an (S)-1-[N-(4-chlorobenzyl)
succinamoyl] pyrrolidine-2-carbaldehyde (aka, ONO-1603), a prolyl
endopeptidase inhibitor and anti-dementia drug.
[0264] In alternative embodiments, two or more or all of the drugs
of a therapeutic, ameliorative and/or pallative combination (which
includes at least one pharmaceutical composition, dosage form or
formulation of this invention) are packaged individually, or are
packaged together, or packaged in any combination, in a single
package, a plurality of packages or packettes, or a blister packet,
lidded blister or blister card or packets, or a shrink wrap.
[0265] In alternative embodiments, the invention provides a
multi-ingredient kit comprising two or more or all of the drugs of
a therapeutic, ameliorative and/or pallative combination (which
includes at least one pharmaceutical composition, dosage form or
formulation of this invention).
[0266] In alternative embodiments, the invention provides an
ingredient or ingredients (which includes at least one
pharmaceutical composition, dosage form or formulation of this
invention) in a multi-ingredient kit of the invention separated by
physical compartmentalization; e.g. in separate compartments that
are part of a kit, where the kit is a multi-compartment kit. In
alternative embodiments, separate compartments, e.g., as found in a
"blister pack" type of packaging, contain different ingredients
(including at least one pharmaceutical composition, dosage form or
formulation of this invention)
[0267] In alternative embodiments, the invention provides a
composition or product of manufacture formulated or made as a
multiparticulate and/or a solid dispersion formulation, e.g., as
described in, e.g., U.S. Patent App. Pub. No. 20080118560, e.g.,
comprising a hydrophobic matrix former which is a water-insoluble,
non-swelling amphiphilic lipid; and a hydrophilic matrix former
which is a meltable, water-soluble excipient. In one embodiment,
the pharmaceutical composition, dosage form or formulation of the
invention, or any composition, drug combination or product of
manufacture of the invention, is/are contained in tablets, pills,
capsules, troches, and the like comprising any combination of a
binder, e.g., as a starch, polyvinyl pyrrolidone, gum tragacanth or
gelatin; a filler, such as microcrystalline cellulose or lactose; a
disintegrating agent, such as crospovidone, sodium starch
glycolate, corn starch, and the like; a lubricant, such as
magnesium stearate, stearic acid, glyceryl behenate; a glidant,
such as colloidal silicon dioxide and talc; a sweetening agent,
such as sucrose or saccharin, aspartame, acesulfame-K; and/or
flavoring agent, such as peppermint, methyl salicylate, or orange
flavoring. When the dosage unit form is a capsule, it also can
comprise a liquid carrier, such as a fatty oil.
[0268] In alternative embodiments, a composition or product of
manufacture of the invention comprises (or is contained or packaged
in) unit dosage formulations having a coating, e.g., a coat
comprising a sugar, shellac, sustained and/or other enteric coating
agents, or any pharmaceutically pure and/or nontoxic agents.
[0269] In alternative embodiments, a composition or product of
manufacture of the invention comprises (or is contained or packaged
in) unit dosage formulations, wherein each different compound of
the composition or product of manufacture is contained in a
different layer of a pill, tablet or capsule, e.g., as described in
U.S. Pat. No. 7,384,653, e.g., having an outer base-soluble layer
and an inner acid-soluble layer. In alternative embodiments, a
composition or product of manufacture of the invention comprises
(or is contained or packaged in) unit dosage formulations, wherein
each different compound of the composition or product of
manufacture is contained in a liquid or a gel of different
viscosity, e.g., described in U.S. Patent App. Pub. No.
20050214223. In alternative embodiments, a composition or product
of manufacture of the invention comprises (or is contained or
packaged in) unit dosage formulations having reduced abuse
potential, e.g., as described in U.S. Patent App. Pub. No.
20040228802, e.g., comprising a bittering agent, a bright
deterrent/indicator dye, or a fine insoluble particulate
matter.
[0270] In alternative embodiments of the invention, a drug
combination of the invention is formulated, packaged or designed
for drug regimen compliance of a particular patient population,
e.g., an Alzheimer's disease patient population, or a group of
patients with dementia, or a pediatric or geriatric population, or
a mentally compromised patient population. In alternative
embodiments of the invention drug combination(s) of the invention
are formulated, packaged or designed for drug regimen compliance of
a particular patient population, e.g., a patient population having
mild or severe mental retardation, slow cognition, dementia,
senility, Alzheimer's disease, traumatic brain injury, chemical
brain damage, mental diseases (e.g., dissociative disorder,
obsessive-compulsive disorder, delusional disorder, schizophrenia,
mania, panic disorder, depression, dyslexia, any learning
disability and the like) posttraumatic stress disorder, traumatic
war neurosis, post-traumatic stress syndrome (PTSS), physical
disability (e.g., blindness).
[0271] In alternative embodiments the invention provides a blister
pack or a plurality of blister packettes, a blister package, a
lidded blister or a blister card or packet, a clamshell, a tray or
a shrink wrap of the invention, or the paper, plastic or cellophane
package or a plurality of packettes, wherein a drug combination
(which includes at least one pharmaceutical composition, dosage
form or formulation of this invention) is formulated, packaged or
designed for drug regimen compliance of a particular patient
population, e.g., an Alzheimer's disease patient population, or a
group of patients with dementia, a pediatric or geriatric
population, or a physically challenged or a mentally compromised
patient population. In alternative embodiments, the invention
provides blister pack or a plurality of blister packettes, a
blister package, a lidded blister or a blister card or packet, a
clamshell, a tray or a shrink wrap of the invention, or a paper,
plastic or cellophane package or a plurality of packettes
comprising a drug combination (which includes at least one
pharmaceutical composition, dosage form or formulation of this
invention) formulated, packaged or designed for drug regimen
compliance of a particular patient population having mild or severe
mental retardation, slow cognition, dementia, senility, Alzheimer's
disease, traumatic brain injury, chemical brain damage, mental
diseases (e.g., dissociative disorder, obsessive-compulsive
disorder, delusional disorder, schizophrenia, mania, panic
disorder, depression, dyslexia, any learning disability and the
like) post-traumatic stress disorder, traumatic war neurosis,
post-traumatic stress syndrome (PTSS), physical disability (e.g.,
blindness).
[0272] In alternative embodiments the invention provides a food or
food supplement and a pharmaceutical composition, dosage form or
formulation of this invention; or, a food or food supplement
comprising a therapeutic combination or a drug combination (the
combination including at least one pharmaceutical composition,
dosage form or formulation of this invention). In alternative
embodiments the invention provides feed or feed supplements
comprising a pharmaceutical composition, dosage form or formulation
of this invention; or, a therapeutic combination of the invention.
In alternative embodiments the invention provides nutraceuticals
comprising a pharmaceutical composition, dosage form or formulation
of this invention; or, a therapeutic combination of the
invention.
Treating Traumatic Brain Injuries
[0273] In alternative embodiments, the invention provides
pharmaceutical compositions comprising compositions, dosage forms
and formulations of the invention for treating traumatic brain
injury (due to, e.g., trauma, infection, surgery, and the like) and
related (e.g., as sequelae or side effects) post-traumatic
injuries, including traumatic war neurosis, post traumatic stress
disorder (PTSD) or post-traumatic stress syndrome (PTSS), and
cognitive, learning or memory impairments resulting therefrom. In
alternative embodiments, the invention provides methods for
treating, preventing, slowing the progression of, reversing or
ameliorating traumatic central nervous system (CNS) (e.g., brain)
injury (due to, e.g., trauma, infection, surgery, and the like) and
related (e.g., as sequelae or side effects) post-traumatic
injuries, including traumatic war neurosis, post traumatic stress
disorder (PTSD) or post-traumatic stress syndrome (PTSS), and
cognitive, learning or memory impairments resulting therefrom.
[0274] While the invention is not limited by any particular
mechanism of action, the compositions, dosage forms and
formulations of the invention can work by inhibiting, decreasing or
slowing the activity of a cysteine protease, e.g., a cathepsin,
e.g., cathepsin B.
[0275] The following examples are provided to further illustrate
the invention but not to limit its scope. Other variants of the
invention will be readily apparent to one of ordinary skill in the
art.
EXAMPLES
[0276] Standard procedures and chemical transformation and related
methods are well known to one skilled in the art, and such methods
and procedures have been described, for example, in standard
references such as Fiesers' Reagents for Organic Synthesis, John
Wiley and Sons, New York, N.Y., 2002; Organic Reactions, vols.
1-83, John Wiley and Sons, New York, N.Y., 2006; March J. and Smith
M., Advanced Organic Chemistry, 6th ed., John Wiley and Sons, New
York, N.Y.; and Larock R. C., Comprehensive Organic
Transformations, Wiley-VCH Publishers, New York, 1999. All texts
and references, patents and patent applications cited herein are
expressly incorporated by reference in their entirety.
[0277] Reactions using compounds having functional groups may be
performed on compounds with functional groups that may be
protected. A "protected" compound or derivatives means derivatives
of a compound where one or more reactive site or sites or
functional groups are blocked with protecting groups. Protected
derivatives are useful in the preparation of the compounds of the
present invention or in themselves; the protected derivatives may
be the biologically active agent. An example of a comprehensive
text listing suitable protecting groups may be found in T. W.
Greene, Protecting Groups in Organic Synthesis, 3rd edition, John
Wiley & Sons, Inc. 1999.
[0278] In alternative embodiments, compounds of Formula I can be
prepared as shown in the following protocols, or schemes; and the
invention encompasses methods for practicing all of the following
protocols and schemes, including routine variations thereof.
Scheme I
[0279] In alternative embodiments, compounds of the invention,
including compounds encompassed by Formula I, can be prepared using
Scheme I, starting with a leucine derivative and an analog of
isoamyl amine. Reaction of the intermediate with an epoxide yielded
a compound of Formula I.
##STR00017##
Example 1. Preparation of Compound 1A
##STR00018##
[0281] In alternative embodiments, compounds of the invention,
including compounds of Formula I, can be prepared as shown in the
following protocol or scheme; and the invention encompasses methods
comprising this scheme and all routine variations thereof:
[0282] N-(tert-butyloxycarbonyl)-L-leucine-d.sub.3 (methyl-d.sub.3)
hydrate (250 mg; 0.99 mmol; C/D/N Isotopes), 1-hydroxybenzotriazole
hydrate (197 mg; 1.29 mmol), and isoamyl amine (95 mg; 1.09 mmol)
were dissolved in CH.sub.2Cl.sub.2 (10 mL) at room temperature
under nitrogen. With stirring,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (288
mg; 1.50 mmol) was added. After stirring for three hours at room
temperature, the reaction was diluted with CH.sub.2Cl.sub.2 (80 mL)
and extracted with saturated NaHCO.sub.3 (2.times.) and brine
(1.times.). The organic layer was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide 313 mg of a
waxy solid. This solid was dissolved in methanol (5 mL) and
p-toluene sulfonic acid hydrate (237 mg; 1.25 mmol) was added with
stirring. The solution was heated to 70.degree. C. for four hours.
After cooling, the reaction was evaporated to dryness and the
residue treated with ether and hexanes. Removal of the volatiles
provided a white foam. To this material was added (2S,
3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (160 mg; 1 mmol;
Peptech) and CH.sub.2Cl.sub.2 (10 ml) to provide a homogeneous
solution. The solution was cooled to 0.degree. C. (ice-water bath)
under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (418 mg; 1.1 mmol) followed by diisopropylethyl
amine (358 uL; 2 mmol) were added to the reaction solution. The
resulting reaction was stirred at 0.degree. C. for one hour and
another hour at room temperature. The reaction was diluted with
CH.sub.2Cl.sub.2 (100 ml) and extracted with saturated NaHCO.sub.3
(2.times.) and brine (1.times.). The organic layer was dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated to provide a
yellow solid. The crude product was filtered through a silica gel
plug eluting with ethyl acetate-hexane (1:9) followed by ethyl
acetate-hexane (1:1). Concentration of the 1:1 ethyl acetate-hexane
elution fractions provided the desired product as a white solid
(295 mg; 0.86 mmol; 86%). Recrystallization from diisopropyl
ether-ethanol yielded Compound 1A, mp 122-123.degree. C. .sup.1H
NMR (CDCl.sub.3, 500 MHz) .delta.6.71 (d, J=8.6 Hz, 1H), 6.12-6.14
(m, 1H), 4.36-4.41 (m, 1H), 4.21-4.29 (m, 2H), 3.67 (d, J=1.4 Hz,
1H), 3.46 (d, J=1.7 Hz, 1H), 3.20-3.28 (m, 2H), 1.56-1.64 (m, 2H),
1.50-1.55 (m, 2H), 1.38 (q, J=7.0 Hz, 2H), 1.30 (t, J=7.0 Hz, 3H),
0.89-0.92 (m, 9H). .sup.13C NMR (CDCl.sub.3, 125 MHz) 171.02,
166.71, 166.22, 62.53, 53.99, 53.11, 51.62, 41.41, 38.49, 38.21,
26.06, 24.81, 22.92, 22.62, 22.58, 22.32, 14.24, LCMS (ESI): m/z
calculated (calcd) for C.sub.17H.sub.27D.sub.3N.sub.2O.sub.5
345.23, [M+H].sup.+ found 346.2.
Example 2. Preparation of Compound 1B
##STR00019##
[0284] In alternative embodiments, compounds of the invention,
including compounds of Formula I, can be prepared as shown in the
following protocol or scheme; and the invention encompasses methods
comprising this scheme and all routine variations thereof:
[0285] The ethyl ester Compound 1A (35 mg; 0.1 mmol) was dissolved
in ethanol (2 mL). The stirred solution was cooled to 0.degree. C.
(ice-water bath) and treated with a solution of 85% KOH in ethanol
(70 uL; 0.11 mmol). The resulting solution was stirred at 0.degree.
C. for 20 minutes and at room temperature for 10 minutes. The
reaction was concentrated to dryness and the residue partitioned
between water and ethyl acetate. The layers were separated and the
aqueous extracted once more with ethyl acetate. The aqueous layer
was acidified to pH 2 with 1N HCl and extracted with ethyl acetate
(3.times.). The combined organic layers were dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide a white
solid, Compound 1B (36 mg; 96%). LCMS (ESI): m/z calcd for
C.sub.15H.sub.23D.sub.3N.sub.2O.sub.5 317.20, [M+H].sup.+ found
318.2.
Example 3. Preparation of Compound 2A
##STR00020##
[0287] In alternative embodiments, compounds of the invention,
including compounds of Formula I, can be prepared as shown in the
following protocol or scheme; and the invention encompasses methods
comprising this scheme and all routine variations thereof:
N-(tert-butyloxycarbonyl)-L-leucine-d.sub.7 (isopropyl-d.sub.7)
hydrate (250 mg; 0.98 mmol; C/D/N Isotopes), 1-hydroxybenzotriazole
hydrate (195 mg; 1.27 mmol), and isoamyl amine (94 mg; 1.07 mmol)
were dissolved in CH.sub.2Cl.sub.2 (10 mL) at room temperature
under nitrogen. With stirring,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (282
mg; 1.47 mmol) was added. After stirring for three hours at room
temperature, the reaction was diluted with CH.sub.2Cl.sub.2 (80 mL)
and extracted with saturated NaHCO.sub.3 (2.times.) and brine
(1.times.). The organic layer was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide 315 mg of a
white semi-solid. This material was dissolved in methanol (5 mL)
and p-toluene sulfonic acid hydrate (237 mg; 1.25 mmol) was added
with stirring. The solution was heated to 70.degree. C. for four
hours. After cooling, the reaction was evaporated to dryness and
the residue treated with ether and hexanes. Removal of the
volatiles provided a white foam. To this material was added (2S,
3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (160 mg; 1 mmol;
Peptech) and CH.sub.2Cl.sub.2 (10 ml) to provide a homogeneous
solution. The solution was cooled to 0.degree. C. (ice-water bath)
under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (418 mg; 1.1 mmol) followed by diisopropylethyl
amine (358 uL; 2 mmol) were added to the reaction solution. The
resulting reaction was stirred at 0.degree. C. for one hour and
another hour at room temperature. The reaction was diluted with
CH.sub.2Cl.sub.2 (100 ml) and extracted with saturated NaHCO.sub.3
(2.times.) and brine (1.times.). The organic layer was dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated to provide a
yellow solid. The crude product was filtered through a silica gel
plug eluting with ethyl acetate-hexane (1:9) followed by ethyl
acetate-hexane (1:1). Concentration of the 1:1 ethyl acetate-hexane
elution fractions provided the desired product as a white solid
(314 mg; 0.9 mmol; 90%). Recrystallization from diisopropyl ether
yielded Compound 2A, mp 122-123.degree. C. .sup.1H NMR (CDCl.sub.3,
500 MHz) .delta.6.68 (d, J=8.6 Hz, 1H), 6.08-6.10 (m, 1H),
4.35-4.40 (m, 1H), 4.21-4.29 (m, 2H), 3.67 (d, J=1.8 Hz, 1H), 3.45
(d, J=1.7 Hz, 1H), 3.20-3.30 (m, 2H), 1.56-1.65 (m, 2H), 1.48-1.52
(m, 1H), 1.38 (q, J=7.3 Hz, 2H), 1.31 (t, J=7.2 Hz, 3H), 0.89-0.91
(d, J=6.6 Hz, 6H). .sup.13C NMR (CDCl.sub.3, 125 MHz)
.delta.171.02, 166.70, 166.22, 62.54, 53.99, 53.11, 51.61, 41.23,
38.49, 38.21, 28.51 (weak), 26.06, 24.11 (m, weak), 22.62, 22.58,
14.24. LCMS (ESI): m/z calcd for
C.sub.17H.sub.23D.sub.7N.sub.2O.sub.5 349.26, [M+H].sup.+ found
350.4.
Example 4. Preparation of Compound 2B
##STR00021##
[0289] In alternative embodiments, compounds of the invention,
including compounds of Formula I, can be prepared as shown in the
following protocol or scheme; and the invention encompasses methods
comprising this scheme and all routine variations thereof:
[0290] The ethyl ester Compound 2A (36 mg; 0.1 mmol) was dissolved
in ethanol (2 mL). The stirred solution was cooled to 0.degree. C.
(ice-water bath) and treated with a solution of 85% KOH in ethanol
(70 uL; 0.11 mmol). The resulting solution was stirred at 0.degree.
C. for 20 minutes and at room temperature for 10 minutes. The
reaction was concentrated to dryness and the residue partitioned
between water and ethyl acetate. The layers were separated and the
aqueous extracted once more with ethyl acetate. The aqueous layer
was acidified to pH 2 with 1N HCl and extracted with ethyl acetate
(3.times.). The combined organic layers were dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide an off-white
powder, Compound 2B (32 mg; 97%). LCMS (ESI): m/z calcd for
C.sub.15H.sub.19D.sub.7N.sub.2O.sub.5 321.23, [M+H].sup.+ found
322.2.
Example 5. Preparation of Compound 3A
##STR00022##
[0292] In alternative embodiments, compounds of the invention,
including compounds of Formula I, can be prepared as shown in the
following protocol or scheme; and the invention encompasses methods
comprising this scheme and all routine variations thereof:
L-Leucine-2-d.sub.1 (188 mg; 1.4 mmol; C/D/N Isotopes) was
suspended in 8 mL of a 1:1 solution of water and tetrahydrofuran.
The suspension was stirred, cooled to 0.degree. C. (ice-water
bath), and treated with sodium bicarbonate (353 mg; 4.2 mmol)
followed by di-tert-butyl dicarbonate (373 mg; 1.7 mmol). The
resulting suspension was stirred at 0.degree. C. for 30 minutes and
then at room temperature for 68 hours. The reaction mixture was
extracted with ethyl acetate (3.times.). The aqueous layer was
acidified to pH=3 with 1 N HCl and extracted with ethyl acetate
(3.times.). All of the ethyl acetate extractions were combined,
washed with brine (3.times.), dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide a viscous
oil (317 mg) of N-(tert-butyloxycarbonyl)-L-leucine-2-di.
N-(tert-butyloxycarbonyl)-L-leucine-2-di (405 mg; 1.74 mmol),
1-hydroxybenzotriazole hydrate (346 mg; 2.26 mmol), and isoamyl
amine (166 mg; 1.91 mmol) were dissolved in CH.sub.2Cl.sub.2 (10
mL) at room temperature under nitrogen. With stirring,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (500
mg; 2.61 mmol) was added. After stirring for three hours at room
temperature, the reaction was diluted with CH.sub.2Cl.sub.2 (80 mL)
and extracted with saturated NaHCO.sub.3 (2.times.) and brine
(1.times.). The organic layer was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide 525 mg of
crude product. This material was dissolved in methanol (9 mL) and
p-toluene sulfonic acid hydrate (414 mg; 2.18 mmol) was added with
stirring. The solution was heated to 70.degree. C. for two hours.
After cooling, the reaction was evaporated to dryness to provide
279 mg of crude product. To this material was added (2S,
3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (279 mg; 1.74 mmol;
Peptech) and CH.sub.2Cl.sub.2 (15 ml) to provide a homogeneous
solution. The solution was cooled to 0.degree. C. (ice-water bath)
under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (722 mg; 1.9 mmol) followed by diisopropylethyl
amine (779 uL; 4.35 mmol) were added to the reaction solution. The
resulting reaction was stirred at 0.degree. C. for one hour and for
two hours at room temperature. The reaction was diluted with
CH.sub.2Cl.sub.2 (150 ml) and extracted with saturated NaHCO.sub.3
(2.times.) and brine (1.times.). The organic layer was dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated. The crude
product was filtered through a silica gel plug eluting with ethyl
acetate-hexane (1:9) followed by ethyl acetate-hexane (1:1).
Concentration of the 1:1 ethyl acetate-hexane elution fractions
provided the desired product as a white solid (468 mg; 78%).
Recrystallization from diisopropyl ether yielded crystalline
material, mp 122-123.degree. C. .sup.1H NMR (CDCl.sub.3, 500 MHz)
.delta.6.95 (s, 1H), 6.43 (bt, J=5.4 Hz, 1H), 4.19-4.27 (m, 2H),
3.67 (d, J=1.8 Hz, 1H), 3.46 (d, J=1.8 Hz, 1H), 3.23-3.30 (m, 1H),
3.14-3.20 (m, 1H), 1.49-1.62 (m, 4H), 1.36 (q, J=7.3 Hz, 2H), 1.29
(t, J=7.2 Hz, 3H), 0.87-0.91 (m, 12H). .sup.13C NMR (CDCl.sub.3,
125 MHz) .delta.171.16, 166.81, 166.20, 62.47, 53.93, 52.96, 51.37
(m, weak), 41.40, 38.42, 38.14, 26.02, 24.99, 22.95, 22.59, 22.54,
22.41, 14.19. LCMS (ESI): m/z calcd for
C.sub.17H.sub.29DN.sub.2O.sub.5 343.22, [M+H].sup.+ found
344.2.
Example 6. Preparation of Compound 3B
##STR00023##
[0294] In alternative embodiments, compounds of the invention,
including compounds of Formula I, can be prepared as shown in the
following protocol or scheme; and the invention encompasses methods
comprising this scheme and all routine variations thereof:
[0295] The ethyl ester from the above preparation (68 mg; 0.2 mmol)
was dissolved in ethanol (3 mL). The stirred solution was cooled to
0.degree. C. (ice-water bath) and treated with a solution of 85%
KOH in ethanol (140 uL; 0.22 mmol). The resulting solution was
stirred at 0.degree. C. for 60 minutes and was concentrated to
dryness. The residue partitioned between water and ethyl acetate.
The layers were separated and the aqueous extracted once more with
ethyl acetate. The aqueous layer was acidified to pH 1 with 1N HCl
and extracted with ethyl acetate (3.times.). The combined organic
layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated to provide an white powder (42 mg; 67%). LCMS (ESI):
m/z calcd for C.sub.15H.sub.25DN.sub.2O.sub.5 315.19, [M+H].sup.+
found 316.2.
Example 6. IC.sub.50 of Metabolically Blocked Analogs
[0296] In alternative embodiments, compounds of the invention,
including compounds of Formula I, can be prepared as shown in the
following protocol or scheme; and the invention encompasses methods
comprising this scheme and all routine variations thereof:
[0297] The IC.sub.50 of Compound 1B, Compound 2B, and Compound 3B
were determined and compared to the IC.sub.50 of E64c in a series
of assays evaluating the inhibition of Cathepsin B in the manner
well known to those of skill in the art. The measured values are
reported in Table 1.
TABLE-US-00001 TABLE 1 Enzymatic activity vs Cathepsin B Compound
IC.sub.50 E64c 1 nM Compound 1B 2 nM Compound 2B 1.5 nM Compound 3B
1.9 nM
Example 7
[0298] In alternative embodiments, compounds of the invention,
including compounds of Formula I, can be prepared as shown in the
following protocols or schemes; and the invention encompasses
methods comprising these schemes and protocols and all routine
variations thereof:
##STR00024##
##STR00025##
##STR00026##
##STR00027##
##STR00028##
##STR00029##
##STR00030##
##STR00031##
##STR00032##
##STR00033##
##STR00034##
Experimental Section
General Experimental Procedures
[0299] All commercially available reagents were used without
further purification unless otherwise specified. Analytical TLC was
performed on EMD precoated silica gel 60 F.sub.254 plates with 0.25
mm thickness. Flash column chromatography was carried out using
Davisil Grade 63 FC Type 60A silica gel (170-400 mesh) or with
pre-packed REVELERIS.TM. (Grace, Deerfield, Ill.) High Resolution
Flash Cartridges. NMR spectra were obtained on a 500 MHz
spectrometer at 500 MHz for .sup.1H NMR and 125 MHz for .sup.13C
NMR with chemical shifts reported in units of parts per million
(ppm). Mass spectra (MS) were recorded on a PE/SCIEX API-150EX.TM.
(AB SCIEX, Foster City, Calif.) mass spectrometer using ESI. The
purity of tested compounds were analyzed on an HPLC equipped with a
Waters 1525.TM. pump, a Waters 2487.TM. dual .lamda. Absorbance
detector, a Waters 717.TM. (Waters, Milford, Mass.) Plus
autosampler and a LUNA.TM. (Phenomenex, Torrance, Calif.) C18(2)
100 mm.times.4.6 mm reverse-phase column using a gradient (see
Table 1) with solvents [A] 0.1% trifluoroacetic acid in water and
[B] 0.1% trifluoroacetic acid in acetonitrile with a flow rate of 1
mL/min.
TABLE-US-00002 TABLE 1 Solvent gradient for condition I Time Flow
Step (min) (ml/min) % A % B 1 0.00 1.00 90.0 10.0 2 5.00 1.00 90.0
10.0 3 15.00 1.00 60.0 40.0 4 45.00 1.00 45.0 55.0 5 47.00 1.00 0.0
100 6 50.00 1.00 0.0 100 7 51.00 1.00 90.0 10.0
##STR00035##
3-(2-tert-Butoxycarbonylamino-4-methylpentanoylamino)-propionic
acid methyl ester (HLI-014-038) (2)
[0300] N-(tert-butyloxycarbonyl)-L-leucine hydrate (1.50 g, 6.0
mmol), 1-hydroxybenzotriazole hydrate (1.11 g, 7.8 mmol), and
B-alanine methyl ester hydrochloride (1) (0.92 g, 6.6 mmol) were
dissolved in CH.sub.2Cl.sub.2 (50 mL) at room temperature under
nitrogen. With stirring, diisopropylethylamine (1.08 mL, 6.0 mmol)
was added into the reaction mixture, followed by
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.73
g, 9.0 mmol) at rt. After stirring for five hours at room
temperature, the reaction was diluted with CH.sub.2Cl.sub.2 (100
mL) and extracted with saturated NaHCO.sub.3 (2.times.) and brine
(2.times.). The organic layer was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide compound 2
(1.90 g, 100%) as a white waxy solid: ESI MS m/z 317.2 (M+H)+.
##STR00036##
[1-(3-Hydroxy-3-methyl-butylcarbamoyl-3-methylbutyl]carbamic acid
tert-butyl ester (HLI-014-044) (3)
[0301] Compound 2 (0.158 g; 0.5 mmol) was dissolved in anhydrous
THF (3 mL) and then treated with a solution of methyl magnesium
bromide in diethyl ether (3.0 M, 1.00 mL, 3.0 mmol) under nitrogen
with stirring. The solution was stirred at room temperature for 20
hours and then quenched with saturated NH.sub.4Cl aqueous solution.
The resulting mixture was extracted with ethyl acetate (3.times.).
The combined ethyl acetate layers were washed with saturated
NH.sub.4Cl aqueous solution (3.times.) and brine (1.times.). The
organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered
and concentrated to provide alcohol 3 as a white foam. The crude
product was filtered through a silica gel plug eluting with ethyl
acetate-hexane (1:9 to 1:1). Concentration of the 1:1 ethyl
acetate-hexane elution fractions provided the pure compound 3 as a
white viscous oil (0.105 g; 0.33 mmol, 66%); .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta. 6.85 (br s, 1H), 4.96 (d, J=8.1 Hz,
1H), 3.98-4.05 (m, 1H), 3.42-3.48 (m, 1H), 3.31-3.38 (m, 1H),
1.77-1.82 (m, 1H), 1.67 (t, J=6.6 Hz, 2H), 1.60-1.68 (m, 2H), 1.43
(s, 9H), 1.26 (s, 3H), 1.25 (s, 3H), 0.91-0.95 (m, 6H); ESI MS m/z
317.2 (M+H).sup.+.
##STR00037##
2-Amino-4-methylpentanoic acid (3-hydroxy-3-methylbutyl)amide
(HLI-014-062) (4)
[0302] Boc protected material 3 (0.080 g, 0.25 mmol) was dissolved
in a mixture of dioxane (3 mL) and water (1 mL) and then treated
with p-toluenesulfonic acid hydrate (0.071 g, 0.38 mmol) with
stirring. The solution was heated to 70.degree. C. for six and half
hours. After cooling, the reaction was concentrated under reduced
pressure at room temperature and the remaining liquid was treated
with 28% NH.sub.4OH aqueous solution to pH 10. The resulting
mixture was extracted with dichloromethane (3.times.) and the
combined extracts were dried over Na.sub.2SO.sub.4, filtered,
concentrated to provide amine 4 as an off-white viscous oil (0.054,
99%); ESI MS m/z 217.2 (M+H).sup.+. This material was used in the
next step without further purification.
##STR00038##
(2S,3S)-3-((S)-[1-(3-Hydroxy-3-methylbutylcarbamoyl)-3-methylbutylcarbamo-
yl]-oxirane-2-carboxylic acid ethyl ester (ALP-###0.000.01;
HLI-014-063) (5)
[0303] To crude amine 4 (0.070 g, 0.32 mmol) was added (2S,
3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (0.054 g, 0.34
mmol, Peptech) and CH.sub.2Cl.sub.2 (4 ml) to provide a homogeneous
solution. The solution was cooled to 0.degree. C. (ice-water bath)
under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (0.13 g, 0.35 mmol) followed by
diisopropylethylamine (0.12 mL, 0.68 mmol) were added to the
reaction solution. The resulting reaction was stirred at 0.degree.
C. for one hour and two and half hours at room temperature. The
reaction was diluted with CH.sub.2Cl.sub.2 (80 ml) and extracted
with saturated NaHCO.sub.3 (2.times.) and brine (1.times.). The
organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered
and concentrated to provide crude 32 as a yellow, viscous oil. The
crude product was filtered through a silica gel plug eluting with
ethyl acetate-hexane (1:9) to pure ethyl acetate. Concentration of
elution fractions provided the title compound as a clear, viscous
oil (0.075 g, 65%). Treatment of this oil with diethyl
ether-hexanes yielded compound 5 as a white solid; .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta. 6.91 (br s, 1H), 6.64 (d, J=8.2 Hz,
1H), 4.32-4.37 (m, 1H), 4.21-4.31 (m, 2H), 3.68 (s, 1H), 3.47 (s,
1H), 3.37-3.44 (m, 2H), 1.91 (s, 1H), 1.67 (t, J=6.5 Hz, 2H),
1.63-1.65 (m, 1H), 1.49-1.59 (m, 2H), 1.31 (t, J=7.1 Hz, 3H), 1.27
(s, 3H), 1.26 (s, 3H), 0.90-0.93 (m, 6H); ESI MS m/z 359.4
(M+H).sup.+.
##STR00039##
(2S,3S)-3-((S)-1-(3-hydroxy-3-methylbutylamino)-4-methyl-1-oxopentan-2-yl-
carbamoyl) oxirane-2-carboxylic acid (ALP-485.000.01; HLI-014-067)
(6)
[0304] Ethyl ester 5 (25 mg, 0.07 mmol) was dissolved in ethanol
(3.0 mL). The stirred solution was treated with a solution of 85%
KOH in ethanol (100 .mu.L, 0.07 mmol). The resulting solution was
stirred at room temperature for 30 minutes. The reaction was
acidified to pH 4 with a solution of HCl in dioxane and ethanol.
The mixture was filtered through a pad of Celite (diatomaceous
earth) and concentrated to afford acid 33 as a colorless viscous
oil (23 mg, 100%); .sup.1H NMR (acetone-d.sub.6, 500 MHz) .delta.
7.65 (br s, 1H), 7.56 (br s, 1H), 4.44 (t, J=7.4 Hz, 1H), 3.66 (s,
1H), 3.56 (s, 1H), 3.31 (t, J=6.2 Hz, 2H), 2.09 (s, 1H), 1.61-1.70
(m, 5H), 1.19 (s, 6H), 0.88-0.93 (m, 6H); ESI MS m/z 331.0
(M+H).sup.+.
##STR00040##
[1-(4-Hydroxy-3-methylbutylcarbamoyl)-3-methylbutyl]-carbamic acid
tert-butyl ester (AKA-016-141) (8)
[0305] To a solution of Boc-Leu-OH.H.sub.2O (2.30 g, 9.23 mmol) in
CH.sub.2Cl.sub.2 (100 mL) under nitrogen at room temperature was
added 1-hydroxybenzotriazole hydrate (1.84 g, 12.00 mmol),
1-ethyl-3(3-dimethyl aminopropyl)carbodiimide hydrochloride (2.65
g, 13.85 mmol) and 4-amino-2-methylbutanol (1.00 g, 9.69 mmol, Ryan
Scientific). After stirring for 5 h at rt, the reaction mixture was
diluted with CH.sub.2Cl.sub.2 (200 mL) and the organics were washed
with saturated NaHCO.sub.3 (2.times.), brine (1.times.), dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated to provide a
white foam. Purification by silica gel chromatography (elution with
33% to 50% to 80% ethyl acetate/hexanes) gave a mixture of
diastereomers 8 (2.53 g, 87%) as a colorless foam: TLC R.sub.f 0.49
(EtOAc; Ninhydrin stain); .sup.1H NMR CDCl.sub.3, 500 MHz) .delta.
6.70 (bs, 1H), 5.07-5.09 (m, 1H), 4.05 (bs, 1), 3.49-3.53 (m, 1H),
3.20-3.43 (m, 3H), 2.71-2.78 (m, 1H), 1.57-1.70 (m, 4H), 1.46-1.48
(m, 2H), 1.42 (s, 9H), 0.89-0.94 (m, 9H); .sup.13C NMR (CDCl.sub.3,
125 MHz) .delta. 172.89, 156.07, 80.26, 67.85, 53.45, 41.56, 37.76
(37.63), 33.76 (33.69), 33.51 (33.45), 28.53, 24.97, 23.10, 22.26,
17.16 (17.07); ESI MS m/z 317.4 (M+H).sup.+.
##STR00041##
3-[1-(4-Hydroxy-3-methylbutylcarbamoyl)-3-methylbutylcarbamoyl]-oxirane-2-
-carboxylic acid ethyl ester (ALP-490.000.01; AKA-016-147) (9)
[0306] To a solution of [1-(4-Hydroxy-3-methylbutyl
carbamoyl)-3-methylbutyl]-carbamic acid tert-butyl ester (8) (2.53
g, 8.01 mmol) in MeOH (65 mL) was added p-toluenesulfonic acid
hydrate (1.90 g; 10.00 mmol) at rt. The mixture was stirred at
70.degree. C. for 2.5 h. After cooling, the reaction mixture was
evaporated to dryness to give a colorless foam. To this material
was added (2S, 3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid
(1.28 g, 8.01 mmol, Peptech) and CH.sub.2Cl.sub.2 (65 mL) at rt
giving a homogeneous solution. The solution was cooled to 0.degree.
C. (ice-water bath) under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (3.35 g; 8.81 mmol) followed by
diisopropylethyl amine (2.87 mL; 16.02 mmol) were added to the
reaction solution. The resulting reaction was stirred at 0.degree.
C. for one hour and another 4 hours at room temperature. The
reaction mixture was diluted with CH.sub.2Cl.sub.2 (400 ml) and the
organics washed with saturated NaHCO.sub.3 (2.times.) and brine
(1.times.). The organic layer was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide a viscous
oil. The crude product was purified by silica gel chromatography
(elution with 17% to 50% to 80% ethyl acetate/hexanes and 100%
ethyl acetate) providing the desired product as a viscous oil (2.32
g, 81%). Recrystallization from ethyl acetate-hexanes (1:1) yielded
crystalline material: mp 92-95.degree. C.; TLC R.sub.f 0.50 (EtOAc;
PMA stain).sup.1H NMR (CDCl.sub.3, 500 MHz) .delta.6.96 (d, J=8.6
Hz, 1H), 6.87 (t, J=5.3 Hz, 1H), 4.39-4.40 (m, 1H), 4.22-4.29 (m,
2H), 3.69 (d, J=1.8, 1H), 3.50-3.54 (m, 1H), 3.48 (d, J=1.7 Hz,
1H), 3.40-3.42 (m, 1H), 3.31-3.35 (m, 1H), 3.21-3.23 (m, 1H), 2.74
(t, J=5.2 Hz, 1H), 1.60-1.68 (m, 3H), 1.51-1.54 (m, 2H), 1.38-1.42
(m, 1H), 1.30 (t, J=7.2 Hz, 3H), 0.89-0.92 (m, 9H); .sup.13C NMR
(CDCl.sub.3, 125 MHz) 3171.41, 166.90, 166.38, 67.85, 62.58, 53.99,
53.02, 51.81, 41.42, 37.95 (37.85), 33.85 (33.74), 33.35 (33.33),
25.04, 23.00, 22.29, 17.09 (17.00), 14.21; ESI MS m/z 359.4
[M+H].sup.+; HPLC Purity: 100% t.sub.R 16.2 min (Condition I, UV215
nm)
##STR00042##
3-[1-(4-Hydroxy-3-methyl-butylcarbamoyl)-3-methyl-butylcarbamoyl]-oxirane-
-2-carboxylic acid (ALP-488.00.01; AKA-016-149) (10)
[0307] Ethyl ester 9 (215 mg, 0.60 mmol) was dissolved in ethanol
(6 mL). The stirred solution was cooled to 0.degree. C. (ice-water
bath) and treated with a 1.0 M solution of 85% KOH in ethanol
(0.613 mL, 0.61 mmol). The resulting solution was stirred at
0.degree. C. for 60 minutes then warmed to rt and then stirred at
rt for 2 hours. The solvent was removed in vacuo and the residue
partitioned between water (4 mL) and CH.sub.2Cl.sub.2 (4 mL). The
aqueous layer was acidified to pH 2 with 2 N HCl and extracted with
ethyl acetate (4.times.). The combined organic layers were washed
with brine (2.times.), dried over anhydrous Na.sub.2SO.sub.4,
filtered and concentrated to provide a viscous oil that was
dissolved in CH.sub.3CN and yielded a colorless foam (103 mg, 52%)
after concentration in vacuo: .sup.1H NMR (500 MHz, acetone-d)
.delta. 7.57 (d, J=8.4 Hz, 1H), 7.48 (bs, 1H), 4.45-4.49 (m, 1H),
3.66 (d, J=1.6 Hz, 1H), 3.58 (d, J=1.7 Hz, 1H), 3.37 (d, J=5.7 Hz,
2H), 3.21-3.28 (m, 2H), 1.58-1.67 (m, 5H), 1.29-1.31 (m, 1H), 0.92
(d, J=6.4 Hz, 3H), 0.89 (d, J=6.3 Hz, 6H); .sup.13C NMR (125 MHz,
acetone-d.sub.6) .delta. 172.22 (172.14), 168.91, 166.40 (166.33),
67.75, 54.39 (54.37), 52.73, 52.46 (52.43), 42.56 (42.52), 38.14
(38.02), 34.47, 34.44, 25.57, 23.48, 22.23, 17.21; ESI MS m/z 331.2
(M+H)+.
##STR00043##
Compound
[1-(4-Hydroxy-3-methylbutylcarbamoyl)-3-methylbutyl]-carbamic acid
tert-butyl ester (HLI-014-080) (12)
[0308] To a solution of Boc-Leu-OH.H.sub.2O (1.27 g, 5.09 mmol) and
(R)-4-amino-2-methylbutanol (0.50 g, 4.85 mmol, Ryan Scientific) in
CH.sub.2Cl.sub.2 (25 mL) at 0.degree. C. was added
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (2.03 g, 5.33 mmol) followed by
diisopropylethyl amine (1.74 mL, 9.70 mmol). The reaction mixture
was stirred for 30 minutes at 0.degree. C. and at rt for 1.5 hours
under nitrogen. The reaction mixture was diluted with
CH.sub.2Cl.sub.2 (100 mL) and the organics were washed with
saturated NaHCO.sub.3 (2.times.), brine (1.times.), dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated. Purification
by silica gel chromatography (elution with 10% to 17% to 50% to 60%
ethyl acetate/hexanes) gave 12 (1.61 g, 87%, 100%) as a viscous
oil: ESI MS m/z 317.4 (M+H).sup.+.
##STR00044##
3-[1-(4-Hydroxy-3-methylbutylcarbamoyl)-3-methylbutylcarbamoyl]-oxirane-2-
-carboxylic acid ethyl ester (ALP-493.000.01; HLI-014-087) (13)
[0309] To a solution of 12 (0.90 g, 2.80 mmol) in MeOH (15 mL) was
added p-toluenesulfonic acid hydrate (0.676 g, 3.60 mmol) at rt.
The reaction mixture was stirred at 65.degree. C. for 1 h. After
cooling, the reaction mixture was evaporated to dryness to give a
viscous oil. To this material was added (2S,
3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (0.480 g, 2.90
mmol, Peptech) and CH.sub.2Cl.sub.2 (25 mL) at rt. The solution was
cooled to 0.degree. C. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (1.17 g, 3.10 mmol) followed by
diisopropylethyl amine (1.00 mL, 5.60 mmol) were added to the
reaction solution. The resulting reaction was stirred at 5.degree.
C. for one hour and another 1 h at room temperature. The reaction
mixture was diluted with CH.sub.2Cl.sub.2 (150 ml) and the organics
washed with saturated NaHCO.sub.3 (2.times.) and brine (2.times.).
The organic layer was dried over anhydrous Na.sub.2SO.sub.4,
filtered and concentrated to provide a viscous oil. The crude
product was purified by silica gel chromatography (elution with 17%
to 25% to 75% to 83% ethyl acetate/hexanes and 100% ethyl acetate)
providing the desired product 13 (0.748 g, 73%) as a white solid.
Recrystallization from ethyl acetate-ethyl ether-hexanes (1:1:1)
yielded crystalline material: mp 106-108.degree. C.; .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta.6.76 (d, J=8.5 Hz, 1H), 6.66 (t, J=5.0
Hz, 1H), 4.35-4.40 (m, 1H), 4.22-4.29 (m, 2H), 3.68 (d, J=1.5 Hz,
1H), 3.52-3.55 (m, 1H), 3.47 (d, J=1.4 Hz, 1H), 3.40-3.45 (m, 1H),
3.37-3.39 (m, 1H), 3.17-3.25 (m, 1H), 2.38 (bs, 1H), 1.60-1.70 (m,
3H), 1.51-1.55 (m, 2H), 1.42-1.47 (m, 1H), 1.31 (t, J=7.2 Hz, 3H),
0.90-0.93 (m, 9H); 6171.27, 166.79, 166.36, 67.96, 62.59, 54.00,
53.11, 51.80, 41.40, 37.89, 33.79, 33.42, 25.07, 23.02, 22.31,
17.06, 14.23; ESI MS m/z 359.4 [M+H].sup.+; HPLC Purity: 99%
t.sub.R 16.1 min (Condition I, UV 215 nm).
##STR00045##
3-[1-(4-Hydroxy-3-methyl-butylcarbamoyl)-3-methyl-butylcarbamoyl]-oxirane-
-2-carboxylic acid (ALP-494.000.01; AKA-016-173) (14)
[0310] Ethyl ester 13 (100 mg, 0.28 mmol) was dissolved in ethanol
(3 mL). The stirred solution was cooled to 0.degree. C. (ice-water
bath) and treated with a 1.0 M solution of 85% KOH in ethanol
(0.285 mL, 0.285 mmol). The resulting solution was stirred at
0.degree. C. for 40 minutes then warmed to rt and then stirred at
rt for 20 minutes. The solvent was removed in vacuo and the residue
partitioned between water (3 mL) and CH.sub.2Cl.sub.2 (4 mL). The
aqueous layer was acidified to pH 2 with 2 N HCl and extracted with
ethyl acetate (5.times.). The combined organics were dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated to provide a
viscous oil that was dissolved in CH.sub.3CN and after
concentration in vacuo yielded 14 as a colorless foam (49 mg, 53%):
.sup.1H NMR (500 MHz, acetone-d.sub.6) .delta. 7.58 (d, J=8.4 Hz,
1H), 7.49 (app t, 1H), 4.45-4.49 (m, 1H), 3.66 (d, J=1.9 Hz, 1H),
3.58 (d, J=1.7 Hz, 1H), 3.37 (d, J=5.7 Hz, 2H), 3.21-3.28 (m, 2H),
1.58-1.66 (m, 5H), 1.29-1.31 (m, 1H), 0.91 (d, J=6.5 Hz, 3H), 0.89
(d, J=6.2 Hz, 6H); 13C NMR (125 MHz, acetone-d.sub.6) .delta.
172.15, 168.94, 166.36, 67.70, 54.38, 52.76, 52.37, 42.51, 38.02,
34.42, 34.16, 25.57, 23.48, 22.23, 17.21; ESI MS m/z 331.4
(M+H).sup.+; HPLC Purity: 95% t.sub.R=23.7 min, respectively
(Condition II, UV 215 nm).
##STR00046##
Compound (AKA-016-128) (16)
[0311] To a pressure vial charged with a stirbar was added
potassium cyanide (0.508 g, 7.80 mmol) and water (1 mL). Gentle
heating was required to dissolve all of the solids. To this
solution was added absolute ethanol (3 mL) and
1-bromo-2-methyl-d.sub.3-propane-2,3,3,3,-d.sub.4 (15) (0.940 g,
6.50 mmol, C/D/N Isotopes). The tube was sealed and heated at
90.degree. C. for 23 h and after an hour a white solid had
precipitated from solution. After cooling to rt, water (4 mL) was
added to the mixture to dissolve the salts that had formed followed
by CHCl.sub.3 (2 mL). The organic phase was washed with brine
(1.times.) and concentrated in vacuo carefully giving nitrile 10
(368 mg, 63%) as an oil: .sup.1H NMR (CDCl.sub.3, 500 MHz)
.delta.2.16 (s, 2H); .sup.13C NMR (CDCl.sub.3, 125 MHz)
.delta.119.1, 26.0, 25.2 (t, J=20 Hz), 20.8 (septet, J=19 Hz).
##STR00047##
Compound (AKA-016-132) (17)
[0312] To a 1 M LiAlH.sub.4 solution (0.775 g, 20.4 mL, 20.4 mmol)
stirring at 0.degree. C. (ice-bath) under nitrogen was added a
solution of nitrile 16 (0.368 g, 4.08 mmol) dropwise over 15
minutes. After stirring for 1 hour at 0.degree. C., the mixture was
warmed to rt and stirring was continued for 19 h. The reaction
mixture was quenched at 0.degree. C. with water (0.775 mL), 15%
NaOH (aq) (0.775 mL) and water (2.34 mL) giving a heterogeneous
mixture. Ethyl ether (10 mL) was added to break up the slurry and
the mixture was stirred for 2 h at rt. The solids were removed by
filtration over a pad of Na.sub.2SO.sub.4 and were rinsed with
Et.sub.2O (4.times.50 mL). The filtrate was cooled to 0.degree. C.
and 4 N HCl in dioxane (3 mL, 12.2 mmol) was added dropwise and the
reaction mixture was stirred at rt for 30 minutes. The solvent was
removed in vacuo to give a clear oil as the crude amine salt. The
residue was treated with CHCl.sub.3 and xylenes and concentrated in
vacuo to yield 17 as a waxy solid, 335 mg (63%): ESI MS m/z 95.4
(M+H).sup.+.
##STR00048##
Compound (AKA-016-136) (18a)
[0313] To a solution of Boc-Leu-OH.H.sub.2O (0.573 g, 2.30 mmol)
and isoamylamine-d.sub.7.HCl (17) (0.335 g, 2.50 mmol) in
CH.sub.2Cl.sub.2 (20 mL) under nitrogen at room temperature was
added 1-hydroxybenzotriazole hydrate (0.459 g, 3.00 mmol),
diisopropylethylamine (0.803 mL, 4.60 mmol) and
1-ethyl-3(3-dimethyl aminopropyl)carbodiimide hydrochloride (0.671
g, 3.50 mmol). After stirring for 6 h at rt, the reaction mixture
was diluted with CH.sub.2Cl.sub.2 (100 mL) and the organics were
washed with saturated NaHCO.sub.3 (2.times.), brine (2.times.),
dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to
provide the crude product. Purification by silica gel
chromatography (12 g Grace Reveleris column, elution with 10% to
20% ethyl acetate/hexanes) gave compound 18a (0.400 g, 57%) as a
waxy, colorless solid: TLC R.sub.f 0.67 (40% EtOAc/hexanes;
Ninhydrin stain); .sup.1H NMR (CDCl.sub.3, 500 MHz) .delta.6.16
(bs, 1H), 4.91 (d, J=7.4 Hz, 1H), 4.04 (bs, 2H), 3.22-3.26 (m, 2H),
1.64-1.66 (m, 3H), 1.43-1.51 (m, 11H), 1.34-1.37 (t, J=7.4 Hz, 2H),
0.91-0.94 (m, 6H). .sup.13C NMR (CDCl.sub.3, 125 2 MHz) m/z 172.56,
155.99, 62.75, 53.38, 41.46, 38.37, 37.94, 29.90, 28.35, 24.99,
23.10, 22.07; ESI MS m/z 308.4 (M+H).sup.+.
##STR00049##
Compound (AKA-016-153) (18b)
[0314] To a solution of L-Leucine-d.sub.7-N-t-Boc.H.sub.2O (0.500
g, 1.95 mmol; C/D/N Isotopes) and isoamylamine-d.sub.7.HCl (17)
(0.280 g, 2.15 mmol) in CH.sub.2Cl.sub.2 (20 mL) under nitrogen at
room temperature was added 1-hydroxybenzotriazole hydrate (0.389 g,
2.54 mmol), diisopropylethylamine (0.681 mL, 3.90 mmol) and
1-ethyl-3(3-dimethyl aminopropyl)carbodiimide hydrochloride (0.562
g, 2.93 mmol). After stirring for 2.5 h at rt, the reaction mixture
was diluted with CH.sub.2Cl.sub.2 (100 mL) and the organics were
washed with saturated NaHCO.sub.3 (2.times.), brine (2.times.),
dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to
provide the crude product. Purification by silica gel
chromatography (20 g SiO2, elution with 10% to 20% to 25% to 30%
ethyl acetate/hexanes) gave compound 18b (0.409 g, 67%) as a waxy,
colorless solid: TLC R.sub.f 0.80 (50% EtOAc/hexanes; Ninhydrin
stain); .sup.1H NMR (CDCl.sub.3, 500 MHz) .delta.5.16 (bs, 1H),
4.91 (app d, J=6.8 Hz), 4.01-4.05 (m, 1H), 3.24 (q, J=6.5 Hz, 2H),
1.62-1.66 (m, 1H), 1.39-1.46 (m, 9H), 1.36 (t, J=7.4 Hz, 2H);
.sup.13C NMR (CDCl.sub.3, 125 MHz) .delta.172.57, 155.98, 80.19,
53.39, 41.22, 38.36, 37.94, 28.63, 28.52; ESI MS m/z 315.2
(M+H).sup.+.
##STR00050##
Compound (ALP-497.000.01; AKA-016-139) (19a)
[0315] To a solution of compound 18a (0.384 g, 1.25 mmol) in MeOH
(10 mL) was added p-toluenesulfonic acid hydrate (0.297 g, 1.56
mmol) with stirring. The solution was heated to 70.degree. C. for
2.5 h. After cooling to rt, the reaction was concentrated to
provide the crude tosic salt. To this material was added (2S,
3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (0.200 g, 1.25
mmol, Peptech) and CH.sub.2Cl.sub.2 (10 mL) to provide a
homogeneous solution. The solution was cooled to 0.degree. C.
(ice-water bath) under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (0.524 g, 1.38 mmol) followed by
diisopropylethylamine (0.448 mL, 2.50 mmol) were added to the
reaction solution and the mixture was stirred at 0.degree. C. for 1
h and 3 h at rt. The reaction was diluted with CH.sub.2Cl.sub.2 (70
mL) and the organics were washed with saturated NaHCO.sub.3
(2.times.) and brine (1.times.). The organic layer was dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The
crude product was passed through a silica gel plug eluting with
ethyl acetate-hexane (1:9) followed by ethyl acetate-hexane (1:5)
and ethyl acetate-hexane (1:1). Concentration of the 1:1 ethyl
acetate-hexane elution fractions provided the desire product 19a
(340 mg, 78%) as a white solid. Recrystallization from diisopropyl
ether-ethanol yielded crystalline material: mp 123-124.degree. C.;
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 6.69 (d, J=8.5 Hz, 1H),
6.10 (bs, 1H), 4.36-4.40 (m, 1H), 4.22-4.28 (m, 2H), 3.67 (s, 1H),
3.46 (s, 1H), 3.19-3.29 (m, 2H), 1.62-1.65 (m, 1H), 1.49-1.53 (m,
2H), 1.37 (t, J=7.5 Hz, 2H), 1.30 (t, J=7.1 Hz, 3H), 0.91 (app t,
J=6.9 Hz, 6H); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 171.01,
166.71, 166.22, 62.53, 53.99, 53.11, 51.61, 41.46, 38.20, 25.05,
23.00 22.40, 14.23; ESI MS m/z 350.4 (M+H)+; HPLC Purity: 100%
t.sub.R=22.6 min (Condition I, UV 215 nm).
##STR00051##
Compound (ALP-499.000.01; AKA-016-156) (19b)
[0316] To a solution of compound 18b (0.389 g, 1.24 mmol) in MeOH
(10 mL) was added p-toluenesulfonic acid hydrate (0.281 g, 1.48
mmol) with stirring. The solution was heated to 70.degree. C. for
2.75 h. After cooling to rt, the reaction was concentrated to
provide the crude tosic salt. To this material was added (2S,
3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (0.208 g, 1.30
mmol, Peptech) and CH.sub.2Cl.sub.2 (11 mL) to provide a
homogeneous solution. The solution was cooled to 0.degree. C.
(ice-water bath) under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (0.543 g, 1.43 mmol) followed by
diisopropylethylamine (0.466 mL, 2.60 mmol) were added to the
reaction solution and the mixture was stirred at 0.degree. C. for 1
h and 2.5 h at rt. The reaction was diluted with CH.sub.2Cl.sub.2
(80 mL) and the organics were washed with saturated NaHCO.sub.3
(2.times.) and brine (1.times.). The organic layer was dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The
crude product was passed through a silica gel plug eluting with
ethyl acetate-hexane (1:9) followed by ethyl acetate-hexane (1:5)
and ethyl acetate-hexane (1:1). Concentration of the 1:1 ethyl
acetate-hexane elution fractions provided the desired product 19b
(400 mg, 86%) as a white solid. Recrystallization from diisopropyl
ether-ethanol yielded crystalline material: mp 121-122.degree. C.;
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 6.72 (d, J=8.7 Hz, 1H),
6.15 (t, J=5.4 Hz, 1H), 4.36-4.40 (m, 1H), 4.22-4.28 (m, 2H), 3.67
(d, J=1.7 Hz, 1H), 3.46 (d, J=1.8 Hz, 1H), 3.18-3.30 (m, 2H),
1.60-1.64 (m, 1H), 1.49-1.53 (m, 1H), 1.37 (t, J=7.5 Hz, 2H), 1.30
(t, J=7.1 Hz, 3H); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta.
171.04, 166.72, 166.22, 62.53, 53.99, 53.10, 51.62, 41.24, 25.25
(m, weak), 24.12 (m, weak), 21.61 (m, weak), 21.44 (m, weak),
38.22, 14.23; ESI MS m/z 357.2 (M+H)+; Anal.
(C.sub.17H.sub.16D.sub.14N.sub.2O.sub.5) C, H, N; HPLC Purity: 99%
t.sub.R=22.2 min (Condition I, UV 215 nm).
##STR00052##
Compound (ALP-498.000.01; AKA-016-142) (20a)
[0317] Ethyl ester 19a (117 mg, 0.334 mmol) was dissolved in
ethanol (3 mL). The stirred solution was cooled to 0.degree. C.
(ice-water bath) and treated with a 1.0 M solution of 85% KOH in
ethanol (0.34 mL, 0.34 mmol). The resulting solution was stirred at
0.degree. C. for 1 h and after warming to rt was concentrated in
vacuo. The residue was partitioned between water (2 mL) and
CH.sub.2Cl.sub.2 (2 mL). The layers were separated and the aqueous
portion was extracted once more with CH.sub.2Cl.sub.2. The aqueous
layer was acidified to pH 2 with 2 N HCl and extracted with ethyl
acetate (3.times.). The combined organic layers were dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated to provide an
oil (94 mg, 88%). Recrystallization from hexanes-ethyl acetate
(1:1) yielded 20a as colorless crystals: mp=150-151.degree. C.;
.sup.1H NMR (500 MHz, acetone-d.sub.6) .delta. 7.52 (d, J=8.4 Hz,
1H), 7.41 (bs, 1H), 4.44-4.49 (m, 1H), 3.66 (d, J=1.8 Hz, 1H), 3.57
(d, J=1.8 Hz, 1H), 3.17-3.24 (m, 2H), 1.57-1.68 (m, 3H), 1.35 (t,
J.sup.=7.3 Hz, 2H), 0.91 (dd, J=6.4, 11.0 Hz, 6H); .sup.13C NMR
(125 MHz, acetone-d.sub.6) .delta. 172.05, 168.85, 166.26, 54.37,
52.72, 52.30 (m), 42.64 (m), 39.07, 38.11 (m), 25.57, 23.47, 22.25;
ESI MS m/z 322.2 (M+H)+; HPLC Purity: 95% t.sub.R=18.0 min
(Condition I, UV 215 nm).
##STR00053##
Compound (ALP-500.000.01; AKA-016-160) (20b)
[0318] Ethyl ester 19b (94 mg, 0.264 mmol) was dissolved in ethanol
(3 mL). The stirred solution was cooled to 0.degree. C. (ice-water
bath) and treated with a 1.0 M solution of 85% KOH in ethanol
(0.269 mL, 0.269 mmol). The resulting solution was stirred at
0.degree. C. for 0.5 h and after warming to rt was concentrated in
vacuo. The residue was partitioned between water (4 mL) and
CH.sub.2Cl.sub.2. The layers were separated and the aqueous layer
was acidified to pH 2 with 2 N HCl and extracted with ethyl acetate
(3.times.). The combined organic layers were washed with brine and
dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated to
provide a white solid (78 mg, 90%). Recrystallization from
hexanes-ethyl acetate (1:1) yielded 20b as colorless crystals:
mp=150-151.degree. C.; .sup.1H NMR (500 MHz, acetone-d.sub.6)
.delta. 4.44-4.47 (m, 1H), 3.66 (d, J=1.9 Hz, 1H), 3.57 (d, J=1.9
Hz, 1H), 3.15-3.25 (m, 2H), 1.57-1.59 (m, 2H), 1.35 (t, J=7.3 Hz,
2H); .sup.13C NMR (125 MHz, acetone-d.sub.6) .delta. 172.09,
168.92, 166.26, 54.37, 52.77, 52.28, 42.38, 39.07, 38.11; ESI MS
m/z 329.4 (M+H)+; Anal. (C.sub.15H.sub.12D.sub.14N.sub.2O.sub.5) C,
H, N; HPLC Purity: 99% t.sub.R=18.0 min (Condition I, UV 215
nm).
##STR00054##
Compound (HLI-014-082) (22)
[0319] To a mixture of sodium azide (455 mg, 7.0 mmol), 4-pentanoic
acid-d.sub.11 (255 mg, 2.0 mmol, C/D/N Isotopes), n-butylammonium
bromide (96 mg, 0.3 mmol) and zinc triflate (24 mg, 0.06 mmol) in a
30-mL pressure vial was added THF (20 mL). To the vial heated to
40.degree. C. with stirring was added (Boc).sub.2O (480 mg, 2.2
mmol) in one portion. The vial was sealed and stirred at 50.degree.
C. for 2 hours. After the reaction mixture was cooled, it was
poured into 10% NaNO.sub.2 (aq) (40 mL). Then ethyl acetate (50 mL)
was added and the mixture was stirred at rt for 20 minutes. The
aqueous layer was separated and extracted with ethyl acetate
(3.times.). The combined organic layers were washed with saturated
NH.sub.4Cl (1.times.), saturated NaHCO.sub.3 (1.times.), brine
(1.times.) and dried over Na.sub.2SO.sub.4, filtered and
concentrated to give an oily residue of an intermediate product.
The residue was dissolved in THF (10 mL) and heated at 85.degree.
C. for 2 hours. The temperature was lowered to 46.degree. C. and 1
N HCl (1 mL) was added to the mixture which was stirred for 6 h.
The reaction mixture was cooled to rt and conc. HCl (0.5 mL) was
added and stirring was continued for 3 days. The solvent was
removed in vacuo and the aqueous phase was extracted with Et.sub.2O
(2.times.). The aqueous phase was basified with 1 N NaOH to pH=12
and extracted with Et.sub.2O (3.times.). The combined organics were
dried over MgSO.sub.4 and filtered. The filtrate was treated with 4
N HCl/dioxane (0.8 mL) and a white solid formed immediately. After
concentration, the crude amine HCl salt was isolated as a
colorless, amorphous solid (111 mg, 41%): ESI MS m/z 99.2
(M+H).sup.+.
##STR00055##
Compound (HLI-014-089) (23)
[0320] To a solution of Boc-Leu-OH.H.sub.2O (226 mg, 0.90 mmol),
isoamylamine-d.sub.11 HCl (22) (111 mg, 0.82 mmol) and
1-hydroxybenzotriazole hydrate (163 mg, 1.07 mmol), in
CH.sub.2Cl.sub.2 (8 mL) under nitrogen at room temperature was
added 1-ethyl-3(3-dimethyl aminopropyl)carbodiimide hydrochloride
(236 mg, 1.23 mmol) followed by the addition of
diisopropylethylamine (0.294 mL, 1.64 mmol). After stirring for 49
h at rt, the reaction mixture was diluted with CH.sub.2Cl.sub.2
(100 mL) and the organics were washed with saturated NaHCO.sub.3
(2.times.), brine (2.times.), dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide an oil (374
mg). Purification by silica gel chromatography (elution with 10% to
20% to 30% ethyl acetate/hexanes) gave amide 23 (124 mg, 49%) as a
waxy, white solid: TLC R.sub.f0.25 (20% EtOAc/hexanes;
phosphomolybdic stain); .sup.1H NMR CDCl.sub.3, 500 MHz)
.delta.6.03 (bs, 1H), 4.86 (bs, 1H), (m, 1H), 4.01-4.05 (m, 1H),
1.57-1.69 (m, 3H), 1.44 (s, 9H), 0.92-0.99 (m, 6H); ESI MS m/z
312.4 (M+H).sup.+.
##STR00056##
Compound (ALP-496.000.01; AKA-016-166) (24)
[0321] To a solution of compound 23 (121 mg, 0.389 mmol) in MeOH (3
mL) was added p-toluenesulfonic acid hydrate (89 mg, 0.467 mmol)
with stirring. The solution was heated to 70.degree. C. for 3 h.
After cooling to rt, the reaction was concentrated to provide the
crude tosic salt. To this material was added (2S,
3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (62 mg, 0.389 mmol;
Peptech) and CH.sub.2Cl.sub.2 (4 mL). The solution was cooled to
0.degree. C. (ice-water bath) under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (163 mg, 0.428 mmol) followed by
diisopropylethylamine (0.140 mL, 0.778 mmol) were added to the
reaction solution and the mixture was stirred at 0.degree. C. for
1.25 h and 30 minutes at rt. The reaction was diluted with
CH.sub.2Cl.sub.2 (50 mL) and the organics were washed with
saturated NaHCO.sub.3 (2.times.) and brine (1.times.). The organic
layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated in vacuo. The crude product was purified by silica gel
chromatography eluting with 10% to 17% to 50% ethyl acetate/hexanes
and provided the desired product 24 (110 mg, 80%) as a white solid.
Recrystallization from ethyl acetate-hexanes (1:1) yielded
crystalline material: mp 123-124.degree. C.; .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 6.53 (d, J=8.6 Hz, 1H), 5.87 (bs, 1H),
4.34-4.38 (m, 1H), 4.23-4.30 (m, 2H), 3.67 (d, J=1.7 Hz, 1H), 3.46
(d, J=1.9 Hz, 1H), 1.62-1.66 (m, 1H), 1.49-1.55 (m, 2H), 1.32 (t,
J=7.0 Hz, 3H), 0.92 (t, J=6.5 Hz, 6H); .sup.13C NMR (125 MHz,
CDCl.sub.3) .delta. 170.94, 166.64, 166.25, 62.57, 54.03, 53.21,
51.59, 41.43, 25.08, 23.03, 22.40, 14.26; ESI MS m/z 354.4 (M+H);
HPLC Purity: 100% t.sub.R=22.6 min (Condition I, UV 215 nm).
##STR00057##
Compound (ALP-495.000.01; AKA-016-171) (25)
[0322] Ethyl ester 24 (48 mg, 0.135 mmol) was dissolved in ethanol
(2 mL). The stirred solution was cooled to 0.degree. C. (ice-water
bath) and treated with a 1.0 M solution of 85% KOH in ethanol
(0.137 mL, 0.137 mmol). The resulting solution was stirred at
0.degree. C. for 0.5 h and then slowly warmed to rt and stirred for
an additional 1.5 h. The solvent was removed in vacuo and the
residue was partitioned between water (4 mL) and CH.sub.2Cl.sub.2
(2 mL). The layers were separated and the aqueous layer was
acidified to pH 2 with 2 N HCl and extracted with ethyl acetate
(3.times.10 mL). The combined organic layers were washed with brine
and dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated to provide a white solid (30 mg, 69%).
Recrystallization from hexanes-ethyl acetate (1:1) yielded 25 as
colorless crystals: mp=152-153.degree. C.; .sup.1H NMR (500 MHz,
acetone-d.sub.6) .delta. 4.44-4.47 (m, 1H), 3.63 (d, J=1.9 Hz, 1H),
3.55 (d, J=1.8 Hz, 1H), 1.61-1.66 (m, 1H), 1.56-1.59 (m, 2H),
0.85-92 (m, 6H); .sup.13C NMR (125 MHz, acetone-d.sub.6) .delta.
171.98, 169.06, 166.36, 54.37, 52.99, 52.26, 42.64, 25.58, 23.49,
22.27; ESI MS m/z 326.4 (M+H); HPLC Purity: 100% t.sub.R=18.3 min
(Condition I, UV 215 nm).
##STR00058##
Compound (HLI-014-090) (27)
[0323] To a mixture of 2-iodopropane-2 di (2.25 g, 13.2 mmol; C/D/N
Isotopes) and ethyl cyanoacetate (1.50 g, 13.2 mmol) in a pressure
vial was added CH.sub.3CN (6 mL) and potassium carbonate (3.64 g,
26.4 mmol). The vial was sealed and the mixture was stirred at rt
for 52 hours. The reaction mixture was heated to 65.degree. C. and
stirred for 48 h. After cooling, the potassium carbonate was
removed by filtration and the solids were rinsed with CH.sub.3CN
(20 mL). The filtrate was concentrated in vacuo to give the
cyanoester as a yellow oil (2.1 g, quantitative yield) which was
used in the following step without further purification. To a
solution of the crude ester dissolved in a mixture THF (25 mL) and
water (8 mL) was added LiOH.H.sub.2O (1.1 g, 26.8 mmol) at rt with
stirring. The reaction mixture was stirred for 71 hours. Then the
solvent was removed in vacuo and the remaining aqueous solution was
diluted with water (10 mL) and extracted with ethyl ether
(2.times.). The aqueous layer was then acidified with 1 N HCl to
pH=1 and extracted with ethyl ether (3.times.). The combined
organic layers were dried over MgSO.sub.4, filtered and
concentrated to give 27 (1.33 g, 78%) as a viscous yellow oil.
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 9.61 (bs, 1H), 3.47 (d,
J=8.5 Hz, 1H), 2.41-2.47 (m, 1H), 1.17 (d, J=6.8 Hz, 3H), 1.30 (d,
J=6.7 Hz, 3H).
##STR00059##
Compound (HLI-014-091) (28)
[0324] Acid 27 (1.32 g, 10.3 mmol) and copper powder (150 mg, 2.4
mmol) were placed into a pressure vial and after it was sealed with
a septum was heated to 200.degree. C. with stirring. The
decarboxylation occurred immediately and the nitrile was distilled
via a canula needle into an empty tube sealed with a septum.
Nitrile 28 (0.88 g, 100%) was obtained as a clear oil. .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta.2.23 (s, 2H), 1.06 (s, 6H); .sup.13C
NMR (125 MHz, CDCl.sub.3) .delta. 119.0, 26.2, 21.8.
##STR00060##
Compound (AKA-016-168) (29)
[0325] To a 1.0 M LiAlH.sub.4 solution (29.9 mL, 29.9 mmol) in THF
at 0.degree. C. under N.sub.2 was added dropwise a solution of
nitrile 28 (0.503 g, 5.98 mmol) in THF (20 mL) over 15 minutes.
After an hour, the ice-bath was removed and stirring was continued
at rt for 22 h. The reaction mixture was cooled to 0.degree. C. and
carefully quenched with water (1.13 mL); then 15% NaOH (aq) (1.13.
mL) was added followed by the addition of water (3.39 mL). Some
Et.sub.2O was added to break-up the slurry and the mixture was
stirred for 2 h at rt. The solids were removed by filtration and
rinsed with Et.sub.2O (4.times.50 mL). The filtrate was cooled to
0.degree. C. and 4 N HCl/dioxane (4.5 mL, 17.94 mmol) was added
dropwise. After stirring at 0.degree. C. for 30 minutes, the
solvent was removed in vacuo. The residue was treated with
CH.sub.2Cl.sub.2 and xylenes and concentrated in vacuo to give 29
as a light-colored waxy solid (quantitative): ESI MS m/z 89.0
(M+H).sup.+.
##STR00061##
Compound (AKA-016-169) (30)
[0326] To a solution of Boc-Leu-OH.H.sub.2O (1.35 g, 5.44 mmol) and
amine salt (29) (0.742 g, 5.98 mmol) in CH.sub.2Cl.sub.2 (50 mL)
under nitrogen at room temperature was added 1-hydroxybenzotriazole
hydrate (1.08 g, 7.07 mmol), diisopropylethylamine (1.90 mL, 10.9
mmol) and 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide
hydrochloride (1.56 g, 8.16 mmol). After stirring for 3 h at rt,
the reaction mixture was diluted with CH.sub.2Cl.sub.2 (200 mL) and
the organics were washed with saturated NaHCO.sub.3 (2.times.),
brine (2.times.), dried over anhydrous Na.sub.2SO.sub.4, filtered
and concentrated to provide the crude product. Purification by
silica gel chromatography (50 g SiO.sub.2, elution with 10% to 15%
to 20% to 30% ethyl acetate/hexanes) gave compound 30 (0.992 g,
60%) as a waxy, colorless solid: TLC R.sub.f 0.45 (40%
EtOAc/hexanes; Ninhydrin stain); .sup.1H NMR (CDCl.sub.3, 500 MHz)
.delta.6.16 (bs, 1H), 4.91 (d, J=7.7 Hz, 1H), 4.04-4.08 (m, 1H),
3.24 (q, J=6.4 Hz, 2H), 1.63-1.68 (m, 2H), 1.43 (s, 9H), 1.35-1.38
(t, J=7.4 Hz, 2H), 0.91-0.96 (m, 7H), 0.88 (s, 6H); ESI MS m/z
302.4 (M+H).sup.+.
##STR00062##
Compound (HLI-014-093) (31)
[0327] To a solution of compound 30 (0.98 g, 3.25 mmol) in MeOH (15
mL) was added p-toluenesulfonic acid hydrate (0.772 g, 4.06 mmol)
with stirring. The solution was heated to 70.degree. C. for 1 h.
After cooling to rt, the reaction was concentrated to provide the
crude tosic salt. To this material was added (2S,
3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (0.520 g, 3.25
mmol, Peptech) and CH.sub.2Cl.sub.2 (20 mL). The solution was
cooled to 5.degree. C. (dry ice-water bath) under nitrogen. With
stirring, 2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (1.36 g, 3.58 mmol) followed by
diisopropylethylamine (1.16 mL, 6.50 mmol) were added to the
reaction solution and the mixture was stirred at 5.degree. C. for 1
h and 1 h at rt. The reaction mixture was diluted with
CH.sub.2Cl.sub.2 (150 mL) and the organics were washed with
saturated NaHCO.sub.3 (2.times.) and brine (1.times.). The organic
layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated in vacuo. The crude product was passed through a
silica gel plug eluting with ethyl acetate-hexane (1:9) followed by
ethyl acetate-hexane (1:5), ethyl acetate-hexane (1:3) and ethyl
acetate-hexane (1:1). Concentration of the 1:1 and 1:3 ethyl
acetate-hexane elution fractions provided the desired product 31
(0.98 g, 88%) as a white solid. Recrystallization from diisopropyl
ether-ethanol yielded crystalline material: .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 6.85 (d, J=8.7 Hz, 1H), 6.29 (t, J=5.4 Hz, 1H),
4.40-4.43 (m, 1H), 4.21-4.28 (m, 2H), 3.67 (d, J=1.6 Hz, 1H), 3.46
(d, J=1.7 Hz, 1H), 3.17-3.28 (m, 2H), 1.60-1.64 (m, 1H), 1.50-1.55
(m, 2H), 1.37 (t, J=7.5 Hz, 2H), 1.30 (t, J=7.1 Hz, 3H), 0.88-0.92
(m, 12H); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 171.09,
166.76, 166.21, 62.49, 53.96, 53.04, 51.65, 41.50, 38.35, 38.16,
25.57 (t, J=19.1H), 25.03, 22.98, 22.43, 22.41, 14.21; ESI MS m/z
344.2 (M+H).sup.+.
##STR00063##
Compound (HLI-014-094) (32)
[0328] Ethyl ester 31 (171 mg, 0.5 mmol) was dissolved in ethanol
(6 mL). The stirred solution was treated with a 0.65 M solution of
85% KOH in ethanol (0.77 mL, 0.5 mmol). The resulting solution was
stirred at rt for an hour. After concentrating in vacuo, the
residue was partitioned between water (10 mL) and ethyl ether. The
layers were separated and the aqueous layer was extracted with
ethyl ether (2.times.) then acidified to pH 2 with 1 N HCl and
extracted with ethyl acetate (3.times.). The combined organic
layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated to provide a white solid that was recrystallized from
hexanes-ethyl acetate (1:2) to yield 32 (30 mg, 19%) as colorless
crystals; .sup.1H NMR (500 MHz, acetone-d.sub.6) .delta. 7.59 (d,
J=8.4 Hz, 1H), 7.46 (bs, 1H), 4.46-4.50 (m, 1H), 3.66 (d, J=1.7
Hz), 3.58 (d, J=1.7 Hz, 1H), 3.18-3.25 (m, 2H), 1.63-1.67 (m, 1H),
1.59 (t, J=6.8 Hz, 2H), 1.36 (t, J=7.3 Hz, 2H), 0.95 (d, J=6.6 Hz,
3H), 0.92 (d, J=5.1 Hz, 3H), 0.88 (s, 6H); .sup.13C NMR (125 MHz,
acetone-d.sub.6) .delta. 172.18, 168.95, 166.30, 54.36, 52.75,
52.30, 42.59, 39.17, 38.14, 25.99 (weak t, J=19.1 Hz), 25.57,
23.46, 22.71, 22.26; ESI MS m/z 316.4 (M+H).sup.+; HPLC Purity: 96%
t.sub.R=17.2 min (Condition I, UV 215 nm).
##STR00064##
Compound (HLI-014-045) (34)
[0329] N-(tert-butyloxycarbonyl)-L-leucine-d.sub.3 (methyl-d.sub.3)
hydrate (33) (250 mg, 0.99 mmol, C/D/N Isotopes),
1-hydroxybenzotriazole hydrate (197 mg, 1.29 mmol), and
isoamylamine (95 mg, 1.09 mmol) were dissolved in CH.sub.2Cl.sub.2
(10 mL) at room temperature under nitrogen. With stirring,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (288
mg, 1.50 mmol) was added. After stirring for three hours at room
temperature, the reaction was diluted with CH.sub.2Cl.sub.2 (80 mL)
and extracted with saturated NaHCO.sub.3 (2.times.) and brine
(1.times.). The organic layer was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide 313 mg of
compound 34 as a waxy solid: ESI MS m/z 304.2 (M+H).sup.+.
##STR00065##
Compound (ALP-###; HLI-014-053) (35)
[0330] Compound 34 was dissolved in methanol (5 mL) and then
treated with p-toluenesulfonic acid hydrate (237 mg, 1.25 mmol)
with stirring. The solution was heated to 70.degree. C. for four
hours. After cooling, the reaction was evaporated to dryness and
the residue treated with ether and hexanes. Removal of the
volatiles provided the tosic salt as a white foam: ESI MS m/z 204.2
(C.sub.11H.sub.22D.sub.3N.sub.2O).sup.+. To this material was added
(2S, 3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (160 mg, 1.0
mmol, Peptech) and CH.sub.2Cl.sub.2 (10 ml) to provide a
homogeneous solution. The solution was cooled to 0.degree. C.
(ice-water bath) under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-phosphate (418 mg, 1.1 mmol) followed by
diisopropylethylamine (358 .mu.L, 2.0 mmol) were added to the
reaction solution. The resulting reaction was stirred at 0.degree.
C. for one hour and another hour at room temperature. The reaction
was diluted with CH.sub.2Cl.sub.2 (100 ml) and extracted with
saturated NaHCO.sub.3 (2.times.) and brine (1.times.). The organic
layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated to provide 35 a yellow solid. The crude product was
filtered through a silica gel plug eluting with ethyl
acetate-hexane (1:9) followed by ethyl acetate-hexane (1:1).
Concentration of the 1:1 ethyl acetate-hexane elution fractions
provided the title compound 35 as a white solid (295 mg, 0.86 mmol,
86%). Recrystallization from diisopropyl ether-ethanol yielded
crystalline material: mp 122-123.degree. C.; .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta.6.71 (d, J=8.6 Hz, 1H), 6.12-6.14 (m,
1H), 4.36-4.41 (m, 1H), 4.21-4.29 (m, 2H), 3.67 (d, J=1.4 Hz, 1H),
3.46 (d, J=1.7 Hz, 1H), 3.20-3.28 (m, 2H), 1.56-1.64 (m, 2H),
1.50-1.55 (m, 2H), 1.38 (q, J=7.0 Hz, 2H), 1.30 (t, J=7.0 Hz, 3H),
0.89-0.92 (m, 9H); .sup.13C NMR (CDCl.sub.3, 125 MHz)
.delta.171.02, 166.71, 166.22, 62.53, 53.99, 53.11, 51.62, 41.41,
38.49, 38.21, 26.06, 24.81, 22.92, 22.62, 22.58, 22.32, 14.24; ESI
MS m/z 346.2 (M+H).sup.+; Anal.
(C.sub.17H.sub.27D.sub.3N.sub.2O.sub.5) C, H, N; HPLC purity:
99.7%. t.sub.R=22.8 min (Condition I, UV 215 nm).
##STR00066##
Compound
(2S,3S)-3-((S)-1-(Isopentylamino)-4-methyl-d.sub.3-1-oxopentan-2-
-ylcarbamoyl)oxirane-2-carboxylic acid (ALP-481.000.01;
HLI-014-057) (36)
[0331] The ethyl ester 35 from the above preparation (35 mg, 0.1
mmol) was dissolved in ethanol (2.0 mL). The stirred solution was
cooled to 0.degree. C. (ice-water bath) and treated with a solution
of 85% KOH in ethanol (70 .mu.L, 0.11 mmol). The resulting solution
was stirred at 0.degree. C. for 20 minutes and at room temperature
for 10 minutes. The reaction was concentrated to dryness and the
residue partitioned between water and ethyl acetate. The organic
layer was separated and the aqueous extracted once more with ethyl
acetate. The aqueous layer was acidified to pH 2 with 1 N HCl and
extracted with ethyl acetate (3.times.). The combined organic
layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated to provide 36 as a white solid (36 mg, 96%).
Recrystallization from ethyl acetate-hexanes yielded acid 36 as a
colorless crystal: mp 152-153.degree. C.; .sup.1H NMR
(acetone-d.sub.6, 500 MHz) .delta.7.60 (d, J=8.5 Hz, 1H), 7.49 (br
s, 1H), 4.48 (q, J=7.5 Hz, 1H), 3.66 (d, J=1.6 Hz, 1H), 3.58 (d,
J=1.8 Hz, 1H), 3.17-3.26 (m, 2H), 1.57-1.66 (m, 4H), 1.37 (q, J=7.2
Hz, 2H), 0.91 (d, J=6.3 Hz, 3H), 0.88 (d, J=6.7 Hz, 6H); .sup.13C
NMR (acetone-d.sub.6, 125 MHz) .delta.172.36, 168.99, 166.37,
54.37, 52.71, 52.41, 52.37 (weak), 52.33 (weak), 42.54, 39.27,
38.29, 38.17, 26.45, 25.32, 23.37, 22.84, 22.82, 22.18; ESI MS m/z
318.2 (M+H)+; HPLC purity: 99.5%. t.sub.R=18.0 min (Condition I, UV
215 nm).
##STR00067##
Compound (HLI-014-048) (38)
[0332] N-(tert-butyloxycarbonyl)-L-leucine-d.sub.7
(isopropyl-d.sub.7) hydrate (37) (250 mg, 0.98 mmol, C/D/N
Isotopes), 1-hydroxybenzotriazole hydrate (195 mg, 1.27 mmol) and
isoamylamine (94 mg, 1.07 mmol) were dissolved in CH.sub.2Cl.sub.2
(10 mL) at room temperature under nitrogen. With stirring,
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (282
mg, 1.47 mmol) was added. After stirring for three hours at room
temperature, the reaction was diluted with CH.sub.2Cl.sub.2 (80 mL)
and extracted with saturated NaHCO.sub.3 (2.times.) and brine
(1.times.). The organic layer was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide 315 mg of 38
as a white semi-solid: ESI MS m/z 308.4 (M+H).sup.+.
##STR00068##
Compound (HLI-014-054) (39)
[0333] Compound 38 was dissolved in methanol (5 mL) and
p-toluenesulfonic acid hydrate (237 mg, 1.25 mmol) was added with
stirring. The solution was heated to 70.degree. C. for four hours.
After cooling, the reaction was evaporated to dryness and the
residue treated with diethyl ether and hexanes. Removal of the
volatiles provided the tosic salt as a white foam: ESI MS m/z 208.0
(C.sub.11H.sub.18D.sub.7N.sub.2O.sup.+). To this material was added
(2S, 3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (160 mg, 1.00
mmol, Peptech) and CH.sub.2Cl.sub.2 (10 ml) to provide a
homogeneous solution. The solution was cooled to 0.degree. C.
(ice-water bath) under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-phosphate (418 mg, 1.1 mmol) followed by
diisopropylethylamine (358 .mu.L, 2.0 mmol) were added to the
reaction solution. The resulting reaction was stirred at 0.degree.
C. for one hour and another hour at room temperature. The reaction
was diluted with CH.sub.2Cl.sub.2 (100 ml) and extracted with
saturated NaHCO.sub.3 (2.times.) and brine (1.times.). The organic
layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated to provide product 39 as a yellow solid. The crude
product was filtered through a silica gel plug eluting with ethyl
acetate-hexane (1:9) followed by ethyl acetate-hexane (1:1).
Concentration of the 1:1 ethyl acetate-hexane elution fractions
provided the desired product 39 as a white solid (314 mg; 0.9 mmol;
90%). Recrystallization from diisopropyl ether-ethanol yielded
crystalline material: mp 122-123.degree. C.; H NMR (CDCl.sub.3, 500
MHz) .delta. 6.68 (d, J=8.6 Hz, 1H), 6.08-6.10 (m, 1H), 4.35-4.40
(m, 1H), 4.21-4.29 (m, 2H), 3.67 (d, J=1.8 Hz, 1H), 3.45 (d, J=1.7
Hz, 1H), 3.20-3.30 (m, 2H), 1.56-1.65 (m, 2H), 1.48-1.52 (m, 1H),
1.38 (q, J=7.3 Hz, 2H), 1.31 (t, J=7.2 Hz, 3H), 0.89-0.91 (d, J=6.6
Hz, 6H); .sup.13C NMR (CDCl.sub.3, 125 MHz) .delta. 171.02, 166.70,
166.22, 62.54, 53.99, 53.11, 51.61, 41.23, 38.49, 38.21, 28.51
(weak), 26.06, 24.11 (m, weak), 22.62, 22.58, 14.24; ESI MS m/z
350.4 (M+H)+; HPLC purity: 99.7%. t.sub.R=22.5 min (Condition I, UV
215 nm).
##STR00069##
Compound
(2S,3S)-3-((S)-4,5,5,5-d.sub.4-1-(isopentylamino)-1-oxo-4-(methy-
l-d.sub.3)pentan-2-ylcarbamoyl) oxirane-2-carboxylic acid
(ALP-480.000.01; HLI-014-058) (40)
[0334] The ethyl ester 39 from the above preparation (36 mg, 0.1
mmol) was dissolved in ethanol (2 mL). The stirred solution was
cooled to 0.degree. C. (ice-water bath) and treated with a solution
of 85% KOH in ethanol (70 .mu.L, 0.11 mmol). The resulting solution
was stirred at 0.degree. C. for 20 minutes and at room temperature
for 10 minutes. The reaction was concentrated to dryness and the
residue partitioned between water and ethyl acetate. The layers
were separated and the aqueous extracted once more with ethyl
acetate. The aqueous layer was acidified to pH 2 with 1 N HCl and
extracted with ethyl acetate (3.times.). The combined organic
layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated to provide 40 as an off-white powder (32 mg, 97%).
Recrystallization from ethyl acetate-hexanes yielded acid 40 as a
colorless crystal: mp 153.degree. C.; .sup.1H NMR (acetone-d.sub.6,
500 MHz) .delta.7.61 (d, J=8.3 Hz, 1H), 7.49 (br s, 1H), 4.48 (q,
J=7.5 Hz, 1H), 3.66 (d, J=1.0 Hz, 1H), 3.58 (s, 1H), 3.17-3.26 (m,
2H), 1.57-1.66 (m, 3H), 1.37 (q, J=7.2 Hz, 2H), 0.88 (d, J=6.6 Hz,
6H); .sup.13C NMR (acetone-d.sub.6, 125 MHz) 172.32, 168.99,
166.33, 54.37, 52.71, 52.41, 52.37 (weak), 52.32 (weak), 52.28
(weak), 42.37, 39.26, 38.29, 38.17, 26.44, 24.95 (m, weak), 22.84,
22.82, 22.75 (m, weak), 21.50 (m, weak); ESI MS m/z 322.2
(M+H).sup.+; HPLC purity: 96.8% t.sub.R=18.1 min (Condition I, UV
215 nm).
##STR00070##
Compound Boc-L-Leu-2-d.sub.1 (HLI-014-055) (41)
[0335] L-Leucine-2-d.sub.1 (188 mg; 1.4 mmol; C/D/N Isotopes) was
suspended in 8 mL of a 1:1 solution of water and tetrahydrofuran.
The suspension was stirred, cooled to 0.degree. C. (ice-water
bath), and treated with sodium bicarbonate (353 mg; 4.2 mmol)
followed by di-tert-butyl dicarbonate (373 mg; 1.7 mmol). The
resulting suspension was stirred at 0.degree. C. for 30 minutes and
then at room temperature for 68 hours. The reaction mixture was
extracted with ethyl acetate (3.times.). The aqueous layer was
acidified to pH=3 with 1 N HCl and extracted with ethyl acetate
(3.times.). All of the ethyl acetate extractions were combined,
washed with brine (3.times.), dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide a viscous
oil (317 mg, 97%) of N-(tert-butyloxycarbonyl)-L-leucine-2-d.sub.1:
ESI MS m/z 233.2 (M+H).sup.+.
##STR00071##
Compound (HLI-014-046) (42)
[0336] N-(tert-butyloxycarbonyl)-L-leucine-2-d.sub.1 (41) (405 mg,
1.74 mmol), 1-hydroxybenzotriazole hydrate (346 mg, 2.26 mmol), and
isoamylamine (166 mg, 1.91 mmol) were dissolved in CH.sub.2Cl.sub.2
(10 mL) at room temperature under nitrogen. With stirring,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (500
mg, 2.61 mmol) was added. After stirring for three hours at room
temperature, the reaction was diluted with CH.sub.2Cl.sub.2 (80 mL)
and extracted with saturated NaHCO.sub.3 (2.times.) and brine
(1.times.). The organic layer was dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide 525 mg of
crude product 42 as a white semi-solid: APCI MS m/z 302.4
(M+H).sup.+.
##STR00072##
[0337] Compound (HLI-014-059) (43).
[0338] Compound 42 was dissolved in methanol (9 mL) and
p-toluenesulfonic acid hydrate (414 mg, 2.18 mmol) was added with
stirring. The solution was heated to 70.degree. C. for two hours.
After cooling, the reaction was evaporated to dryness to provide
279 mg of the tosic salt as a white semi-solid: ESI MS m/z 202.2
(C.sub.11H.sub.24DN.sub.2O.sup.+). To this material was added (2S,
3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (279 mg, 1.74 mmol;
Peptech) and CH.sub.2Cl.sub.2 (15 ml) to provide a homogeneous
solution. The solution was cooled to 0.degree. C. (ice-water bath)
under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate (722 mg, 1.90 mmol) followed by
diisopropylethyl amine (779 .mu.L, 4.35 mmol) were added to the
reaction solution. The resulting reaction was stirred at 0.degree.
C. for one hour and for two hours at room temperature. The reaction
was diluted with CH.sub.2Cl.sub.2 (150 ml) and extracted with
saturated NaHCO.sub.3 (2.times.) and brine (1.times.). The organic
layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated. The crude product was filtered through a silica gel
plug eluting with ethyl acetate-hexane (1:9) followed by ethyl
acetate-hexane (1:1). Concentration of the 1:1 ethyl acetate-hexane
elution fractions provided the desired product 43 as a white solid
(468 mg, 78%). Recrystallization from diisopropyl ether-ethanol
yielded crystalline material: mp 122-123.degree. C. .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta.6.95 (s, 1H), 6.43 (br t, J=5.4 Hz,
1H), 4.19-4.27 (m, 2H), 3.67 (d, J=1.8 Hz, 1H), 3.46 (d, J=1.8 Hz,
1H), 3.23-3.30 (m, 1H), 3.14-3.20 (m, 1H), 1.49-1.62 (m, 4H), 1.36
(q, J=7.3 Hz, 2H), 1.29 (t, J=7.2 Hz, 3H), 0.87-0.91 (m, 12H);
.sup.13C NMR (CDCl.sub.3, 125 MHz) .delta.171.16, 166.81, 166.20,
62.47, 53.93, 52.96, 51.37 (m, weak), 41.40, 38.42, 38.14, 26.02,
24.99, 22.95, 22.59, 22.54, 22.41, 14.19; APCI MS m/z 344.2
(M+H).sup.+; HPLC purity: 98.6% t.sub.R=22.7 min (Condition I, UV
215 nm).
##STR00073##
Compound
(2S,3S)-3-((S)-2-d.sub.1-1-(isopentylamino)-4-methyl-1-oxopentan-
-2-ylcarbamoyl)oxirane-2-carboxylic acid (ALP-479.000.01;
HLI-014-060) (44)
[0339] The ethyl ester 43 from the above preparation (68 mg, 0.2
mmol) was dissolved in ethanol (3 mL). The stirred solution was
cooled to 0.degree. C. (ice-water bath) and treated with a solution
of 85% KOH in ethanol (140 .mu.L, 0.22 mmol). The resulting
solution was stirred at 0.degree. C. for 1 h and was concentrated
to dryness. The residue partitioned between water and ethyl
acetate. The layers were separated and the aqueous extracted once
more with ethyl acetate. The aqueous layer was acidified to pH 1
with 1 N HCl and extracted with ethyl acetate (3.times.). The
combined organic layers were dried over anhydrous Na.sub.2SO.sub.4,
filtered and concentrated to provide a white powder (42 mg, 67%).
Recrystallization from ethyl acetate-hexanes yielded acid 44 as a
colorless crystal: mp 152-153.degree. C.; .sup.1H NMR
(acetone-d.sub.6, 500 MHz) .delta.7.62 (br s, 1H), 7.50 (br s, 1H),
3.66 (d, J=1.7 Hz, 1H), 3.58 (d, J=1.6 Hz, 1H), 3.17-3.26 (m, 2H),
1.57-1.68 (m, 4H), 1.37 (q, J=7.1 Hz, 2H), 0.87-0.96 (m, 12H);
.sup.13C NMR (acetone-d.sub.6, 125 MHz) .delta. 172.37, 169.0,
166.33, 54.36, 54.34, 52.71, 52.13 (m, weak), 42.50, 42.46, 39.26,
38.29, 38.17, 26.45, 25.54, 23.45, 22.84, 22.82, 22.26; APCI MS m/z
316.2 (M+H).sup.+; HPLC purity: 94.0% t.sub.R=18.2 min (Condition
I, UV 215 nm).
##STR00074##
Compound Boc-L-Leu-3,3-d.sub.2 (HLI-014-065) (45)
[0340] L-Leucine-3,3-d.sub.2 (250 mg, 1.9 mmol, C/D/N Isotopes) was
suspended in 10 mL of a 1:1 solution of water and tetrahydrofuran.
The suspension was stirred, cooled to 0.degree. C. (ice-water
bath), and treated with sodium bicarbonate (474 mg, 5.6 mmol)
followed by di-tert-butyl dicarbonate (492 mg, 2.3 mmol). The
resulting suspension was stirred at 0.degree. C. for 30 minutes and
then at room temperature for 42 hours. The reaction mixture was
extracted with ethyl acetate (3.times.). The aqueous layer was
acidified to pH=2 with 1 N HCl and extracted with ethyl acetate
(3.times.). All of the ethyl acetate extractions were combined,
washed with brine (3.times.), dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide a viscous
oil (440 mg, 100%) of
N-(tert-butyloxycarbonyl)-L-leucine-3,3-d.sub.2: APCI MS m/z 234.2
(M+H).sup.+.
##STR00075##
Compound (HLI-014-071) (46)
[0341] N-(tert-butyloxycarbonyl)-L-leucine-3,3-d.sub.2 (45) (440
mg, 1.88 mmol), 1-hydroxybenzotriazole hydrate (374 mg, 2.44 mmol),
and isoamylamine (176 mg, 2.01 mmol) were dissolved in
CH.sub.2Cl.sub.2 (10 mL) at room temperature under nitrogen. With
stirring, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (540 mg, 2.82 mmol) was added. After stirring for 18
hours at room temperature, the reaction was diluted with
CH.sub.2Cl.sub.2 (80 mL) and extracted with saturated NaHCO.sub.3
(2.times.) and brine (1.times.). The organic layer was dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated to provide
525 mg of crude product 46 as a white foam: APCI MS m/z 303.4
(M+H).sup.+.
##STR00076##
(2S,3S)-Ethyl
3-((S)-3,3-d.sub.2-1-(isopentylamino)-4-methyl-1-oxopentan-2-ylcarbamoyl)
oxirane-2-carboxylate (HLI-014-075) (47)
[0342] This material was dissolved in methanol (10 mL) and
p-toluenesulfonic acid hydrate (447 mg, 2.35 mmol) was added with
stirring. The solution was heated to 70.degree. C. for two hours.
After cooling, the reaction was evaporated to dryness to provide
789 mg of tosic salt as a off-white foam: APCI MS m/z 203.2
(C.sub.11H.sub.23D.sub.2N.sub.2O.sup.+). To this material was added
(2S, 3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (304 mg, 1.90
mmol; Peptech) and CH.sub.2Cl.sub.2 (15 ml) to provide a
homogeneous solution. The solution was cooled to 0.degree. C.
(ice-water bath) under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate (794 mg, 2.10 mmol) followed by
diisopropylethyl amine (681 .mu.L, 3.80 mmol) were added to the
reaction solution. The resulting reaction was stirred at 0.degree.
C. for one hour and for two hours at room temperature. The reaction
was diluted with CH.sub.2Cl.sub.2 (100 ml) and extracted with
saturated NaHCO.sub.3 (2.times.) and brine (1.times.). The organic
layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and
concentrated. The crude product 47 was filtered through a silica
gel plug eluting with ethyl acetate-hexane (1:9) followed by ethyl
acetate-hexane (1:1). Concentration of the 1:1 ethyl acetate-hexane
elution fractions provided the desired product 47 as a white solid
(649 mg, 99%). Recrystallization from t-butylmethyl ether-ethanol
yielded crystalline material: .sup.1H NMR (CDCl.sub.3, 500 MHz)
.delta.6.89 (d, J=8.7 Hz, 1H), 6.36 (br t, J=5.4 Hz, 1H), 4.40 (d,
J=8.7 Hz, 1H), 4.19-4.28 (m, 2H), 3.67 (d, J=1.8 Hz, 1H), 3.46 (d,
J=1.7 Hz, 1H), 3.23-3.30 (m, 1H), 3.15-3.21 (m, 1H), 1.49-1.62 (m,
2H), 1.37 (q, J=7.3 Hz, 2H), 1.29 (t, J=7.1 Hz, 3H), 0.88-0.91 (m,
12H); .sup.13C NMR (CDCl.sub.3, 125 MHz) .delta.171.14, 166.79,
166.21, 62.49, 53.95, 53.00, 51.55, 40.70 (m, weak), 38.44, 38.16,
26.03, 24.85, 22.91, 22.60, 22.55, 22.35, 14.20; APCI MS m/z 345.2
(M+H)+; HPLC purity: 99.5% t.sub.R=22.78 min (Condition I, UV 215
nm).
##STR00077##
Compound
(2S,3S)-3-((S)-3,3-d.sub.2-1-(isopentylamino)-4-methyl-1-oxopent-
an-2-ylcarbamoyl)oxirane-2-carboxylic acid (ALP-487.000.01;
HLI-014-078) (48)
[0343] The ethyl ester 47 (101 mg, 0.29 mmol) was dissolved in
ethanol (4 mL). The stirred solution was treated with a solution of
85% KOH in ethanol (450 .mu.L, 0.29 mmol) at room temperature. The
resulting solution was stirred at room temperature for 1 h and was
concentrated to dryness. The residue was partitioned between water
and dichloromethane. The layers were separated and the aqueous
extracted once more with dichloromethane. The aqueous layer was
acidified to pH 2 with 1 N HCl and extracted with ethyl acetate
(3.times.). The combined organic layers were dried over anhydrous
Na.sub.2SO.sub.4, filtered and concentrated to provide acid 48 as a
white solid (85 mg, 92%). Recrystallization from ethyl
acetate-hexanes yielded acid 48 as a colorless crystal: mp
149-150.degree. C.; .sup.1H NMR (acetone-d.sub.6, 500 MHz)
.delta.7.63 (d, J=8.5 Hz, 1H), 7.50 (br s, 1H), 4.48 (d, J=8.6 Hz,
1H), 3.66 (d, J=1.7 Hz, 1H), 3.58 (d, J=1.8 Hz, 1H), 3.17-3.26 (m,
2H), 1.57-1.67 (m, 2H), 1.37 (q, J=7.2 Hz, 2H), 0.87-0.96 (m, 12H);
.sup.13C NMR (acetone-d.sub.6, 125 MHz) .delta. 172.39, 169.00,
166.41, 54.35, 54.33, 52.71, 52.32, 52.24 (m, weak), 41.50 (br m,
weak), 39.25, 38.30, 38.17, 26.44, 25.39, 23.39, 22.83, 22.81,
22.20; APCI MS m/z 317.2 (M+H).sup.+; HPLC purity: 97.4%
t.sub.R=18.1 min (Condition I, UV 215 nm).
##STR00078##
Compound (HLI-014-066) (50)
[0344] N-(tert-butyloxycarbonyl)-L-leucine-do hydrate (49) (250 mg,
0.96 mmol, C/D/N Isotopes), 1-hydroxybenzotriazole hydrate (191 mg,
1.25 mmol), and isoamylamine (92 mg, 1.06 mmol) were dissolved in
CH.sub.2Cl.sub.2 (10 mL) at room temperature under nitrogen. With
stirring, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (276 mg, 1.44 mmol) was added. After stirring for 18
hours at room temperature, the reaction was diluted with
CH.sub.2Cl.sub.2 (100 mL) and extracted with saturated NaHCO.sub.3
(2.times.) and brine (1.times.). The organic layer was dried over
anhydrous Na.sub.2SO.sub.4, filtered and concentrated to provide
313 mg of crude product 50 as a viscous oil: APCI MS m/z 311.4
(M+H).sup.+.
##STR00079##
Compound
(2S,3S)-3-((S)-2,3,3,4,5,5,5-d.sub.7-1-(isopentylamino)-1-oxo-4--
(methyl-d.sub.3)pentan-2-ylcarbamoyl) oxirane-2-carboxylate
(HLI-014-070) (51)
[0345] This material was dissolved in methanol (10 mL) and
p-toluenesulfonic acid hydrate (237 mg, 1.25 mmol) was added with
stirring. The solution was heated to 70.degree. C. for three hours.
After cooling, the reaction was evaporated to dryness to provide
440 mg of the tosic salt as a viscous oil: APCI MS m/z 211.2
(C.sub.11H.sub.15D.sub.10N.sub.2O.sup.+). To this material was
added (2S, 3S)-3-(ethoxycarbonyl)oxirane-2-carboxylic acid (160 mg,
1.00 mmol, Peptech) and CH.sub.2Cl.sub.2 (15 ml) to provide a
homogeneous solution. The solution was cooled to 0.degree. C.
(ice-water bath) under nitrogen. With stirring,
2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate (418 mg, 1.10 mmol) followed by
diisopropylethylamine (358 .mu.L, 2.00 mmol) were added to the
reaction solution. The resulting reaction was stirred at 0.degree.
C. for 30 minutes and for one hour at room temperature. The
reaction was diluted with CH.sub.2Cl.sub.2 (80 ml) and extracted
with saturated NaHCO.sub.3 (2.times.) and brine (1.times.). The
organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered
and concentrated. The crude product 51 was filtered through a
silica gel plug eluting with ethyl acetate-hexane (1:9) followed by
ethyl acetate-hexane (1:1). Concentration of the 1:1 ethyl
acetate-hexane elution fractions provided the desired product 51 as
a white solid (345 mg, 98%). Recrystallization from diisopropyl
ether-ethanol yielded crystalline material: .sup.1H NMR
(CDCl.sub.3, 500 MHz) .delta.6.84 (s, 1H), 6.43 (br t, J=5.3 Hz,
1H), 4.19-4.29 (m, 2H), 3.67 (d, J=1.7 Hz, 1H), 3.46 (d, J=1.7 Hz,
1H), 3.24-3.30 (m, 1H), 3.16-3.23 (m, 1H), 1.58 (septet, J=6.7 Hz,
1H), 1.37 (q, J=7.3 Hz, 2H), 1.30 (t, J=7.3 Hz, 3H), 0.89 (d, J=6.6
Hz, 6H); .sup.13C NMR (CDCl.sub.3, 125 MHz) .delta.171.13, 166.78,
166.22, 62.50, 53.96, 53.03, 51.24 (t, weak), 41.05 (br, weak),
38.45, 38.16, 26.04, 24.00 (br, weak), 22.61, 22.56, 14.21; APCI MS
m/z 353.4 (M+H)+; HPLC purity: 99.3% t.sub.R=22.6 min (Condition I,
UV 215 nm).
##STR00080##
Compound
(2S,3S)-3-((S)-2,3,3,4,5,5,5-d.sub.7-1-(isopentylamino)-1-oxo-4--
(methyl-d.sub.3)pentan-2-ylcarbamoyl) oxirane-2-carboxylic acid
(ALP-486.000.01; HLI-014-076) (52)
[0346] The ethyl ester 51 from the above preparation (105 mg, 0.3
mmol) was dissolved in ethanol (4 mL). The stirred solution was
treated with a solution of 85% KOH in ethanol (448 .mu.L; 0.3 mmol)
at room temperature. The resulting solution was stirred at room
temperature for 45 minutes and was concentrated to dryness. The
residue was partitioned between water and dichloromethane. The
layers were separated and the aqueous extracted once more with
dichloromethane. The aqueous layer was acidified to pH 2 with 1N
HCl and extracted with ethyl acetate (3.times.). The combined
organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered
and concentrated to provide acid 52 as a white solid (78 mg, 81%).
Recrystallization from ethyl acetate-hexanes yielded acid 52 as a
colorless crystal: mp 150-151.degree. C.; .sup.1H NMR
(acetone-d.sub.6, 500 MHz) .delta.7.62 (br s, 1H), 7.50 (br s, 1H),
3.66 (d, J=1.8 Hz, 1H), 3.58 (d, J=1.6 Hz, 1H), 3.17-3.27 (m, 2H),
1.59 (septet, J=6.7 Hz, 1H), 1.37 (q, J=7.2 Hz, 2H), 0.88 (d, J=6.7
Hz, 6H); .sup.13C NMR (acetone-d.sub.6, 125 MHz) .delta.172.41,
169.01, 166.38, 54.36, 52.70, 52.25 (br, weak), 41.50 (br, weak),
39.25, 38.28, 38.16, 26.44, 22.83, 22.81; APCI MS m/z 325.4
(M+H).sup.+; HPLC purity: 99.3% t.sub.R=18.0 min (Condition I, UV
215 nm).
Example 8: Modulation of A.beta. Levels in an Animal Model of
A.beta. Production
[0347] This example demonstrates the successful reduction of levels
of brain A1340 and A.beta.42 in wild-type guinea pigs, an animal
model of AP production. Wild-type guinea pigs are an art-accepted
model for normal A.beta. production and are known to produce
A.beta. peptides that are immunologically identical to human
A.beta.. Wild-type guinea pigs represent an animal model for a
physiological hallmark of Alzheimer's disease.
[0348] Oral Dosing of AB-007 in Guinea Pigs in DMSO Solution
[0349] Guinea Pigs #70 (3 weeks of age, Charles River Labs) were
gavaged once daily for 7 days with AB-007 suspended in DMSO (500
uL). 10 animals were administered each of the following doses: 0
mg/kg/day, 0.1 mg/kg/day, 0.5 mg/kg/day, 1 mg/kg/day, 5 mg/kg/day,
10 mg/kg/day, or 50 mg/kg/day.
[0350] Preparation of Samples
[0351] On the 7.sup.th day of administration of AB-007, 4 hours
after the final dosing, the animals were sacrificed (isoflurane
anesthesia with exsanguination), the brains removed and frozen in
liquid nitrogen. In preparation for testing, the frozen hemi-brains
were weighed, and homogenized at 1 mL/100 mg in 1 mL THB+PIC
(Tissue Homogenization Buffer: 250 mM sucrose, 20 mM TRIS base, 1
mM EDTA, 1 mM EGTA in ddH.sub.2O, pH 7.4). Homogenized brain
samples were snap frozen in liquid nitrogen.
[0352] Measurement of Brain A.beta.40 and Brain A.beta.42
(pg/mg)
[0353] Duplicate ELISA assays were run on thawed undiluted
homogenized brain samples (using Amyloid-.beta. (1-40) ELISA and
Amyloid-.beta. (1-42) ELISA kits and following instructions
provided by IBL International GmbH, Hamburg, Germany) to measure
the concentration of brain A.beta.40 and brain A.beta.42 in pg/mg
brain sample. As shown in FIG. 1, after 7 days of 10 mg/kg/day
dosing reduces brain A.beta.40 and of brain A.beta.42 by 42%
relative to vehicle control (0 mg/kg/day dosing). Measurement of
brain A.beta.40 and brain A.beta.42 (pg/mg) for each of the doses
from Example 8 are provided in FIG. 2 and FIG. 3 respectively.
[0354] Measurement of Cathepsin B Activity
[0355] Whole tissue brain homogenates (5 uL) were added to cold
1.times. cell Extraction Buffer (245 uL) (final protein
concentration of roughly 2.0 mg/mL). The lysate was incubated on
ice and centrifuged. The supernatant was transferred to a new tube
and kept on ice (cell lysate had approximate protein concentration
of 2 mg/mL). The Cathepsin B activity was measured using the
Cathepsin B Activity Assay Kit (Fluorometric) (ab65300, abeam,
Cambridge, Mass.). Measurement of Cathepsin B activity for each of
the doses from Example 8 are provided in FIG. 4.
[0356] Measurement of .beta.-Secretase Activity (BACE-1)
[0357] Whole tissue brain homogenates (5 uL) were added to cold
1.times. cell Extraction Buffer (245 uL) (final protein
concentration of roughly 2.0 mg/mL). The lysate was incubated on
ice and centrifuged. The supernatant was transferred to a new tube
and kept on ice (cell lysate had approximate protein concentration
of 2 mg/mL). The .beta.-secretase activity was measured using
.beta.-secretase Activity Kit (Catalog Number: FP002, R&D
Systems, Minneapolis, Minn.). Fluorescence between 495 and 510 nm
was measured, following EDANS excitation between 335 and 355 nm.
Measurement of BACE-1 for each of the doses from Example 8 are
provided in FIG. 5.
Example 9: Modulation of A.beta. Levels in an Animal Model of
A.beta. Production
[0358] This example presents data and analysis demonstrating that
oral administration of AB-007 (E64d, loxistatin) results in a dose
response reduction in brain A.beta.(40) and brain A.beta.(42)
peptides, CSF A3 and CSF A.beta.(42) peptides, and plasma
A.beta.(40) and A.beta.(42) peptides. These data demonstrate that
oral administration of AB-007 (E64d, loxistatin) to guinea pigs
results in a dose response reduction of brain cathepsin B activity,
and that brain cathepsin B inhibition reduces brain A.beta.(40) and
A.beta.(42) peptides. These data demonstrate that oral
administration of the invention's exemplary composition of the
invention "E64d7" (a hepta-deuterated isoform of E64d, illustrated
above) to guinea pigs results in a dose response reduction in brain
A.beta.(40) and brain A.beta.(42) peptides, cerebrospinal fluid
(CSF) AP and CSF A.beta.(42) peptides, and plasma A.beta.(40) and
A.beta.(42) peptides. These data demonstrate that once-a-day oral
administration of "E64d7" to guinea pigs results in a dose response
reduction of brain cathepsin B activity; and that brain cathepsin B
inhibition reduces brain A.beta.(40) and A.beta.(42) peptides.
These data demonstrate that feeding mice E64d-doped chow for 1 or 3
months caused a significant reduction in brain A.beta.(40) and
A.beta.(42) peptides in both the young and old mice relative to
age-matched controls.
[0359] This example presents data and analysis demonstrating that
that feeding APPlon mice E64d-doped chow for either 1 month or 3
months to either age group significantly reduced the mean latency
period relative to age-matched controls; thus, this treatment
improved the memory deficit in animals having either mild or severe
spatial memory deficit; and the data demonstrates that oral
administration of E64d is effective for improving the early and
late memory deficit that develops in Alzheimer's disease.
[0360] This example presents data and analysis demonstrating that
in general brain A.beta.(40) and A.beta.(42) is highly positively
correlated with spatial memory deficit and that treating the
animals with E64d results in about the same or slightly less
positive correlation in the young animals and a much greater
positive correlation in old animals relative to age-matched
controls. These data also illustrate the efficacy of E64d in
improving memory deficit in aged animals; and while the invention
is not limited by any particular mechanism of action, these also
data illustrate the efficacy of E64d in decreasing levels of brain
A.beta.(40) and A.beta.(42) peptide.
Materials and Methods
[0361] AB-007 (E64d, loxistatin)
[0362] AB-007 (E64d, loxistatin) was synthesized by American Life
Science Pharmaceuticals (San Diego, Calif.). Purity was determined
to be 99% by reverse-phase high-pressure liquid chromatograph
(RP-HPLC) and the identity of the compound confirmed by .sup.1H
nuclear magnetic resonance (NMR), melting point, elemental analysis
and liquid chromatography mass spectroscopy (LCMS). E64d exhibited
excellent stability with 99% of the compound remaining after a
5-week stability test at 60.degree. C.
[0363] Animal Models
[0364] Guinea Pigs. Guinea Pigs (Harley strain, average weight 400
g corresponding to animals about 6 weeks old) were obtained from
Charles River (Wilmington, Mass.). The animals were given free
access to standard chow and water before and during the
experiment.
[0365] Transgenic AD Mice. Transgenic mice expressing human APP
containing the wt .beta.-secretase site and the London mutant
.gamma.-secretase site sequences (also called "transgenic APPlon
mice", or "London APP mice") were generated at the Ralph A. Johnson
VA Medical Center (Charleston, S.C.) by methods previously
described, see e.g., Hook, et al. (2008) J. Biol. Chem.
283:7745-7753. Briefly, APP.sub.Ln was generated using
site-directed mutagenesis to insert the V7171 London mutation into
human APP cDNA. The mouse strain was created in a C57BL/6 mouse
background using the Thy-1.2 expression cassette driven by the
Thy-1 promoter containing an SV40 polyadenylation site. Mice were
given free access to food and water before and during the
experiment.
Oral Formulations
[0366] Gavage Formulation: Delivering a drug by gavage offers the
advantage of accurate dosing but is traumatic and thus only
suitable for relatively short dosing periods. Gavage delivery was
used for the guinea pig work. E64d was resuspended in DMSO at
indicated concentrations (see Figures, and below) and guinea pigs
were gavaged daily using a feeding tube. Vehicle control animals
were provided DMSO alone.
[0367] Mouse Chow Formulation: Delivering a drug by doped chow
feeding allows for extended dosing periods without trauma but
results in less accurate dosing because individual animals consume
variable amounts of chow. Doped chow feeding was used for the
transgenic AD mice experiments. The guinea pig data found that a 10
mg/kg/day E64d oral dose was maximally effective at lowering brain
A.beta. and cathepsin B activity and, therefore, that dose was used
for the doped chow studies to increase the chances of a positive
outcome. E64d doped mouse chow was prepared in the mouse chow
routinely used to feed the transgenic AD mice (2018, Teklad Global
18% Protein Rodent Diet, Harlan Laboratories, Inc. of Madison,
Wis.). Briefly, 40 milligrams (mg) of E64d per kilogram (kg) of
chow was processed into standard 0.5 inch pellets according to the
manufacturer's procedure. The resulting doped chow was analyzed by
RP-HPLC and found to contain 20 mg of E64d per kg of chow. Studies
of chow spiked with E64d found that RP-HPLC spike recovery of about
50%, suggesting that the chow contained 40 mg E64d per kg of chow.
The mice consume on average about 5 grams (gm) of chow per day and
had an average body weight of about 20 gm. Thus, an average mice
ate a calculated daily E64d dose of 10 mg/kg.
[0368] Oral Dosing
[0369] Guinea Pig Gavage Dosing: Guinea pigs were gavaged daily for
one week. Guinea pigs were lightly anesthetized with isoflurane
prior to gavaging. 250 .mu.l of compound solution was gavaged daily
per animal and animals were sacrificed 4 hrs after the last
dosing.
[0370] Mouse Chow Dosing: Two age groups of transgenic AD mice
treated with two dose regimes were studied. One group of animals
aged for 6 months and the other for 12 months before beginning E64d
treatment, which consisted of either feeding the animals E64d doped
chow for 1 or 3 months (controls received normal chow for the same
time periods). Thus, all animals were fed normal chow up to the
appropriate age and then the experimental animals switched to
feeding on E64d doped chow. At all times and for both formulations,
the mice were fed ad libitum. There was no difference in the amount
of E64d doped and normal chow consumed.
[0371] Brain A.beta. Analysis
[0372] The brain AP analysis method was that previously described
for guinea pigs and transgenic AD mice, see e.g., Hook (2007) Biol.
Chem. 388:979-983. Animals were sacrificed and brain extracts were
prepared as previously described, see e.g., Hook (2008) J. Biol.
Chem. 283:7745-7753. Briefly, brain extracts were homogenized (1:3
weight/volume of buffer) in buffer of 5 M guanidine HCl in 50 mM
Tris-HCl, pH 7.6, 150 mM NaCl, plus protease inhibitors (Sigma).
Homogenates were diluted to 0.5 M guanidine and centrifuged
(200,000 g for 20 min), and supernatant and pellet fractions were
collected. Protein content was determined by the Bradford method.
Enzyme-linked immunosorbent assays (ELISAs) measured A.beta.
peptides by methods previously described, see e.g., Hook (2007)
Biol. Chem. 388:247-252. The pellet from the brain extract
procedure was sonicated in 6 M guanidine and centrifuged at 200,000
g for 20 min at 4.degree. C., and the supernatant was diluted to
0.5 M guanidine. The two supernatants were combined, and A.beta.40
and A.beta.42 were determined using ELISA kits specific for each
peptide (IBL, JP27718 and JP27711).
[0373] Cerebrospinal Fluid (CSF) A.beta.
[0374] The CSF A.beta. in guinea pigs was measured as follows: CSF
was collected from the guinea pig brains and protease inhibitors
were added and A.beta.40 and A.beta.42 were determined using ELISA
kits specific for each peptide (IBL, JP27718 and JP27711).
[0375] Plasma A.beta.
[0376] The plasma A.beta. in guinea pigs was measured as follows:
Plasma samples were collected from the guinea pigs following
sacrifice, protease inhibitors were added and A.beta.40 and
A.beta.42 were determined using ELISA kits specific for each
peptide (IBL, JP27718 and JP27711).
[0377] Brain Cathepsin B Analysis
[0378] The brain cathepsin B activity in guinea pigs was measured
as follows: Cathepsin B activity was determined using a
fluorometric assay kit from Abcam, Inc. (catalog ab65300;
Cambridge, Mass.) as described by the manufacturer.
[0379] Brain BACE1 Analysis
[0380] The brain BACE1 activity in guinea pigs was measured by Beta
secretase activity was determined using a fluorometric assay kit
from Abcam (ab65357) as described by the manufacturer.
[0381] Spatial Memory Deficit
[0382] The memory deficit in transgenic APP.sub.ln mice (APPlon
mice) was measured using the Morris water maze test. Briefly, the
spatial memory capability of each animal was assessed by the Morris
water maze test as described e.g., by S. Mueller-Steiner (2006)
Neuron 51:703-714; using catalog part no. 700-0718-4W, of SD
Instruments, San Diego, Calif. The Morris water maze test evaluates
memory in a swimming test. Mice were individually trained in a 1.2
meter open field water maze in a pool filled with water to a depth
of 30 cm and maintained at 25.degree. C. An escape platform (10 cm
square) was placed 1 cm below the surface of the water. All animals
underwent nonspatial pretraining for 3 consecutive days, which
prepared the animals for the final behavioral test to determine the
retention of memory to find the platform. Two days following the
nonspatial pretraining, the hidden platform was placed in the
center of one quadrant of the pool, the animal was released facing
the pool wall in a random fashion, the time was recorded (latency
period), and the distance traveled to reach the platform was
measured using video recording (Smart Video Tracking System; SD
Instruments).
[0383] Animal treatment: Animal studies were conducted according to
regulations by the NIH and as approved by the IACUC at the Medical
University of South Carolina and Ralph H. Johnson VA Medical
Center.
[0384] FIG. 6 graphically illustrates data showing that once-a-day
for one week oral administration of E64d to guinea pigs results in
a dose response reduction in FIG. 6(A) brain A.beta.(40), FIG. 6
(B) brain A.beta.(42), FIG. 6 (C) CSF A.beta., FIG. 6 (D) CSF
A.beta.(42), FIG. 6 (E) plasma A.beta.(40) and FIG. 6 (F) plasma
A.beta.(42). Similar biphasic reductions in AP occurred for both
peptide forms and in all compartments. Stars represent means that
are statistically different from the no E64d dose group (student's
t test * p=0.02, *** p<0.0001).
[0385] FIG. 7A (upper graph) graphically illustrates data showing
that once-a-day for one week oral administration of AB-007 (E64d,
loxistatin) to guinea pigs results in a dose response reduction
brain cathepsin B activity, which is similar to the biphasic
lowering of A.beta., e.g., as demonstrated in FIG. 6. In contrast,
as illustrated in FIG. 7(B) (lower graph), that same treatment
results in an increase in brain BACE1 activity. Stars represent
means that are statistically different from the no E64d dose group
(student's t test ** p<0.006, *** p<0.001).
[0386] FIG. 8 graphically illustrates data showing paired guinea
pig data for brain A.beta.(40) or A.beta.(42) vs brain cathepsin B
or BACE1 activity, respectively for the combined AB-007 (E64d,
loxistatin) dose groups. FIG. 8(A) Brain A.beta.(40) vs brain
cathepsin B activity shows a significant positive correlation
(linear regression analysis, r squared 0.98, 95% confidence
interval for the slope is 0.21 to 0.22, which is a significant
non-zero slope at p<0.0001). This result is consistent with the
hypothesis that brain cathepsin B inhibition reduces brain
A.beta.(40). FIG. 8(B) Brain A.beta.(42) vs brain cathepsin B
activity also has a significant positive correlation (r squared
0.87, 95% confidence interval for the slope is 0.45 to 0.56, which
is a significant non-zero slope at p<0.0001). This data is also
consistent with brain cathepsin B inhibition reducing brain
A.beta.(42). FIG. 8(C): Brain A.beta.(40) vs brain BACE1 activity
demonstrates a small negative correlation (r squared 0.45, 95%
confidence interval for the slope is -0.06 to -0.03, which is a
significant non-zero slope at p<0.0001). These data are not
consistent with brain BACE1 inhibition decreasing brain
A.beta.(40). FIG. 8(D) Brain A.beta.(42) vs brain BACE1 activity
has a negative correlation (r squared 0.36, 95% confidence interval
the slope is -0.14 to -0.06, which is a significant non-zero slope
at p<0.001). These data are also not consistent with brain BACE1
inhibition causing the decrease in A.beta.(42).
[0387] FIG. 9 graphically illustrates data showing paired guinea
pig data for brain A.beta.(40) vs A.beta.(42) and brain cathepsin B
vs BACE1 activity for all AB-007 (E64d, loxistatin) dose groups
combined. FIG. 9(A) Brain A.beta.(40) vs A.beta.(42) shows a
significant positive correlation (linear regression analysis, r
squared 0.89, 95% confidence interval for the slope is 0.34 to
0.42, which is a significant non-zero slope at p<0.0001). This
result is consistent with both forms of A.beta. being reduced in
each animal at a ratio of about 1 to 0.4 A.beta.(40) to
A.beta.(42). FIG. 9(B) Brain cathepsin B vs BACE1 activity shows a
slight negative correlation (r squared 0.47, 95% confidence
interval for the slope is -0.27 to -0.14, which is a significant
non-zero slope at p<0.0001). These data are consistent with
brain BACE1 activity compensating for decreased cathepsin B
activity.
[0388] FIG. 10 graphically illustrates data showing that once-a-day
for a week oral administration of the invention's exemplary
composition of the invention "E64d7" (a hepta-deuterated isoform of
E64d, illustrated above) to guinea pigs results in a dose response
reduction in FIG. 10(A) brain A.beta.(40), FIG. 10(B) brain
A.beta.(42), FIG. 10(C) CSF A.beta., FIG. 10(D) CSF A.beta.(42),
FIG. 10(E) plasma A.beta.(40) and FIG. 10(F) plasma A.beta.(42).
Similar biphasic reductions in A3 occurred for both peptide forms
and in all compartments. Stars represent means that are
statistically different from the no E64d dose group (student's t
test ** p<0.008, *** p<0.0001).
[0389] FIG. 11A (upper graph) graphically illustrates data showing
that once-a-day oral administration of the exemplary composition of
the invention "E64d7" to guinea pigs results in a dose response
reduction of brain cathepsin B activity, which is similar to the
biphasic lowering of A.beta.. In contrast, FIG. 11B (lower graph)
graphically illustrates data showing that same treatment results in
an increase in brain BACE1 activity. Stars represent means that are
statistically different from the no E64d dose group (student's t
test * p<0.04, ** p<0.005).
[0390] FIG. 12 graphically illustrates data showing paired guinea
pig data for brain A.beta.(40) or A.beta.(42) vs brain cathepsin B
or BACE1 activity, respectively for the combined "E64d7" dose
groups. FIG. 12(A) Brain A.beta.(40) vs brain cathepsin B activity
shows a significant positive correlation (linear regression
analysis, r squared 0.97, 95% confidence interval for the slope is
0.20 to 0.22, which is a significant non-zero slope at
p<0.0001). This result is consistent with the hypothesis that
brain cathepsin B inhibition reduces brain A.beta.(40). FIG. 12(B)
Brain A.beta.(42) vs brain cathepsin B activity also has a
significant positive correlation (r squared 0.87, 95% confidence
interval for the slope is 0.43 to 0.54, which is a significant
non-zero slope at p<0.0001). This data is also consistent with
brain cathepsin B inhibition reducing brain A.beta.(42). FIG. 12(C)
Brain A.beta.(40) vs brain BACE1 activity demonstrates a small
negative correlation (r squared 0.24, 95% confidence interval for
the slope is -0.05 to -0.02, which is a significant non-zero slope
at p=0.0003). These data are not consistent with brain BACE1
inhibition decreasing brain A.beta.(40). FIG. 12(D) Brain
A.beta.(42) vs brain BACE1 activity has a negative correlation (r
squared 0.28, 95% confidence interval the slope is -0.14 to -0.05,
which is a significant non-zero slope at p<0.001). These data
are also not consistent with brain BACE1 inhibition causing the
decrease in A.beta.(42).
[0391] FIG. 13 graphically illustrates data showing paired guinea
pig data for brain A.beta.(40) vs A.beta.(42) and brain cathepsin B
vs BACE1 activity for all "E64d7" dose groups combined. FIG. 13(A)
Brain A.beta.(40) vs A.beta.(42) shows a significant positive
correlation (linear regression analysis, r squared 0.90, 95%
confidence interval for the slope is 0.34 to 0.42, which is a
significant non-zero slope at p<0.0001). This result is
consistent with both forms of A.beta. being reduced in each animal
at a ratio of about 1 to 0.4 A.beta.(40) to A.beta.(42). FIG. 13(B)
Brain cathepsin B vs BACE1 activity shows a slight negative
correlation (r squared 0.22, 95% confidence interval for the slope
is -0.25 to -0.07, which is a significant non-zero slope at
p=0.0007). These data are consistent with brain BACE1 activity
compensating for decreased cathepsin B activity.
[0392] FIG. 14 graphically illustrates data showing the brain
A.beta.(40) and A.beta.(42) data from the AB-007 (E64d,
loxistatin)-doped mouse chow experiments. The results from two
experimental groups are shown. The group labeled "Young mice" were
fed either E64d doped chow beginning at 6 months of age as
indicated by the arrow. The "Old mice" group began feeding on E64d
doped chow beginning at 12 months of age as indicated by the arrow.
Both groups were fed E64d doped chow for either 1 or 3 months after
which they were sacrificed. Prior to those ages, experimental and
control mice were fed control chow and control mice continued on
that diet during the experimental period. FIG. 14(A) Feeding the
E64d doped chow for 1 or 3 months caused a significant reduction in
brain A.beta.(40) in both the young and old mice relative to
age-matched controls (*** p<0.0001, student's t test). FIG.
14(B) The E64d doped chow fed for 1 or 3 months also caused a
significant reduction in brain A.beta.(42) in both the young and
old mice relative age-matched controls (** p<0.003, ***
p<0.0001, student's t test, n=10 animals per group). These data
show that oral AB-007 (E64d, loxistatin) administration can reduce
brain AP in an animal model of Alzheimer's disease.
[0393] FIG. 15 graphically compares the data shown in FIG. 14, for
brain A.beta.(40) and A.beta.(42) levels in young and old animals
feed AB-007 (E64d, loxistatin)-doped doped chow for 1 or 3 months.
In all cases, brain A.beta. was significantly higher in the old
mice treated with either regime. FIG. 15(A) The brain A.beta.(40)
data from 1 month feeding, FIG. 15(B) the brain A.beta.(40) data
from 3 month feeding, FIG. 15(C) the brain A.beta.(42) data from 1
month feeding, and FIG. 15(D) the brain A.beta.(42) data from 3
month feeding are shown (*** p<0.0001, student's t test).
[0394] FIG. 16 graphically illustrates data showing the effects of
AB-007 (E64d, loxistatin)-doped chow on the spatial memory deficit
that develops in transgenic APPlon (London APP) mice, see above
description, and e.g. Hook, et al. (2008) supra. The Morris water
maze test was used to measure the latency period, which is the time
it takes an animal to swim to a submerged and not visible platform
after having been previously trained as to the location of that
platform. The longer the latency period, the poorer the spatial
memory. Two groups of animals were evaluated, young and old mice.
Young mice began feeding on E64d doped chow at 6 months of age
(arrow), which is when only mild memory deficit develops. Old mice
began feeding on E64d doped chow at 12 month of age (arrow), which
is when severe memory deficit develops. Both experimental and
control mice were transgenic APPlon mice. The data show that
feeding the mice the E64d-doped chow for either 1 month or 3 months
to either age group significantly reduced the mean latency period
relative to age-matched controls (*** p<0.0001, student's t
test, n=10 animals per group). Thus, the treatment improved the
memory deficit in animals having either mild or severe spatial
memory deficit. The data suggest that oral administration of AB-007
(E64d, loxistatin) is effective for improving the early and late
memory deficit that develops in Alzheimer's disease.
[0395] FIG. 17 graphically illustrates data showing a paired data
analysis from the mouse experiments between brain A.beta.(40) or
A.beta.(42) peptides and latency period for young and old mice
either treated or not with AB-007 (E64d, loxistatin). FIG. 17(A)
graphically illustrates the effect of feeding young mice either the
control or E64d-doped chow for 1 month on brain A.beta.(40) peptide
and latency period is shown. Both control and E64d E64d-doped chow
fed mice exhibited a positive correlation between brain A.beta.(40)
peptide and latency period (r squared 0.98 and 0.96 for control and
treated, respectively). The slope of the linear regression analysis
for the control was slightly more positive than the treated and
that was statistically different (95% confidence interval for the
slope was 0.14 to 0.16 and 0.095 and 0.12 for the control and
treated respectively, difference in the slopes p<0.0001).
[0396] FIG. 17(B) graphically illustrates the effect of the
E64d-doped chow feeding on young animals on brain A.beta.(42)
peptide vs. latency period is shown. Both control and E64d-fed mice
exhibited a positive correlation between brain A.beta.(42) peptide
and latency period (r squared 0.97 and 0.86 for the control and
treated, respectively). The slope of the linear regression analysis
for the control and treated animals was not significantly different
(95% confidence interval was 0.80 to 1.00 and 0.68 to 1.03 for the
control and treated, respectively).
[0397] FIG. 17(C) graphically illustrates the effect of the
E64d-doped chow feeding on old animals on brain A.beta.(40) peptide
and latency period is shown. Both control and E64d-fcd mice had a
positive correlation between on brain A.beta.(40) and latency
period (r squared 0.90 and 0.98 for control and treated,
respectively) (in other words, the more brain A.beta.(40) peptide
the longer the latency period, where a longer latency period
indicates more brain function deficit, i.e., a poorer memory). The
slope of the linear analysis for the control was significantly less
than that for the E64d treated animals (95% confidence interval was
0.06 to 0.82 and 0.12 to 0.13 for the control and treated,
respectively, p<0.0001).
[0398] FIG. 17(D) graphically illustrates the effect of the
E64d-doped chow feeding on old animals on brain A.beta.(42) and
latency period is shown. Both control and E64d fed mice had a
positive correlation between on brain A.beta.(42) peptide and
latency period (r squared 0.92 and 0.93 for control and treated,
respectively) (in other words, the more brain A.beta.(42) peptide
the longer the latency period, where a longer latency period
indicates more brain function deficit, i.e., a poorer memory). The
slope of the linear analysis for the control was significantly less
than that for the E64d treated animals (95% confidence interval was
0.06 to 0.11 to 0.15 and 0.25 to 0.33 for the control and treated,
respectively, p<0.0001).
SUMMARY Mouse Chow Data Analysis
Percent Reduction in A.beta.3
TABLE-US-00003 [0399] Mouse Age Mean Control Mean treated
Difference % Control A.beta. 40 I month feeding Young 95.2 47.7
47.5 50 Old 230.0 87.1 142.9 62 A.beta. 40 3 month feeding Young
126.1 38.7 87.4 69 Old 286.5 91.2 195.3 68 Summary A.beta. 40 (1
& 3 month feeding) Mouse Age Mean % Control Young 60 Old 65 All
Ages 64
TABLE-US-00004 Mouse Age Mean Control Mean treated Difference %
Control A.beta. 42 1 month feeding Young 11.5 5.5 6 52 Old 58.5
31.9 26.6 45 A.beta. 42 3 month feeding Young 16.2 4.5 11.7 72 Old
99.8 61.3 38.5 39 Summary A.beta. 42 (1 & 3 month feeding)
Mouse Age Mean % Control Young 62 Old 42 All Ages 52
[0400] In summary, these data show that in general brain
A.beta.(40) and A.beta.(42) peptides are highly positively
correlated with spatial memory deficit and that treating the
animals with E64d results in about the same or slightly less
positive correlation in the young animals and a much greater
positive correlation in old animals relative to age-matched
controls. These data illustrate the efficacy of E64d in improving
memory deficit in aged animals; and while the invention is not
limited by any particular mechanism of action, these also data
illustrate the efficacy of E64d in decreasing levels of brain
A.beta.(40) and A.beta.(42) peptide.
[0401] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this application that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims. A number
of aspects of the invention have been described. Nevertheless, it
will be understood that various modifications may be made without
departing from the spirit and scope of the invention. Accordingly,
other aspects are within the scope of the following claims.
[0402] All publications, databases, patents, and patent
applications cited in this specification are herein expressly
incorporated by reference as if each was specifically and
individually indicated to be incorporated by reference.
* * * * *