U.S. patent application number 11/912293 was filed with the patent office on 2009-09-17 for n-acetylcysteine amide (nac amide) for the treatment of diseases and conditions associated with oxidative stress.
Invention is credited to Glenn A. Goldstein.
Application Number | 20090234011 11/912293 |
Document ID | / |
Family ID | 37215387 |
Filed Date | 2009-09-17 |
United States Patent
Application |
20090234011 |
Kind Code |
A1 |
Goldstein; Glenn A. |
September 17, 2009 |
N-ACETYLCYSTEINE AMIDE (NAC AMIDE) FOR THE TREATMENT OF DISEASES
AND CONDITIONS ASSOCIATED WITH OXIDATIVE STRESS
Abstract
Methods and compositions comprising N-acetylcysteine amide (NAC
amide) and derivatives thereof are used in treatments and therapies
for human and non-human mammalian diseases, disorders, conditions
and pathologies. Pharmaceutically or physiologically acceptable
compositions of NAC amide or derivatives thereof are administered
alone, or in combination with other suitable agents, to reduce,
prevent, or counteract oxidative stress and free radical oxidant
formation and overproduction in cells and tissues, as well as to
provide a new source of glutathione.
Inventors: |
Goldstein; Glenn A.; (New
York, NY) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
37215387 |
Appl. No.: |
11/912293 |
Filed: |
April 21, 2006 |
PCT Filed: |
April 21, 2006 |
PCT NO: |
PCT/US06/15548 |
371 Date: |
November 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60673561 |
Apr 21, 2005 |
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60705967 |
Aug 5, 2005 |
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Current U.S.
Class: |
514/563 ;
435/375; 435/6.16; 506/7; 562/567 |
Current CPC
Class: |
A61P 11/06 20180101;
A61P 33/06 20180101; A61P 43/00 20180101; A61P 31/18 20180101; A61P
35/00 20180101; A61P 3/10 20180101; A61K 31/16 20130101; A61P 25/00
20180101; Y02A 50/411 20180101; A61P 9/00 20180101; A61P 31/14
20180101; A61P 11/00 20180101; A61P 27/02 20180101; Y02A 50/30
20180101; A61P 11/16 20180101; A61P 31/04 20180101; A61P 19/08
20180101; C07C 323/41 20130101; A61P 19/04 20180101; A61P 39/06
20180101; C07C 233/36 20130101; A61P 31/20 20180101; A61P 39/02
20180101; A61P 7/00 20180101; A61P 9/04 20180101; C07C 233/18
20130101; A61P 21/00 20180101; C07C 323/60 20130101; A61P 19/10
20180101; A61P 7/06 20180101; A61P 17/02 20180101; A61P 31/00
20180101; A61P 29/00 20180101 |
Class at
Publication: |
514/563 ; 435/6;
506/7; 435/375; 562/567 |
International
Class: |
A61K 31/195 20060101
A61K031/195; C12Q 1/68 20060101 C12Q001/68; C40B 30/00 20060101
C40B030/00; C12N 5/02 20060101 C12N005/02; C07C 229/00 20060101
C07C229/00 |
Claims
1. A pharmaceutical composition for increasing glutathione levels
to reduce overproduction of oxidants in cells and tissues,
comprising N-acetylcysteine amide (NAC amide), or a
pharmaceutically acceptable salt, ester, or derivative thereof.
2. A method of increasing antioxidant levels in cells and tissues
of an organism, comprising administering NAC amide to the organism
in an amount effective to increase antioxidant levels.
3. The method according to claim 2, wherein said organism is a
human being suffering from a condition, disease, disorder, or
pathology associated with over production of oxidants.
4. The method according to claim 3, wherein said condition,
disease, disorder, or pathology is selected from the group
consisting of AIDS, diabetes, macular degeneration, congestive
heart failure, cardiovascular disease, coronary artery restenosis,
lung disease, inflammatory disease, asthma, RNA virus infection,
DNA virus infection, sepsis, sepsis, osteoporosis, bone disease,
infection by microorganisms, toxin exposure, radiation exposure,
burn trauma, prion disease, neurological disease, blood disease,
blood cell disease, arterial disease and muscle disease.
5. The method according to claim 4, wherein the condition, disease,
disorder, or pathology is malaria.
6. The method according to claim 4, wherein the condition, disease,
disorder, or pathology is tuberculosis.
7. The method according to claim 4, wherein the blood disease is
sickle cell anemia.
8. A method of protecting an organism from radiation-induced
oxidative stress, comprising administering a radioprotective amount
of NAC amide or a derivative of NAC amide to the organism.
9. The method according to claim 8, wherein NAC amide or derivative
of NAC amide is administered orally in an amount of 500 mg/kg.
10. A method of increasing levels of thiol antioxidants in an
organism, comprising administering NAC amide or derivative of NAC
amide to the organism in an amount effective to increase thiol
antioxidant levels.
11. The method according to claim 10, wherein the thiol antioxidant
is glutathione or cysteine.
12. The method according to claim 10, wherein the levels of thiol
antioxidant increase in liver and plasma.
13. The method according to claim 10, wherein NAC amide or
derivative of NAC amide is administered orally in an amount of 500
mg/kg.
14. A method of killing or inhibiting the growth of bacteria in
cells of an infected host, comprising: providing NAC amide or
derivative of NAC amide in an amount effective to induce the
production of HIF-1 or HIF-1 .alpha. in white blood cells of the
host, thereby enhancing the capacity of the white blood cells to
kill or inhibit the growth of the microorganisms.
15. A method of blocking the effects of Rac1b-induced ROS
production associated with metalloproteinase activity, comprising:
administering or introducing NAC amide or derivative of NAC amide
to cells, tissues, and/or a subject in need thereof, thereby
targeting molecules in the pathway leading to tissue damage and
degradation.
16. A method of blocking or inhibiting the effects of MMP-3
metalloproteinase on Rac1b-induced ROS production, comprising:
administering or introducing NAC amide or derivative of NAC amide
to cells, tissues, and/or a subject in need thereof, to block or
inhibit the activity of MMP-3 which leads to tissue damage and
degradation.
17. A method of stimulating endogenous production of cytokines and
hematopoietic factors, comprising: administering or introducing NAC
amide or derivative of NAC amide to cells, tissues, and/or a
subject in need thereof for a period of time to stimulate the
endogenous production to obtain a pre-determined, desired
therapeutic effect.
18. A method of detecting NAC-amide responsive changes in gene
expression in a cell, tissue, and/or a subject, comprising:
administering or introducing NAC amide or derivative of NAC amide
to the cell, tissue, and/or subject for a period of time to induce
changes in gene expression and detecting the changes in gene
expression.
19. The method of claim 18, wherein cell is an endothelial
cell.
20. The method of claim 18, wherein the tissue is vascular
tissue.
21. The method of claim 18, wherein the changes in gene expression
are detected by microarray analysis, RT-PCR, Northern Blotting,
immunofluorescence, immunoblotting, or enzyme-linked immunosorbent
assay.
22. The method according to claim 18, comprising administering or
providing NAC amide or derivative of NAC amide coupled to
nanoparticles.
23. A method of directed delivery of NAC amide or derivative of NAC
amide to host cells expressing high levels of surface receptor for
a ligand, comprising: a) coupling NAC amide or derivative of NAC
amide to the surface receptor ligand to form a NAC amide-ligand
conjugate; b) adsorbing the NAC amide-ligand conjugate onto
nanoparticles; and c) introducing the nanoparticles of step (b)
into the host.
24. A method of directed delivery of NAC amide or derivative of NAC
amide to host cells expressing high levels of surface receptor for
a ligand, comprising: a) conjugating acetylated dendritic
nanopolymers to a ligand; b) coupling the conjugated ligand of step
(a) to NAC amide or derivative of NAC amide to form NAC
amide-ligand nanoparticles; and c) introducing the nanoparticles of
step (b) into the host.
25. The method according to claim 23 or claim 24, wherein the
ligand is folic acid or glutathione.
26. The method according to claim 23 or claim 24, wherein the
nanoparticles are PAMAM dendritic polymers.
27. A compound of formula I: ##STR00017## wherein: R.sub.1 is OH,
SH, or S--S-Z; X is C or N; Y is NH.sub.2, OH, CH.sub.3--C.dbd.O,
or NH--CH.sub.3; R.sub.2 is absent, H, or .dbd.O R.sub.3 is absent
or ##STR00018## wherein: R.sub.4 is NH or O; R.sub.5 is CF.sub.3,
NH.sub.2, or CH.sub.3 and wherein: Z is ##STR00019## with the
proviso that if R.sub.1 is S--S-Z, X and X' are the same, Y and Y'
are the same, R.sub.2 and R.sub.6 are the same, and R.sub.3 and
R.sub.7 are the same.
28. The compound of claim 27, wherein R.sub.1 is S, X is C, Y is
NH.sub.2, R.sub.2 is .dbd.O, R.sub.3 is ##STR00020## R.sub.4 is O,
and R.sub.5 is CH.sub.3.
29. The compound of claim 28, wherein the compound is chiral and is
selected from the group consisting of a D-isomer, a L-isomer, and a
racemic mixture of D- and L-isomers.
30. The compound of claim 27, wherein R.sub.1 is S, X is C, Y is
NH--CH.sub.3, R.sub.2 is H, R.sub.3 is ##STR00021## R.sub.4 is O,
and R.sub.5 is CH.sub.3.
31. The compound of claim 30, wherein the compound is chiral and is
selected from the group consisting of a D-isomer, a L-isomer, and a
racemic mixture of D- and L-isomers.
32. The compound of claim 27, wherein R.sub.1 is S, X is N, Y is
CH.sub.3--C.dbd.O, R.sub.2 is H, and R.sub.3 is absent.
33. The compound of claim 27, wherein R.sub.1 is S, X is C, Y is
NH.sub.2, R.sub.2 is .dbd.O, R.sub.3 is ##STR00022## R.sub.4 is O,
and R.sub.5 is CF.sub.3.
34. The compound of claim 33, wherein the compound is chiral and is
selected from the group consisting of a D-isomer, a L-isomer, and a
racemic mixture of D- and L-isomers.
35. The compound of claim 27, wherein R.sub.1 is O, X is C, Y is
NH.sub.2, R.sub.2 is .dbd.O, R.sub.3 is ##STR00023## R.sub.4 is O,
and R.sub.5 is CH.sub.3.
36. The compound of claim 35, wherein the compound is chiral and is
selected from the group consisting of a D-isomer, a L-isomer, and a
racemic mixture of D- and L-isomers.
37. The compound of claim 27, wherein R.sub.1 is S, X is C, Y is
OH, R.sub.2 is absent, R.sub.3 is ##STR00024## R.sub.4 is O, and
R.sub.5 is CH.sub.3.
38. The compound of claim 37, wherein the compound is chiral and is
selected from the group consisting of a D-isomer, a L-isomer, and a
racemic mixture of D- and L-isomers.
39. The compound of claim 27, wherein R.sub.1 is S, X is C, Y is
NH.sub.2, R.sub.2 is .dbd.O, R.sub.3 is ##STR00025## R.sub.4 is NH,
and R.sub.5 is NH.sub.2.
40. The compound of claim 39, wherein the compound is chiral and is
selected from the group consisting of a D-isomer, a L-isomer, and a
racemic mixture of D- and L-isomers.
41. The compound of claim 27, wherein R.sub.1 is O, X is C, Y is
OH, R.sub.2 is absent, R.sub.3 is ##STR00026## R.sub.4 is O, and
R.sub.5 is CH.sub.3.
42. The compound of claim 41, wherein the compound is chiral and is
selected from the group consisting of a D-isomer, a L-isomer, and a
racemic mixture of D- and L-isomers.
43. The compound of claim 27, wherein R.sub.1 is S--S-Z, X is C, Y
is NH.sub.2, R.sub.2 is .dbd.O, R.sub.3 is ##STR00027## R.sub.4 is
O and R.sub.5 is CH.sub.3.
44. The compound of claim 43, wherein the compound is chiral and is
selected from the group consisting of a D-isomer, a L-isomer, and a
racemic mixture of D- and L-isomers.
45. A process for preparing an L-isomer of the compound of claim
27, comprising: (a) adding a base to L-cystine diamide
dihydrochloride to produce a first mixture, and subsequently
heating the first mixture under vacuum; (b) adding a methanolic
solution to the heated first mixture; (c) acidifying the mixture
with alcoholic hydrogen chloride to obtain a first residue; (d)
dissolving the first residue in a first solution comprising
methanol saturated with ammonia; (e) adding a second solution to
the dissolved first residue to produce a second mixture; (f)
precipitating and washing the second mixture; (g) filtering and
drying the second mixture to obtain a second residue; (h) mixing
the second residue with liquid ammonia and an ethanolic solution of
ammonium chloride to produce a third mixture; and (i) filtering and
drying the third mixture, thereby preparing the L-isomer
compound.
46. The process of claim 45, wherein the base comprises liquid
ammonia or methylamine.
47. The process of claim 45, wherein the second solution comprises
water, an acetate salt, and an anhydride.
48. The process of claim 47, wherein the acetate salt comprises
sodium acetate or sodium trifluoroacetate.
49. The process of claim 47, wherein the anhydride comprises acetic
anhydride or trifluoroacetic anhydride.
50. The process of claim 45, wherein the second solution comprises
dichloromethane, triethylamine, and
1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.
51. The process of claim 45, wherein step (h) further comprises
mixing the second residue in the presence of sodium metal.
52. The process of claim 45, further comprising the steps of (j)
dissolving the L-isomer compound in ether; (k) adding to the
dissolved L-isomer compound an ethereal solution of lithium
aluminum hydride, ethyl acetate, and water to produce a fourth
mixture; and (l) filtering and drying the fourth mixture, thereby
preparing the L-isomer compound.
53. A process for preparing an D-isomer of the compound of claim
27, comprising: (a) adding a base to D-cystine diamide
dihydrochloride to produce a first mixture, and subsequently
heating the first mixture under vacuum; (b) adding a methanolic
solution to the heated first mixture; (c) acidifying the mixture
with alcoholic hydrogen chloride to obtain a first residue; (d)
dissolving the first residue in a first solution comprising
methanol saturated with ammonia; (e) adding a second solution to
the dissolved first residue to produce a second mixture; (f)
precipitating and washing the second mixture; (g) filtering and
drying the second mixture to obtain a second residue; (h) mixing
the second residue with liquid ammonia, sodium metal, and an
ethanolic solution of ammonium chloride to produce a third mixture;
and (i) filtering and drying the third mixture, thereby preparing
the L-isomer compound.
54. The process of claim 53, wherein the base comprises liquid
ammonia or methylamine.
55. The process of claim 53, wherein the second solution comprises
water, an acetate salt, and an anhydride.
56. The process of claim 55, wherein the acetate salt comprises
sodium acetate or sodium trifluoroacetate.
57. The process of claim 55, wherein the anhydride comprises acetic
anhydride or trifluoroacetic anhydride.
58. The process of claim 53, wherein the second solution comprises
dichloromethane, triethylamine, and
1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.
59. The process of claim 53, wherein step (h) further comprises
mixing the second residue in the presence of sodium metal.
60. The process of claim 53, further comprising the steps of (j)
dissolving the D-isomer compound in ether; (k) adding to the
dissolved D-isomer compound an ethereal solution of lithium
aluminum hydride, ethyl acetate, and water to produce a fourth
mixture; and (l) filtering and drying the fourth mixture, thereby
preparing the D-isomer compound.
61. A process for preparing an L-isomer of the compound of claim
27, comprising: (a) mixing S-benzyl-L-cysteine methyl ester
hydrochloride or O-benzyl-L-serine methyl ester hydrochloride with
a base to produce a first mixture; (b) adding ether to the first
mixture; (c) filtering and concentrating the first mixture; (d)
repeating steps (c) and (d), to obtain a first residue; (e) adding
ethyl acetate and a first solution to the first residue to produce
a second mixture; (f) filtering and drying the second mixture to
produce a second residue; (g) mixing the second residue with liquid
ammonia, sodium metal, and an ethanolic solution of ammonium
chloride to produce a third mixture; and (h) filtering and drying
the third mixture, thereby preparing the L-isomer compound.
62. The process of claim 61, wherein the base comprises a
methanolic solution of ammonia or methylamine.
63. The process of claim 61, wherein the second solution comprises
water, an acetate salt, and an anhydride.
64. The process of claim 63, wherein the acetate salt comprises
sodium acetate or sodium trifluoroacetate.
65. The process of claim 63, wherein the anhydride comprises acetic
anhydride or trifluoroacetic anhydride.
66. The process of claim 61, wherein the second solution comprises
dichloromethane, triethylamine, and
1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.
67. The process of claim 61, further comprising the steps of (j)
dissolving the L-isomer compound in ether; (k) adding to the
dissolved L-isomer compound an ethereal solution of lithium
aluminum hydride, ethyl acetate, and water to produce a fourth
mixture; and (l) filtering and drying the fourth mixture, thereby
preparing the L-isomer compound.
68. A process for preparing an D-isomer of the compound of claim
27, comprising: (a) mixing S-benzyl-D-cysteine methyl ester
hydrochloride or O-benzyl-D-serine methyl ester hydrochloride with
a base to produce a first mixture; (b) adding ether to the first
mixture; (c) filtering and concentrating the first mixture; (d)
repeating steps (c) and (d), to obtain a first residue; (e) adding
ethyl acetate and a first solution to the first residue to produce
a second mixture; (f) filtering and drying the second mixture to
produce a second residue; (g) mixing the second residue with liquid
ammonia, sodium metal, and an ethanolic solution of ammonium
chloride to produce a third mixture; and (h) filtering and drying
the third mixture, thereby preparing the D-isomer compound.
69. The process of claim 68, wherein the base comprises a
methanolic solution of ammonia or methylamine.
70. The process of claim 68, wherein the second solution comprises
water, an acetate salt, and an anhydride.
71. The process of claim 70, wherein the acetate salt comprises
sodium acetate or sodium trifluoroacetate.
72. The process of claim 70, wherein the anhydride comprises acetic
anhydride or trifluoroacetic anhydride.
73. The process of claim 68, wherein the second solution comprises
dichloromethane, triethylamine, and
1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.
74. The process of claim 68, further comprising the steps of (j)
dissolving the D-isomer compound in ether; (k) adding to the
dissolved D-isomer compound an ethereal solution of lithium
aluminum hydride, ethyl acetate, and water to produce a fourth
mixture; and (l) filtering and drying the fourth mixture, thereby
preparing the D-isomer compound.
75. A process for preparing a compound of claim 27, comprising: (a)
mixing cystamine dihydrochloride with ammonia, water, sodium
acetate, and acetic anhydride to produce a first mixture; (b)
allowing the first mixture to precipitate; (c) filtering and drying
the first mixture to produce a first residue; (d) mixing the second
residue with liquid ammonia, sodium metal, and an ethanolic
solution of ammonium chloride to produce a second mixture; (e)
filtering and drying the second mixture, thereby preparing the
compound.
76. A NAC amide compound or derivative selected from the group
consisting of: ##STR00028## ##STR00029##
77. A food additive comprising NAC amide or a NAC amide derivative
of claim 76.
Description
FIELD OF THE INVENTION
[0001] The present invention generally relates to the treatment of
mammalian, including human, diseases with antioxidants. More
particularly, the invention relates to treatments and therapies of
a variety of diseases and conditions involving the administration
of N-acetylcysteine amide (NAC amide) or a derivative thereof,
alone or in combination with another agent, to a mammal in need
thereof.
BACKGROUND OF THE INVENTION
[0002] Oxidative stress plays an important role in the progression
of neurodegenerative and age-related diseases, causing damage to
proteins, DNA, and lipids. Low molecular weight, hydrophobic
antioxidant compounds are useful in treating conditions of
peripheral tissues, such as acute respiratory distress syndrome,
amyotrophic lateral sclerosis, atherosclerotic cardiovascular
disease, multiple organ dysfunctions and central nervous system
neurodegenerative disorders, e.g., Parkinson's disease, Alzheimer's
disease and Creutzfeldt-Jakob's disease. Oxidative stress has been
causally linked to the pathogenesis of Parkinson's disease,
Alzheimer's disease and Creutzfeldt-Jakob's disease, as well as
other types of disorders. (U.S. Pat. No. 6,420,429 to D. Atlas et
al.).
[0003] A deficiency of cellular antioxidants may lead to excess
free radicals, which cause macromolecular breakdown, lipid
peroxidation, buildup of toxins and ultimately cell death. Because
of the importance of antioxidant compounds in preventing this
cellular oxidation, natural antioxidants, such as glutathione (GSH)
(.gamma.-glutamyl cysteinyl glycine) are continuously supplied to
the tissues. GSH is synthesized by most cells and is one of the
primary cellular antioxidants responsible for maintaining the
proper oxidation state within the body. When oxidized, GSH forms a
dimer, GSSG, which may be recycled in organs producing glutathione
reductase. In human adults, reduced GSH is produced from GSSG,
primarily in the liver, and to a smaller extent, by skeletal muscle
and red and white blood cells, and is distributed through the blood
stream to other tissues in the body.
[0004] However, under certain conditions, the normal, physiologic
supplies of GSH are insufficient, its distribution is inadequate or
local oxidative demands are too high to prevent cellular oxidation.
Under other conditions, the production of and demand for cell
antioxidants, such as GSH, are mismatched, thus leading to
insufficient levels of these molecules in the body. In other cases,
certain tissues or biological processes consume the antioxidants so
that their intracellular levels are suppressed. In either case,
increased serum levels of antioxidant, e.g., glutathione, leads to
increased amounts of the antioxidant that can be directed into
cells. In facilitated transport systems for cellular uptake, the
concentration gradient that drives uptake is increased.
[0005] Glutathione N-acetylcysteine amide (NAC amide), the amide
form of N-acetylcysteine (NAC), is a low molecular weight thiol
antioxidant and a Cu.sup.2+ chelator. NAC amide provides protective
effects against cell damage. NAC amide was shown to inhibit
tert.-butylhydroxyperoxide (BuOOH)-induced intracellular oxidation
in red blood cells (RBCs) and to retard BuOOH-induced thiol
depletion and hemoglobin oxidation in the RBCs. This restoration of
thiol-depleted RBCs by externally applied NAC amide was
significantly greater than that found using NAC. Unlike NAC, NAC
amide protected hemoglobin from oxidation. (L. Grinberg et al.,
Free Radic Biol Med., 2005 Jan. 1, 38(1):136-45). In a cell-free
system, NAC amide was shown to react with oxidized glutathione
(GSSG) to generate reduced glutathione (GSH). NAC amide readily
permeates cell membranes, replenishes intracellular GSH, and, by
incorporating into the cell's redox machinery, protects the cell
from oxidation. Because of its neutral carboxyl group, NAC amide
possesses enhanced properties of lipophilicity and cell
permeability. (See, e.g., U.S. Pat. No. 5,874,468 to D. Atlas et
al.). NAC amide is also superior to NAC and GSH in crossing the
cell membrane, as well as the blood-brain barrier.
[0006] NAC amide may function directly or indirectly in many
important biological phenomena, including the synthesis of proteins
and DNA, transport, enzyme activity, metabolism, and protection of
cells from free-radical mediated damage. NAC amide is a potent
cellular antioxidant responsible for maintaining the proper
oxidation state within the body. NAC amide can recycle oxidized
biomolecules back to their active reduced forms and may be as
effective, if not more effective, than GSH as an antioxidant.
[0007] Glutamate, an excitatory amino acid, is one of the major
neurotransmitters in the central nervous system (CNS). Elevated
levels of extracellular glutamate have been shown to be responsible
for acute neuronal damage as well as many CNS disorders, including
hyperglycemia, ischemia, hypoxia (Choi, D. W., Neuron, 1(8):623-34,
1988), and chronic disorders such as Huntington's, Alzheimer's, and
Parkinson's diseases (Meldrum B. and Garthwaite J., Trends
Pharmacol Sci., 11(9):379-87, 1990; and Coyle J. T. and Puttfarcken
P., Science, 262(5134):689-95, 1993). Two mechanisms have been
proposed for glutamate toxicity. The first mechanism explains the
excitotoxicity of glutamate as being mediated through three types
of excitatory amino acid receptors (Monaghan D. T. et al., Annu Rev
Pharmacol Toxicol., 29:365-402, 1989). In addition to
receptor-mediated glutamate excitotoxicity, it has also been
proposed that elevated levels of extracellular glutamate inhibits
cystine uptake, which leads to a marked decrease in cellular GSH
levels, resulting in the induction of oxidative stress (Murphy T.
H. et al., Neuron, 2(6):1547-58, 1989).
[0008] Cysteine is a critical component for intracellular GSH
synthesis. Because of redox instability, almost all of the
extracellular cysteine is present primarily in its oxidized state,
cystine, which is taken up by cells via a cystine/glutamate
transporter, the X c-system. Studies indicate that glutamate and
cystine share the same transporter; therefore, elevated levels of
extracellular glutamate competitively inhibit cystine transport,
which leads to depletion of intracellular GSH. (Bannai S, and
Kitamura E., J Biol. Chem. 255(6):2372-6, 1980; and Bannai S.,
Biochem Biophys Acta., 779(3):289-306, 1984). Depletion of reduced
glutathione results in decreased antioxidant capacity of the cell,
accumulation of ROS (reactive oxygen species), and ultimately
apoptotic cell death. Several studies have demonstrated the
induction of oxidative stress by glutamate in various cell lines
including immature cortical neurons (Murphy T. H. et al., FASEB J.,
4(6):1624-33, 1990; and Sagara J. et al., J. Neurochem.,
61(5):1667-71, 1993), oligodendroglia (Oka A. et al., J. Neurosci.,
13(4):1441-53, 1993), cultured rat astrocytes (Cho Y. and Bannai
S., J. Neurochem., 55(6):2091-7, 1990), neuroblastoma cells (Murphy
T. H. et al., Neuron., 2(6):1547-58, 1989), and PC12 cells
(Froissard P. and Duval D., Neurochem Int., 24(5):485-93,
1994).
[0009] Certain antioxidants such as NAC, lipoic acid (LA), (Han D.
et al., Am J. Physiol., 273:1771-8, 1997), tocopherol (Pereira C.
M. and Oliveira C. R., Free Radic Biol Med., 23(4):637-47, 1997),
and probucol (Naito M. et al., Neurosci Lett., 186(2-3):211-3,
1995) can protect against glutamate cytotoxicity, mostly by
replenishing GSH. However, in certain neurological diseases, such
as cerebral ischemia and Parkinson's disease, enhancement of tissue
GSH in brain regions cannot be attained, because these antioxidant
agents have been obstructed by the blood-brain barrier (Panigrahi
M. et al., Brain Res., 717(1-2):184-8, 1996; and Gotz M. E. et al.,
J Neural Transm Suppl., 29:241-9, 1990).
[0010] In addition to neurodegenerative diseases, such as those
which affect the brain and/or peripheral nervous tissues, other
diseases, such as asthma, respiratory-related diseases and
conditions, e.g., acute respiratory distress syndrome (ARDS),
amyotrophic lateral sclerosis (ALS or Lou Gerhig's disease),
atherosclerotic cardiovascular disease and multiple organ
dysfunction, are related to the overproduction of oxidants or
reactive oxygen species by cells of the immune system.
[0011] A number of other disease states have been specifically
associated with reductions in the levels of antioxidants such as
GSH. Depressed antioxidant levels, either locally in particular
organs or systemically, have been associated with a number of
clinically defined diseases and disease states, including HIV/AIDS,
diabetes and macular degeneration, all of which progress because of
excessive free radical reactions and insufficient antioxidants.
Other chronic conditions may also be associated with antioxidant
deficiency, oxidative stress, and free radical formation, including
heart failure and associated conditions and pathologies, coronary
arterial restenosis following angioplasty, diabetes mellitus and
macular degeneration.
[0012] Clinical and pre-clinical studies have demonstrated the
linkage between a range of free radical disorders and insufficient
antioxidant levels. It has been reported that diabetic
complications are the result of hyperglycemic episodes that promote
glycation of cellular enzymes and thereby inactivate the synthetic
pathways of antioxidant compounds. The result is antioxidant
deficiency in diabetics, which may be associated with the
prevalence of cataracts, hypertension, occlusive atherosclerosis,
and susceptibility to infections in these patients.
[0013] High levels of antioxidants, such as GSH, have been
demonstrated to be necessary for proper functioning of platelets,
vascular endothelial cells, macrophages, cytotoxic T-lymphocytes,
and other immune system components. Recently it has been discovered
that patients infected with the human immunodeficiency virus, HIV,
exhibit low GSH levels in plasma, other body fluids, and in certain
cell types, such as macrophages. These low GSH levels do not appear
to be due to defects in GSH synthesis. Antioxidant deficiency has
been implicated in the impaired survival of patients with HIV.
(1997, PNAS USA, Vol. 94, pp. 1967-1972). Raising antioxidant
levels in cells is widely recognized as being important in HIV/AIDS
and other disorders, because the low cellular antioxidant levels in
these disease types permit more and more free radical reactions to
fuel and exacerbate the disorders.
[0014] HIV is known to start pathologic free radical reactions,
which lead to the destruction of antioxidant molecules, as well as
their exhaustion and the destruction of cellular organelles and
macromolecules. In mammalian cells, oxidative stresses, e.g. low
intracellular levels of reduced antioxidants and relatively high
levels of free radicals, activate certain cytokines, including
NF-.kappa.B and TNF-.alpha., which, in turn, activate cellular
transcription of the DNA to mRNA, resulting in translation of the
mRNA to a polypeptide sequence. In a virus-infected cell, the viral
genome is transcribed, resulting in viral RNA production, generally
necessary for viral replication of RNA viruses and retroviruses.
These processes require a relatively oxidized state of the cell, a
condition which results from stress, low antioxidant levels, or the
production of reduced cellular products. The mechanism which
activates cellular transcription is evolutionarily highly
conserved, and therefore it is unlikely that a set of mutations
would escape this process, or that an organism in which mutated
enzyme and receptor gene products in this pathway would be well
adapted for survival. Thus, by maintaining a relatively reduced
state of the cell (redox potential), viral transcription, a
necessary step in late stage viral replication, is impeded.
[0015] The amplification effect of oxidative intracellular
conditions on viral replication is compounded by the actions of
various viruses and viral products, which degrade antioxidants,
such as GSH. For example, gp120, an HIV surface glycoprotein having
a large number of disulfide bonds, is normally present on the
surface of infected cells. gp120 oxidizes GSH, resulting in reduced
intracellular GSH levels. On the other hand, GSH reduces the
disulfide bonds of gp120, thus reducing or eliminating its
biological activity that is necessary for viral infectivity.
Antioxidants such as GSH therefore interfere with the production of
such oxidized proteins and degrade them once formed. In a cell that
is actively replicating viral gene products, a cascade of events
may occur which can allow the cell to pass from a relatively
quiescent stage with low viral activity to an active stage with
massive viral replication and cell death. This is accompanied by a
change in redox potential. By maintaining adequate levels of
antioxidant, this cascade may be impeded.
[0016] HIV is transmitted through two predominant routes, namely,
contaminated blood and/or sexual intercourse. In pediatric cases,
approximately one half of the newborn individuals are infected in
utero and one half are infected at delivery. This circumstance
permits a study of prevention of transmission since the time of
spread is known. Initially, there is an intense viral infection
simulating a severe case of the flu, with massive replication of
the virus. Within weeks, this acute phase passes spontaneously as
the body mounts a largely successful immune defense. Thereafter,
the individual has no outward manifestations of the infection.
However, the virus continues to replicate within immune system
cells and tissues, e.g., lymph nodes, lymphoid nodules, macrophages
and certain multidendritic cells that are found in various body
cavities.
[0017] Such stealthy and widespread infection is not just a viral
problem. The virus, in addition to replicating, causes excessive
production of various free radicals and various cytokines in toxic
or elevated levels. The cytokines are normally occurring
biochemical substances that signal numerous reactions and that
typically exist in minuscule concentrations. Eventually, after an
average of 7-10 years of seemingly quiescent HIV infection, the
corrosive free radicals and the toxic levels of cytokines begin to
cause outward symptoms in infected individuals and failures in the
immune system begin. Substances like 15-HPETE are immunosuppressive
and TNF-.alpha. causes muscle wasting, among other toxic factors.
The numbers of viral particles increase and the patient develops
the Acquired Immune Deficiency Syndrome, AIDS, which may last 2 to
4 years before the individual's demise. AIDS, therefore, is not
merely a virus infection, although the viral infection is believed
to be an integral part of the etiology of the disease.
[0018] Further, HIV has a powerful ability to mutate. It is this
capability that makes it difficult to create a vaccine or to
develop long-term, antiviral pharmaceutical treatments. As more
people fail to be successfully treated by the present complex
regimens, the number of resistant viral strains is increasing.
Resistant strains of HIV are a particularly dangerous population of
the virus and pose a considerable health threat. These resistant
HIV mutants also add to the difficulties in developing vaccines
that will be able to inhibit the activity of highly virulent viral
types. Further, the continuing production of free radicals and
cytokines that may become largely independent of the virus
perpetuate the dysfunctions of the immune system, the
gastrointestinal tract, the nervous system, and many other organs
in patients with AIDS. The published scientific literature
indicates that many of these diverse organ system dysfunctions are
due to systemic deficiencies of antioxidant compounds that are
engendered by the virus and its free radicals. For example, GSH is
consumed in HIV infections because it is the principal, bulwark
antioxidant versus free radicals. An additional cause of erosion of
GSH levels is the presence of numerous disulfide bonds in HIV
proteins, such as the gp120 cell surface protein. Disulfide bonds
react with GSH and oxidize it. Thus, there is a need for other
antioxidants to be used to replace antioxidants such as GSH whose
normal function is adversely affected by HIV infection.
[0019] The current HIV/AIDS pharmaceuticals take good advantage of
the concept of pharmaceutical synergism, wherein two different
targets in one process are affected simultaneously. The effect is
more than additive. The drugs now in use were selected to inhibit
two very different points in the long path of viral replication.
The pathway of viral replication as understood by skilled
practitioners in the art is described in U.S. Pat. No. 6,420,429.
New anti-HIV/AIDS therapies include additional drugs in the classes
of Reverse Transcriptase inhibitors and protease inhibitors. Also,
drugs are in development to block the integrase enzyme of the
virus, which integrates the HIV DNA into the infected cell's DNA,
analogous to splicing a small length of wire into a longer wire.
Vaccine development also continues, although prospects seem poor
because HIV appears to be a moving target and seems to change
rapidly. Vaccine development is also impaired by the immune cell
affinity of the virus.
[0020] Individuals infected with HIV have lowered levels of serum
acid-soluble thiols and antioxidants such as GSH in plasma,
peripheral blood monocytes and lung epithelial lining fluid. In
addition, it has been shown that CD4.sup.+ and CD8.sup.+ T cells
with high intracellular GSH levels are selectively lost as HIV
infection progresses. This deficiency may potentiate HIV
replication and accelerate disease progression, especially in
individuals with increased concentrations of inflammatory
cytokines, because such cytokines stimulate HIV replication more
efficiently in cells in which antioxidant compounds are depleted.
In addition, the depletion of antioxidants, such as GSH, is also
associated with a process known as apoptosis, or programmed cell
death. Thus, intercellular processes which artificially deplete GSH
may lead to cell death, even if the process itself is not
lethal.
[0021] Diabetes mellitus ("diabetes") is found in two forms:
childhood or autoimmune (Type I, IDDM) and late-onset or
non-insulin dependent (Type II, NIDDM). Type I constitutes about
30% of the cases of diabetes. The rest of the cases are represented
by Type II. In general, the onset of diabetes is sudden for Type I
and insidious or chronic for Type II. Symptoms include excessive
urination, hunger and thirst, with a slow and steady loss of weight
associated with Type I. Obesity is often associated with Type II
and has been thought to be a causal factor in susceptible
individuals. Blood sugar is often high and there is frequent
spilling of sugar in the urine. If the condition goes untreated,
the victim may develop ketoacidosis with a foul-smelling breath
similar to some who has been drinking alcohol. The immediate
medical complications of untreated diabetes can include nervous
system symptoms, and even diabetic coma.
[0022] Because of the continuous and pernicious occurrence of
hyperglucosemia (very high blood sugar levels), a non-enzymatic
chemical reaction, called glycation, frequently occurs inside cells
and causes a chronic inactivation of essential enzymes. One of the
most critical enzymes, .gamma.-glutamyl-cysteine synthetase, is
glycated and readily inactivated. This enzyme is involved in a
critical step in the biosynthesis of glutathione in the liver. The
net result of this particular glycation is a deficiency in the
production of GSH in diabetics.
[0023] GSH is in high demand throughout the body for multiple,
essential functions, for example, within all mitochondria, to
produce chemical energy called ATP. With a deficiency or absence of
GSH, brain cells, heart cells, nerve cells, blood cells and many
other cell types are not able to function properly and can be
destroyed through apoptosis associated with oxidative stress and
free radical formation. GSH is the major antioxidant in the human
body and the only one that can be synthesized de novo. It is also
the most common small molecular weight thiol in both plants and
animals. Without GSH the immune system cannot function, and the
central and peripheral nervous systems become aberrant and then
cease to function. Because of the dependence on GSH as the carrier
of nitric oxide, a vasodilator responsible for control of vascular
tone, the cardiovascular system does not function well and
eventually fails. Since all epithelial cells seem to require GSH,
without GSH, intestinal lining cells also do not function properly
and valuable micronutrients are lost, nutrition is compromised, and
microbes are given portals of entry to cause infections.
[0024] In diabetes, the use of GSH precursors cannot help to
control GSH deficiency due to the destruction of the rate-limiting
enzyme by glycation. As GSH deficiency becomes more profound, the
well-known sequelae of diabetes progress in severity. The
complications that develop in diabetics are essentially due to
runaway free radical damage since the available GSH supplies in
diabetics are insufficient. For example, a diabetic individual
becomes more susceptible to infections because the immune system
approaches collapse when GSH levels fall, analogous to the
situation in HIV/AIDS. In addition, peripheral vasculature becomes
comprised and blood supply to the extremities is severely
diminished because GSH is not available in sufficient amounts to
stabilize nitric oxide to effectively exert its vascular dilation
(relaxation) property. Gangrene is a common sequel and successive
amputations often result in later years. Peripheral neuropathies,
the loss of sensation commonly of the feet and lower extremities
develop and are often followed by aberrant sensations like
uncontrollable burning or itching. Retinopathy and nephropathy are
later events that are actually due to icroangiopathy, i.e.,
excessive budding and growth of new blood vessels and capillaries,
which often will bleed due to weakness of the new vessel walls.
This bleeding causes damage to the retina and kidneys with
resulting blindness and renal shutdown, which requires dialysis
treatment. Further, cataracts occur with increasing frequency as
the GSH deficiency deepens. Large and medium sized arteries become
sites of accelerated severe atherosclerosis, with myocardial
infarcts at early ages, and of a more severe degree. If coronary
angioplasty is used to treat the severe atherosclerosis, diabetics
are much more likely to have re-narrowing of cardiac vessels,
termed restenosis.
[0025] Macular degeneration as a cause of blindness is a looming
problem as the population ages. Age-related macular degeneration
(ARMD) is characterized by either a slow (dry form) or rapid (wet
form) onset of destruction and irrevocable loss of rods and cones
in the macula of the eye. The macula is the approximate center of
the retina wherein the lens of the eye focuses its most intense
light. The visual cells, known as the rods and cones, are an
outgrowth and active part of the central nervous system. They are
responsible and essential for the fine visual discrimination
required to see clear details such as faces and facial expression,
reading, driving, operation of machinery and electrical equipment
and general recognition of surroundings. Ultimately, the
destruction of the rods and cones leads to functional, legal
blindness. Since there is no overt pain associated with the
condition, the first warnings of onset are usually noticeable loss
of visual acuity. This may already signal late stage events. It is
now thought that one of the very first events in this pathologic
process is the formation of a material called "drusen", which first
appears as either patches or diffuse drops of yellow material
deposited upon the surface of the retina in the macula lutea or
yellow spot. This is the area of the retina where sunlight is
focused by the lens and which contains the highest density of rods
for acuity. Although cones, which detect color, are lost as well in
this disease, it is believed to be loss of rods, which causes the
blindness. Drusen has been chemically analyzed and found to be
composed of a mixture of lipids that are peroxidized by free
radical reactions.
[0026] It is believed that the loss of retinal pigmented epithelial
(RPE) cells occurs first in ARMD. Once an area of the retinal
macula is devoid of RPE cells, loss of rods, and eventually some
cones, occurs. Finally, budding of capillaries begins and typical
microangiopathy associated with late stage ARMD occurs. It is also
known that RPE cells require large quantities of GSH for their
proper functioning. When GSH levels drop severely in cultures of
RPE cells, the RPE cells begin to die. When cultures of these cells
are supplemented with GSH in the medium, they thrive. There is
increasing evidence that progression of the disease is paced by a
more profound deficiency in GSH within the retina and probably
within these cells, as indicated by cell culture studies.
[0027] It is generally believed that "near" ultraviolet (UVB) and
visual light of high intensity primarily from sunlight is a strong
contributing factor of ARMD. People with light-colored irises
constitute a high risk population for macular degeneration, as do
those with jobs that keep them outdoors and those in equatorial
areas where sunlight is most intense. Additional free radical
insults, e.g., smoking, adds to the risk of developing ARMD.
Several approaches have been unsuccessfully tested to combat ARMD,
including chemotherapy. Currently, there is no effective therapy to
treat ARMD. Laser therapy has been developed which has been used
widely to slow the damage produced in the slow onset form of the
disease by cauterizing neovascular growth. However the eventual
outcome of the disease, once it has started to progress, is
certain.
[0028] The importance of thiols and especially of GSH to lymphocyte
function has been known for many years. Adequate concentrations of
GSH are required for mixed lymphocyte reactions, T-cell
proliferation, T- and B-cell differentiation, cytotoxic T-cell
activity, and natural killer cell activity. Adequate GSH levels
have been shown to be necessary for microtubule polymerization in
neutrophils. Intraperitoneally administered GSH augments the
activation of cytotoxic T-lymphocytes in mice, and dietary GSH was
found to improve the splenic status of GSH in aging mice, and to
enhance T-cell mediated immune responses. The presence of
macrophages can cause a substantial increase of the intracellular
GSH levels of activated lymphocytes in their vicinity. Macrophages
consume cystine via a strong membrane transport system, and
generate large amounts of cysteine, which they release into the
extracellular space. It has been demonstrated that macrophage GSH
levels (and therefore cysteine equivalents) can be augmented by
exogenous GSH. T-cells cannot produce their own cysteine, and it is
required by T-cells as the rate-limiting precursor of GSH
synthesis. The intracellular GSH level and the DNA synthesis
activity in mitogenically-stimulated lymphocytes are strongly
increased by exogenous cysteine, but not cystine. In T-cells, the
membrane transport activity for cystine is ten-fold lower than that
for cysteine. As a consequence, T-cells have a low baseline supply
of cysteine, even under healthy physiological conditions. The
cysteine supply function of the macrophages is an important part of
the mechanism which enables T-cells to shift from a GSH-poor to a
GSH-rich state.
[0029] The importance of the intracellular GSH concentration for
the activation of T-cells is well established. It has been reported
that GSH levels in T-cells rise after treatment with GSH; it is
unclear whether this increase is due to uptake of the intact GSH or
via extracellular breakdown, transport of breakdown products, and
subsequent intracellular GSH synthesis. Decreasing GSH by 10-40%
can completely inhibit T-cell activation in vitro. Depletion of
intracellular GSH has been shown to inhibit the
mitogenically-induced nuclear size transformation in the early
phase of the response. Cysteine and GSH depletion also affects the
function of activated T-cells, such as cycling T-cell clones and
activated cytotoxic T-lymphocyte precursor cells in the late phase
of the allogeneic mixed lymphocyte culture. DNA synthesis and
protein synthesis in IL-2 dependent T-cell clones, as well as the
continued growth of preactivated CTL precursor cells and/or their
functional differentiation into cytotoxic effector cells are
strongly sensitive to GSH depletion.
[0030] Glutathione status is a major determinant of protection
against oxidative injury. GSH acts on the one hand by reducing
hydrogen peroxide and organic hydroperoxides in reactions catalyzed
by glutathione peroxidases, and on the other hand by conjugating
with electrophilic xenobiotic intermediates capable of inducing
oxidant stress. The epithelial cells of the renal tubule have a
high concentration of GSH, no doubt because the kidneys function in
toxin and waste elimination, and the epithelium of the renal tubule
is exposed to a variety of toxic compounds. GSH, transported into
cells from the extracellular medium, substantially protects
isolated cells from intestine and lung against
t-butylhydroperoxide, menadione or paraquat-induced toxicity.
Isolated kidney cells also transport GSH, which can supplement
endogenous synthesis of GSH to protect against oxidant injury.
Hepatic GSH content has also been reported to increase (i.e. to
double) in the presence of exogenous GSH. This may be due either to
direct transport, as has been reported for intestinal and alveolar
cells, or via extracellular degradation, transport, and
intracellular resynthesis.
[0031] The nucleophilic sulfur atom of the cysteine moiety of GSH
serves as a mechanism to protect cells from harmful effects induced
by toxic electrophiles. It is well established that glutathione
S-conjugate biosynthesis is an important mechanism of drug and
chemical detoxification. GSH conjugation of a substrate generally
requires both GSH and glutathione-S-transferase activity. The
existence of multiple glutathione-S-transferases with specific, but
also overlapping, substrate specificities enables the enzyme system
to handle a wide range of compounds. Several classes of compounds
are believed to be converted by glutathione conjugate formation to
toxic metabolites. For example, halogenated alkenes, hydroquinones,
and quinones have been shown to form toxic metabolites via the
formation of S-conjugates with GSH. The kidney is the main target
organ for compounds metabolized by this pathway. Selective toxicity
to the kidney is the result of the kidney's ability to accumulate
intermediates formed by the processing of S-conjugates in the
proximal tubular cells, and to bioactivate these intermediates to
toxic metabolites.
[0032] The administration of morphine and related compounds to rats
and mice results in a loss of up to approximately 50% of hepatic
GSH. Morphine is known to be biotransformed into morphinone, a
highly hepatotoxic compound, which is 9 times more toxic than
morphine in mouse by subcutaneous injection, by morphine
6-dehydrogenase activity. Morphinone possesses an
.alpha.,.beta.-unsaturated ketone, which allows it to form a
glutathione S-conjugate. The formation of this conjugate correlates
with loss of cellular GSH. This pathway represents the main
detoxification process for morphine. Pretreatment with GSH protects
against morphine-induced lethality in the mouse.
[0033] The deleterious effects of methylmercury on mouse
neuroblastoma cells are largely prevented by co-administration of
GSH. GSH may complex with methylmercury, prevent its transport into
the cell, and increase cellular antioxidant capabilities to prevent
cell damage. Methylmercury is believed to exert its deleterious
effects on cellular microtubules via oxidation of tubulin
sulfhydryls, and by alterations due to peroxidative injury. GSH
also protects against poisoning by other heavy metals such as
nickel and cadmium.
[0034] Because of its known role in renal detoxification and its
low toxicity, GSH has been explored as an adjunct therapy for
patients undergoing cancer chemotherapy with nephrotoxic agents
such as cisplatin, in order to reduce systemic toxicity. In one
study, GSH was administered intravenously to patients with advanced
neoplastic disease, in two divided doses of 2,500 mg, shortly
before and after doses of cyclophosphamide. GSH was well tolerated
and did not produce unexpected toxicity. The lack of bladder
damage, including microscopic hematuria, supports the protective
role of this compound. Other studies have shown that
co-administration of GSH intravenously with cisplatin and/or
cyclophosphamide combination therapy, reduces associated
nephrotoxicity, while not unduly interfering with the desired
cytotoxic effect of these drugs.
[0035] GSH has an extremely low toxicity, and oral LD.sub.50
measurements are difficult to perform due to the sheer mass of GSH,
which has to be ingested by the animal in order to see any toxic
effects. GSH can be toxic, especially in cases of ascorbate
deficiency, and these effects may be demonstrated in, for example,
ascorbate deficient guinea pigs given 3.75 mmol/kg daily (1,152
mg/kg daily) in three divided doses, whereas in non-ascorbate
deficient animals, toxicity was not seen at this dose, but were
seen at double this dose.
[0036] There is a need in the art for other compounds and
therapeutic aspects to treat a number of diseases that are linked
to oxidative stress and the presence of free oxygen radicals and
associated disease pathogenesis in cells and tissues. Needed are
antioxidant compounds, other than GSH, that are safe and even more
potent, to overcome high oxidative stress in the pathogenesis of
diseases. Ideally, such compounds should readily cross the
blood-brain barrier and easily permeate the cell membrane.
Antioxidants such as vitamins E and C are not completely effective
at decreasing oxidative stress, particularly because, in the case
of vitamin E, they do not effectively cross through the cell
membrane to reach the cytoplasm so as to provide antioxidant
effects.
SUMMARY OF THE INVENTION
[0037] The present invention provides the use of a potent
antioxidant N-acetylcysteine amide (NAC amide) or derivatives
thereof, or a physiologically acceptable derivative, salt, or ester
thereof, in new applications to treat disorders, conditions,
pathologies and diseases that result from, or are associated with,
the adverse effects of oxidative stress and/or the production of
free radicals in cells, tissues and organs of the body. NAC amide
and its derivatives are provided for use in methods and
compositions for improving and treating such disorders, conditions,
pathologies and diseases.
[0038] As used herein, a "subject" within the context of the
present invention encompasses, without limitation, mammals, e.g.,
humans, domestic animals and livestock including cats, dogs, cattle
and horses. A "subject in need thereof" is a subject having one or
more manifestations of disorders, conditions, pathologies, and
diseases as disclosed herein in which administration or
introduction of NAC amide or its derivatives would be considered
beneficial by those of ordinary skill in the art.
[0039] In an aspect of the present invention, methods and
compositions comprising NAC amide provide an antioxidant to cells
and tissues to reduce oxidative stress, and the adverse effects of
cellular oxidation, in an organism. The invention provides a method
of reducing oxidative stress associated with the conditions,
diseases, pathologies as described herein, by administering a
pharmaceutically acceptable formulation of NAC amide or derivatives
thereof to a human or non-human mammal in an amount effective to
reduce oxidative stress.
[0040] In another aspect of the present invention, NAC amide and
its derivatives are provided to treat an organism having a
disorder, condition, pathology, or disease that is associated with
the overproduction of oxidants and/or oxygen free radical species.
According to this invention NAC amide treatment can be prophylactic
or therapeutic.
[0041] "Therapeutic treatment" or "therapeutic effect" means any
improvement in the condition of a subject treated by the methods of
the present invention, including obtaining a preventative or
prophylactic effect, or any alleviation of the severity of signs or
symptoms of a disorder, condition, pathology, or disease or its
sequelae, including those caused by other treatment methods (e.g.,
chemotherapy and radiation therapy), which can be detected by means
of physical examination, laboratory, or instrumental methods and
considered statistically and/or clinically significant by those
skilled in the art.
[0042] "Prophylactic treatment" or "prophylactic effect" means
prevention of any worsening in the condition of a subject treated
by the methods of the present invention, as well as prevention of
any exacerbation of the severity of signs and symptoms of a
disorder, condition, pathology, or disease or its sequelae,
including those caused by other treatment methods (e.g.,
chemotherapy and radiation therapy), which can be detected by means
of physical examination, laboratory, or instrumental methods and
considered statistically and/or clinically significant by those
skilled in the art.
[0043] In another aspect of the present invention, NAC amide is
used in the treatment and/or prevention of cosmetic conditions and
dermatological disorders of the skin, hair, nails, and mucosal
surfaces when applied topically. In accordance with the invention,
compositions for topical administration are provided that include
(a) NAC amide, or derivatives thereof, or a suitable salt or ester
thereof, or a physiologically acceptable composition containing NAC
amide or its derivatives; and (b) a topically acceptable vehicle or
carrier. The present invention also provides a method for the
treatment and/or prevention of cosmetic conditions and/or
dermatological disorders that entails topical administration of NAC
amide- or NAC-amide derivative-containing compositions to an
affected area of a patient.
[0044] In yet another of its aspects the present invention provides
methods and compositions useful for cancer and pre-cancer therapy
utilizing NAC amide or a derivative thereof, or its
pharmaceutically acceptable salts or esters. The present invention
particularly relates to methods and compositions comprising NAC
amide or a derivative thereof in which apoptosis is selectively
induced in cells of cancers or precancers.
[0045] In another aspect, the present invention provides
compositions and methods comprising NAC amide or a derivative
thereof for the suppression of allograft rejection in recipients of
allografts.
[0046] In another aspect, the present invention provides a NAC
amide or a derivative thereof in a method of supporting or
nurturing the growth of stem cells for stem cell transplants,
particularly stem cells cultured in vitro prior to introduction
into a recipient animal, including humans.
[0047] In another aspect, the present invention provides methods of
inhibiting, preventing, treating, or both preventing and treating,
central nervous system (CNS) injury or disease, traumatic brain
injury, neurotoxicity or memory deficit in a subject, involving the
administration of a therapeutically effective amount of NAC amide,
or derivative thereof or a pharmaceutically acceptable composition
thereof.
[0048] In another of its aspects, the present invention provides a
method of killing or inhibiting the growth of microorganisms by
providing NAC amide in an amount effective to increase cellular
levels of HIF-1 or HIF-1.alpha. to enhance the capacity of white
blood cells to kill or inhibit the growth of the microorganisms.
Also in accordance with the invention, NAC amide is used as a
countermeasure for biodefensive purposes, e.g., in killing or
growth inhibiting microorganisms, viruses, mycoplasma, etc., and in
treating resulting diseases and conditions, as further described
herein.
[0049] In another aspect, the present invention provides a method
of preventing tissue destruction resulting from the effects of
metalloproteinases, such as MMP-3, which has been found to cause
normal cells to express the Rac1b protein, an unusual form of Rho
GTPase that has previously been found only in cancers. Rac1b
stimulates the production of highly reactive oxygen species (ROS),
which can promote cancer by activating major genes that elicits
massive tissue disorganization. In accordance with the present
invention NAC amide is used to block the effects of Rac1b-induced
ROS production by administering or introducing NAC amide to cells,
tissues, and/or the body of a subject in need thereof, to target
molecules in the pathways leading to tissue damage and degradation.
Thus, NAC amide can be used to inhibit MMP-3 and its adverse
functions, to target ROS indirectly or directly via the processes
by which ROS activates genes to induce the EMT.
[0050] Another aspect of the present invention provides a method of
stimulating endogenous production of cytokines and hematopoietic
factors, comprising administering or introducing NAC amide to
cells, tissues, and/or a subject in need thereof for a period of
time to stimulate the endogenous production. NAC amide can be used
to stimulate production of cytokines and hematopoietic factors,
such as but not limited to, TNF-.alpha., IFN-.alpha., IFN-.beta.,
IFN-.gamma., IL-1, IL-2, IL-6, IL-10, erythropoietin, G-CSF, M-CSF,
and GM-CSF, which are factors that modulate the immune system and
whose biological activities are involved in various human diseases,
such as neoplastic and infectious diseases, as well as those
involving hematopoiesis and immune depressions of various origin
(such as, without limitation, erythroid, myeloid, or lymphoid
suppression). Stimulation of endogenous production of these
cytokines and hematopoietic factors by NAC amide is particularly
advantageous, since exogenous administration of these cytokines and
hematopoietic factors have limitations associated with the lack of
acceptable formulations, their exhorbitant cost, short half-life in
biological media, difficulties in dose-determination, and numerous
toxic and allergic effects.
[0051] In another embodiment, the present invention encompasses
methods and composition comprising NAC amide for detecting
NAC-amide responsive changes in gene expression in a cell, tissue,
and/or a subject, comprising administering or introducing NAC amide
or derivative of NAC amide to the cell, tissue, and/or subject for
a period of time to induce changes in gene expression and detecting
the changes in gene expression. NAC amide and derivatives thereof
can induce changes in gene expression such as genes involved in
apoptosis, angiogenesis, chemotaxis, among others.
[0052] In another aspect, the present invention provides directed
delivery of NAC amide to cells, such as cancer cells that express
high levels of receptors for folic acid (folate) or glutathione.
According to this aspect, NAC amide ("NACA") is coupled to a ligand
for the receptor (e.g., folic acid or glutathione) to form a
conjugate, and then this NACA-ligand conjugate is coated or
adsorbed onto readily injectable nanoparticles using procedures
known to those skilled in the art. According to this aspect, the
nanoparticles containing NAC amide ("nano-NACA particles") may be
preferentially taken up by cancer or tumor cells where the NAC
amide will exert its desired effects. Accordingly, the present
invention provides a method of directed delivery of NAC amide to
host cells expressing high levels of surface receptor for a ligand,
in which the method involves (a) coupling NAC amide to the surface
receptor ligand to form a NAC amide-ligand conjugate; (b) adsorbing
the NAC amide-ligand conjugate onto nanoparticles; and (c)
introducing the nanoparticles of (b) into the host. The invention
further provides a method of directed delivery of NAC amide to host
cells expressing high levels of surface receptor for a ligand, in
which the method involves (a) conjugating acetylated dendritic
nanopolymers to a ligand; (b) coupling the conjugated ligand of (a)
to NAC amide to form NAC amide-ligand nanoparticles; and c)
introducing the nanoparticles of (b) into the host.
[0053] Another aspect of the present invention provides a compound
of the formula I:
##STR00001##
[0054] wherein: [0055] R.sub.1 is OH, SH, or S--S-Z; [0056] X is C
or N; [0057] Y is NH.sub.2, OH, CH.sub.3--C.dbd.O, or NH--CH.sub.3;
[0058] R.sub.2 is absent, H, or .dbd.O [0059] R.sub.3 is absent
or
##STR00002##
[0060] wherein: R.sub.4 is NH or O; [0061] R.sub.5 is CF.sub.3,
NH.sub.2, or CH.sub.3
[0062] and wherein: Z is
##STR00003##
with the proviso that if R.sub.1 is S--S-Z, X and X' are the same,
Y and Y' are the same, R.sub.2 and R.sub.6 are the same, and
R.sub.3 and R.sub.7 are the same.
[0063] The present invention also provides a NAC amide compound and
NAC amide derivatives comprising the compounds disclosed
herein.
[0064] In another aspect, a process for preparing an L- or D-isomer
of the compounds of the present invention are provided, comprising
adding a base to L- or D-cystine diamide dihydrochloride to produce
a first mixture, and subsequently heating the first mixture under
vacuum; adding a methanolic solution to the heated first mixture;
acidifying the mixture with alcoholic hydrogen chloride to obtain a
first residue; dissolving the first residue in a first solution
comprising methanol saturated with ammonia; adding a second
solution to the dissolved first residue to produce a second
mixture; precipitating and washing the second mixture; filtering
and drying the second mixture to obtain a second residue; mixing
the second residue with liquid ammonia and an ethanolic solution of
ammonium chloride to produce a third mixture; and filtering and
drying the third mixture, thereby preparing the L- or D-isomer
compound.
[0065] In some embodiments, the process further comprises
dissolving the L- or D-isomer compound in ether; adding to the
dissolved L- or D-isomer compound an ethereal solution of lithium
aluminum hydride, ethyl acetate, and water to produce a fourth
mixture; and filtering and drying the fourth mixture, thereby
preparing the L- or D-isomer compound.
[0066] Another aspect of the invention provides a process for
preparing an L- or D-isomer of the compounds disclosed herein,
comprising mixing S-benzyl-L- or D-cysteine methyl ester
hydrochloride or O-benzyl-L- or D-serine methyl ester hydrochloride
with a base to produce a first mixture; adding ether to the first
mixture; filtering and concentrating the first mixture; repeating
steps (c) and (d), to obtain a first residue; adding ethyl acetate
and a first solution to the first residue to produce a second
mixture; filtering and drying the second mixture to produce a
second residue; mixing the second residue with liquid ammonia,
sodium metal, and an ethanolic solution of ammonium chloride to
produce a third mixture; and filtering and drying the third
mixture, thereby preparing the L- or D-isomer compound.
[0067] Yet another aspect of the invention provides a process for
preparing a compound as disclosed herein, comprising mixing
cystamine dihydrochloride with ammonia, water, sodium acetate, and
acetic anhydride to produce a first mixture; allowing the first
mixture to precipitate; filtering and drying the first mixture to
produce a first residue; mixing the second residue with liquid
ammonia, sodium metal, and an ethanolic solution of ammonium
chloride to produce a second mixture; filtering and drying the
second mixture, thereby preparing the compound.
[0068] The present invention also provides a food additive
comprising NAC amide or a NAC amide derivative as disclosed
herein.
[0069] Additional aspects, features and advantages afforded by the
present invention will be apparent from the detailed description
and exemplification hereinbelow.
BRIEF DESCRIPTION OF THE FIGURES
[0070] FIG. 1A presents the structure of N acetyl cysteine. FIG. 1B
presents the structure of N-acetylcysteine amide (NAC amide).
[0071] FIGS. 2A-2D show the cytotoxic response of PC12 cells to
glutamate and protection by NAC amide. PC12 cells were plated at a
density 25.times.10.sup.3 cells/well in a 24 well plate and grown
for 24 h in culture medium. They were treated or not (control) with
10 mM Glu with or without NAC amide, as described in Example 1.
Twenty-four hours later, cells were examined and photographed. FIG.
2A: Control; FIG. 2B: NAC amide (NACA) only; FIG. 2C: Glutamate
only; FIG. 2D: Glutamate and NACA.
[0072] FIG. 3 shows the protective effect of NAC amide against
glutamate cytotoxicity. Cells were plated and grown for 24 hours in
a culture medium; then they were treated or not (control) with 10
mM Glu, with or without NAC amide. Twenty-four hours later, the %
LDH release was determined using LDH analysis. Values represent
means.+-.SD. Statistically different values of * P<0.0001 and **
P<0.05 were determined, compared to control. * P<0.0001
compared to glutamate-treated group.
[0073] FIG. 4 shows the effect of NAC amide on glutamate-induced
cytotoxicity. Cells were exposed to 10 mM Glu, with or without NAC
amide, for 24 hours; the effects were compared to the control. Cell
viability was quantified by the MTS assay. Values represent
means.+-.SD. Statistically different values of *P<0.0005 and **
P<0.05 were determined, compared to control. *** P<0.05
compared to glutamate-treated group.
[0074] FIG. 5 shows the effects of NAC amide [NAC amide] on
cysteine levels in PC12 cells. Cells were plated and grown for 24
hours, and then they were exposed to glutamate (10 mM) in the
presence or absence of NAC amide (750 .mu.M). Twenty-four hours
later, the cells were harvested and cysteine levels were measured.
Values represent means.+-.SD. Statistically different values of *
P<0.005 and ** P<0.05 were determined, compared to control.
*** P<0.05 compared to glutamate-treated group.
[0075] FIG. 6 is a graph depicting a comparison of survival rates
of Sprague-Dawley rats after X-ray irradiation treatment in
combination with pre-treatment or post-treatment with NAC or NAC
amide (TOVA).
DETAILED DESCRIPTION OF THE INVENTION
[0076] The present invention involves the use of an effective and
potent antioxidant, glutathione N-acetylcysteine amide (NAC amide),
(FIG. 1), or a physiologically or pharmaceutically acceptable
derivative or salt or ester thereof, for use in a variety of
disorders, conditions, pathologies and diseases in which oxidative
stress and/or free radical formation cause damage, frequently
systemic damage, to cells, tissues and organs of the body. The
invention encompasses a pharmaceutically acceptable composition
comprising NAC amide, e.g., water-soluble NAC amide, or
physiologically acceptable derivatives, salts, or esters thereof,
which can be used in treatment and therapeutic methods in
accordance with this invention.
[0077] Glutathione N-acetylcysteine amide (NAC amide), the amide
form of N-acetylcysteine (NAC), is a novel low molecular weight
thiol antioxidant and a Cu.sup.2+ chelator. NAC amide provides
protective effects against cell damage in its role as a scavenger
of free radicals. In mammalian red blood cells (RBCs), NAC amide
has been shown to inhibit tert.-butylhydroxyperoxide
(BuOOH)-induced intracellular oxidation and to retard BuOOH-induced
thiol depletion and hemoglobin oxidation in the RBCs. This
restoration of thiol-depleted RBCs by externally applied NAC amide
was significantly greater than that found using NAC. Unlike NAC,
NAC amide protected hemoglobin from oxidation. (L. Grinberg et al.,
Free Radic Biol Med., 2005 Jan. 1, 38(1):136-45). In a cell-free
system, NAC amide was shown to react with oxidized glutathione
(GSSG) to generate reduced glutathione (GSH). NAC amide readily
permeates cell membranes, replenishes intracellular GSH, and, by
incorporating into the cell's redox machinery, protects the cell
from oxidation. Because of its neutral carboxyl group, NAC amide
possesses enhanced properties of lipophilicity and cell
permeability. (See, e.g., U.S. Pat. No. 5,874,468 to D. Atlas et
al.). NAC amide is also superior to NAC and GSH in crossing the
cell membrane, as well as the blood-brain barrier. NAC amide can be
prepared as described in U.S. Pat. No. 6,420,429 to D. Atlas et
al., the contents of which are incorporated by reference
herein.
[0078] NAC amide may function directly or indirectly in many
important biological phenomena, including the synthesis of proteins
and DNA, transport, enzyme activity, metabolism, and protection of
cells from free-radical mediated damage. NAC amide is a potent
cellular antioxidant responsible for maintaining the proper
oxidation state within cells. NAC amide is synthesized by most
cells and can recycle oxidized biomolecules back to their active
reduced forms. As an antioxidant, NAC amide may be as effective, if
not more effective, than GSH.
[0079] In one embodiment, the present invention encompasses methods
and compositions comprising NAC amide for preventing, reducing,
protecting, or alleviating glutamate-induced cytotoxicity in
neurodegenerative diseases, particularly in neuronal cells and
tissues (See, e.g., Example 1). In this embodiment, NAC amide can
protect cells of the nervous system from the effects of oxidative
toxicity induced by glutamate. Without wishing to be bound by
theory, NAC amide treatment can function to supply GSH as a
substrate for GSH peroxidase activity in affected cells. In
accordance with the present invention, NAC amide can inhibit lipid
peroxidation, scavenge for reactive oxygen species (ROS) and
enhance intracellular levels of GSH to combat and overcome
oxidative stress. In addition, NAC amide can chelate lead and
protect against lead-induced oxidative stress. NAC amide is
particularly beneficial and advantageous for neurological disorders
and diseases affecting the brain and associated parts thereof,
because it more readily crosses the blood-brain barrier to enter
the brain and provide its antioxidant effects.
[0080] Different neurodegenerative conditions and diseases that can
be treated according to this embodiment include cerebral ischemia,
Parkinson's disease. NAC amide can be used in the reduction of
brain damage during seizures; to provide resistance to induced
epileptic seizures; for protection during traumatic brain injury
through the effect on mitochondrial function, reduction of
inflammation and attenuation of and improvement in re-profusion
with decreased re-profusion injury; for reduction of traumatic
brain injury; and for treating prion disease, such as
Creutzfeldt-Jakob disease and mad cow disease, by acting as an NMDA
receptor antagonist, by enhancing intracellular levels of the
anti-apoptotic protein Bcl-2; and by increasing antioxidants to
glutathione. NAC amide can be used in neural protection,
mitochondrial preservation and therapy potential after nerve
injury, particularly to prevent primary sensory neuronal death.
[0081] In another embodiment, the invention embraces methods and
compositions comprising NAC amide for protecting cells and tissues
from radiation-induced oxidative stress. In accordance with this
embodiment, NAC amide is superior to NAC in protecting tissues from
radiation-induced oxidative stress. (Example 2). The medical crisis
following the Chernobyl incident and the threat of a terrorist
nuclear attack have raised awareness that high-dose total body
irradiation may occur and result in death due to the induction of
three potentially lethal cerebrovascular, gastrointestinal and
hematopoietic clinical syndromes, which result from high dose
radiation exposure. The combination of the prodromal syndrome
followed by the gastrointestinal syndrome and bone marrow death
induces dehydration, anemia, and infection that lead to
irreversible shock. Current treatment for the subacute
gastrointestinal and hematopoietic syndromes includes supportive
therapy such as plasma volume expansion, platelets, and antibiotics
to prevent dehydration and infection and promote bone marrow
repopulation. Human total body exposure to a radiation dose above
10 Gy has been regarded as uniformly fatal. With therapeutic
intervention, survival may be possible up to 15 Gy of total body
irradiation, but beyond 20 Gy the symptoms would not be
manageable.
[0082] The systemic damage observed following irradiation is
partially due to the overproduction of reactive oxygen species
(ROS), which disrupt the delicate pro-oxidant/antioxidant balance
of tissues leading to protein, lipid and DNA oxidation. For
example, oxidation of the glucosamine synthetase active site
sulfhydryl groups is a key factor in the toxicity of the
gastrointestinal syndrome. Polyunsaturated fatty acids, when
exposed to ROS, can also be oxidized to hydroperoxides that
decompose in the presence of metals to hydrocarbons and aldehydes
such as malondialdehyde (MDA). This lipid peroxidation can cause
severe impairment of membrane function through increased membrane
permeability and membrane protein oxidation. DNA oxidation can lead
to strand breakage and consequent mutation or cell death. GSH is
the principal intracellular thiol responsible for scavenging ROS
and maintaining the oxidative balance in tissues, such as plasma,
brain, kidney, liver and lung. In accordance with this embodiment,
NAC amide significantly improves GSH levels in these tissues after
radiation exposure. (Example 2). The prevention of spinal cord
damage resulting from radiation exposure is also encompassed by the
use of NAC amide.
[0083] In another embodiment, the present invention encompasses
methods and compositions comprising NAC amide for stimulating
endogenous production of cytokines and hematopoietic factors,
comprising administering or introducing NAC amide to cells,
tissues, and/or a subject in need thereof for a period of time to
stimulate the endogenous production. NAC amide can be used to
stimulate production of cytokines and hematopoietic factors, such
as but not limited to, TNF-.alpha., IFN-.alpha., IFN-.beta.,
IFN-.gamma., IL-1, IL-2, IL-6, IL-10, erythropoietin, G-CSF, M-CSF,
and GM-CSF, which are factors that modulate the immune system and
whose biological activities are involved in various human diseases,
such as neoplastic and infectious diseases, as well as those
involving hematopoiesis and immune depressions of various origin
(such as, without limitation, erythroid, myeloid, or lymphoid
suppression).
[0084] As used herein, "endogenous" means naturally occurring
within a cell, tissue, or organism, or within a subject.
[0085] In another embodiment, the present invention encompasses
methods and composition comprising NAC amide for detecting
NAC-amide responsive changes in gene expression in a cell, tissue,
and/or a subject, comprising administering or introducing NAC amide
or derivative of NAC amide to the cell, tissue, and/or subject for
a period of time to induce changes in gene expression and detecting
the changes in gene expression. The cell can be an endothelial
cell, smooth muscle cell, immune cell such as erythroid, lymphoid,
or myeloid cell, progenitors of erythroid, lymphoid, or myeloid
cells, epithelial cell, fibroblasts, neuronal cell and the like.
The tissue can be any tissue of the subject, such as hair, skin, or
nail tissue, vascular tissue, brain tissue, among many others.
Preferably, the changes in gene expression are detected by
microarray analysis, but other detection means can encompass,
without limitation, reverse-transcription polymerase chain reaction
(RT-PCR), Northern Blotting, immunofluorescence, immunoblotting, or
enzyme-linked immunosorbent assay, all of which are familiar
techniques to those skilled in the art.
[0086] NAC amide and derivatives of NAC amide can induce changes
in, for example, endothelial cells that are indicative of an
anti-angiogenic effect. NAC has been shown to inhibit chemotaxis of
endothelial cells in culture, and produce anti-angiogenic effects,
such as modulation of genes responsible for blood vessel growth and
differentiation, through its antioxidant effects and upregulation
of angiostatin (Pfeffer, U. et al, (2005) Mut. Res. 591: 198-211).
Thus, NAC amide and NAC amide derivatives can be used to inhibit
angiogenesis as an anti-cancer agent, for example, by preventing or
inhibiting tumor growth and metastasis.
[0087] Cells, tissues, and/or a subject can be exposed to stimuli
in the presence of NAC amide or derivatives of NAC amide. Stimuli
include, for example, cells cultured in the presence of chemotactic
or chemoattractant agents, like chemokines CXCL1-16, CCL1-27, XCL1,
XCL2, RANTES, MIP 1-5 (alpha, beta, and gamma isoforms), MCP-1
through 5, and the like. Cells, tissues, and subjects can also be
stimulated with pharmaceutical agents, drugs, or treatment
modalities. After stimulation, DNA, RNA, or protein can be isolated
from the cells, tissues, and/or subject, and changes in gene
expression can be detected. For example, total RNA can be isolated
from cells according to standard techniques known in the art and
resultant cDNAs can be synthesized and subsequently hybridized to a
solid support, such as a silicon chip for microarray analysis.
Expression data and changes in the expression of genes in response
to the stimuli can then be analyzed using computer software
programs, such as GeneSpring (Silicon Genetics).
[0088] Non-limiting examples of such genes that exhibit changes in
their expression include genes involved in or pertaining to
cellular adhesion, apoptosis, chemokine and cytokine biosynthesis,
synthesis of extracellular matrix components, endothelium,
inflammation, MAP kinases, metalloproteinases, NF-.kappa.B, nitric
oxide, transforming growth factor (TGF) signaling, and blood
vessels. Pfeffer et al reported that a plurality of NAC-responsive
genes that are modulated (i.e., up- or downregulated) include HSP40
(heat shock protein 40; DnaJ homolog), SERCA2 (Ca2+ transporting
ATPase in cardiac muscle), MKP2 (MAP kinase phosphatase), TIP30
(HIV-1 Tat interactive protein 2), BTG1 (B-cell translocation gene
1), TXL (thioredoxin-like), CRADD (Death receptor adaptor protein),
WSX1 (Class I cytokine receptor), EMAP2 (endothelial
monocyte-activating protein), Jagged 1 (ligand for Notch receptor),
MEA5 (hyaluronoglucosaminidase), VRNA (Integrin .alpha.V), COL4A1
(Type IV collagen .alpha.1), uPA (urokinase plasminogen activator),
CPE (carboxypeptidase E), TSPAN-6 (transmembrane 4 superfamily
member 6), FGFB (basic fibroblast growth factor), I-TRAF (TRAF
interacting factor), CDHH (cadherin 13), IL10RB (Interleukin-10
receptor .beta.), MAP-1 (modulator of apoptosis 1), hCOX-2
(cyclooxygenase-2), CAS-L (Cas-like docking protein), CED-6 (CED-6
protein), CX37 (gap junction protein .alpha.4), ABCG1 (ATP-binding
cassette protein, subfamily G), TRAIL (TNF ligand superfamily
member 10), and ESEL (endothelial adhesion molecule 1; Selectin E),
as well as CHOP (DNA-damage-inducible transcript 3), PIM2 (pim-2
oncogene, MIF-1 (homocysteine-inducible protein), PIG-A
(phosphatidylinositol glycan, class A), K1AA0062, HK2 (hexokinase
2), UDPGDH (UDP-glucose dehydrogenase), ERF2 (Zinc finger protein
36, C3H type-like 2), RAMP (Zinc finger protein 198), Doc1
(Downregulated in ovarian cancer 1), GBP-1 (Guanylate-binding
protein 1, interferon-inducible), GR (glucocorticoid receptor), ENH
(LIM protein-enigma homolog), Id-2H (Inhibitor of DNA binding 2),
BPGM (2,3-bisphosphoglycerate mutase), HOXA4 (Homeobox A10), EFNB2
(ephrin-B2), ART4 (Dombrock blood group), KIAA0740 (Rho-related BTB
domain containing protein 1).
[0089] In another embodiment, the present invention encompasses
methods and compositions comprising NAC amide for stimulating
macrophages and neutrophils to phagocytize infectious agents and
other foreign bodies and to eliminate microorganisms, mediated by
reactive oxygen species and proteases. NAC amide can be used to
improve macrophage function by increasing glutathione availability,
which, in turn, will improve alveolar function in fetal alcohol
syndrome and to augment premature alveolar macrophage function.
[0090] In another embodiment, the invention encompasses methods and
compositions comprising NAC amide to increase levels of
intracellular reduced glutathione levels, which blocks the
formation of irreversibly sickled cell red blood cells. Methods
involving the administration of NAC amide to prevent and treat
sickle cell anemia and thalassemia are provided.
[0091] In another embodiment, the invention encompasses methods and
compositions comprising NAC amide to treat leishmania through the
mechanism of histopathological modulation, in which cytokine
pattern is modified as demonstrated by a sustained higher frequency
of interferon-.gamma. (IFN-.gamma.) and tumor necrosis factor alpha
producing cells. NAC amide is used in the modulation of effector
responses in animals, in conjunction with bi-glutathione.
[0092] In an embodiment, NAC amide is used to down-regulate
cytokine synthesis, activation and downstream processes and/or to
exert an antagonistic effect on pro-inflammatory signals. Such an
effect is beneficial in the treatment of many diseases in which
cytokines participate in the pathophysiology of the disease. For
example, cytokines, which are mediators of oxidative stress, can
alter the redox equilibrium by affecting GSH/oxidized glutathione
disulfide (GSSG) shuttling and recycling. (For a review of the
glutathione-mediated regulation of cytokines and the role of
antioxidants, see, J. J. Haddad, 2005, Mol. Immunol.,
42(9):987-1014; and J. J. Haddad, 2002, Cellular Signalling,
14(11):879-897). Additionally, liver injury related to the
administration of certain drugs can be initiated or intensified by
inflammation states that stimulate unregulated production of
proinflammatory cytokines or growth factors, such as interferon
.gamma., which leads to the down-regulation of enzymes and proteins
involved in drug metabolism and elimination. NAC amide, or
derivative thereof as an agent that can decrease proinflammatory
cytokine levels, is thus useful for preventing and/or managing
drug-induced hepatocytoxicity.
[0093] In another embodiment, the invention encompasses methods and
compositions comprising NAC amide or a derivative thereof for use
as a chemoprotectant against bone marrow toxicity after or during
chemotherapy, including alkylators with or without glutathione
depletion.
[0094] In another embodiment, the invention encompasses methods and
compositions comprising NAC amide or a derivative thereof to treat
various aspects of sepsis, particularly bacterial sepsis and septic
shock, including gram-negative septic shock. NAC amide and its
derivatives can act as an inhibitor of the nuclear factor
NF-.kappa.B, which prevents staphylococcal enterotoxin A (SCC)
fever by acting through the human peripheral blood mononuclear
cells to block the stimulation and synthesis or release pyrogenic
cytokines and to block inflammatory sponsors through the regulation
of genes in coding for proinflammatory cytokines. In accordance
with this embodiment, NAC amide or a derivative thereof is used to
block lipid peroxidation and to improve the disease status in
children with acute purulent meningitis and encephalitis. NAC amide
and its derivatives can be used to block pertussis toxin secretion
by Bordetella pertussis and for the treatment of lethal sepsis by
limiting inflammation and potentiating host defense. Because
decreased bacterial colonies improve survival, migration of
neutrophils to the site of infection and to a distant site is
upregulated and optimal GSH levels are important for an efficient
response to sepsis. In addition, ROS release by immune cells are
important mediators in sepsis and septic shock. During a normal
immune response antioxidant serves to down-regulate the ongoing
immune response mostly through modulation of proinflammatory
mediators.
[0095] In another embodiment, methods and compositions comprising
NAC amide or a derivative thereof can be used in the treatment of
infection and disease caused by microorganisms and the like, such
as bacteria, parasites, nematodes, yeast, fungi, plasmodia,
mycoplasma, spores, and the like, e.g., malarial infections and
tuberculosis and rickettsia infection. In a related aspect, it has
recently been found that infection by a number of types of
bacteria, such as Streptococcus, Staphylococcus, Salmonella,
Bacillus (Tubercule bacillus) etc., which cause diseases in humans,
induce a direct response by leukocytes (i.e., white blood cells) in
the body, to increase their levels of hypoxia inducible
transcription factor-1, or HIF-1. The HIF-1 protein binds to
cellular DNA and activates specific genes to help cells function in
a low oxygen environment. HIF-1, in turn, stimulates the white
blood cells to produce and release antimicrobial compounds, e.g.,
small proteins, enzymes and nitric oxide, that work together to
kill bacteria. In addition, it has been found that low oxygen
levels, which occur at the site of an infection, activate HIF-1 in
macrophages and neutrophils, which typically ingest and destroy
invading microorganisms. The greater the increase in HIF-1 levels
in the white blood cells, the greater their anti-bacterial
activity. In accordance with this aspect of the invention and in
view of the influence of HIF-1 in regulating the killing functions
of white blood cells, an alternative to the direct killing of
bacteria, etc. is to use agents, e.g., small molecules, that
promote HIF-1 activity in white blood cells to boost their
bacterial killing ability, thereby promoting a resolution to
infection through the actions of the immune system's natural
defense mechanisms. One such agent is NAC amide, which can be used
in a method of killing or inhibiting the growth of microorganisms
by increasing cellular levels of HIF-1, i.e., HIF-1, thereby
enhancing the capacity of white blood cells, such as macrophages,
to kill the microorganisms. Because N-acetyl-L-cysteine, NAC, a
glutathione (GSH) precursor and a ROS scavenger, which does not
possess the enhanced properties of lipophilicity and cell
permeability of NAC amide, has been shown to induce HIF-1.alpha. in
epithelial cells (J. J. E. Haddad et al., 2000, J. Biol. Chem.,
275:21130-21139), the use of NAC amide to modulate HIF-1 .alpha.
production in white blood cells in order to activate the bacterial
killing potential of these cells is embraced as an improved
antioxidant treatment provided by the present invention. The
present invention is further directed to the use of NAC amide or a
derivative thereof as a bacteriostatic agent when used as a
treatment for bacterial infection, particularly antibiotic
resistant, or multi-antibiotic resistant bacteria such as
tuberculosis-causing microorganisms.
[0096] In a related embodiment, the present invention is directed
to the use of NAC amide or a derivative thereof as a biodefensive
agent for inducing the killing of infecting or contaminating
microorganisms. These types of microorganisms may pose a severe
health threat if they should be disseminated to the public and/or
genetically altered so as to be antibiotic resistant. The following
lists set forth categories of microorganisms, viruses, diseases and
agents for which NAC amide or its derivative is provided as a
suitable countermeasure, used alone, or in combination with other
active compounds, agents and substances to treat affected organisms
and/or cells thereof:
[0097] Infectious Diseases: Aflatoxins, Alphavirus Eastern equine
encephalitis virus, Alphavirus Venezuelan equine encephalitis
virus, Antibiotic-resistant Mycobacterium tuberculosis, Arenavirus
Junin Virus, Arenavirus Lassa Virus, Ascaris lumbricoides
(roundworm), Avian influenza, Bacillus anthracis (anthrax),
Borrelia, Brucella, Burkholderia mallei (glanders), Chlamydia
psittaci (parrot fever), Chlamydia trachiomitis (Trachoma),
Clostridium botulinum (botulism), Clostridium perfringens (gas
gangrene), Coccidioidomycosis immitis, Coxiella burnetti (Q fever),
Cryptosporidium parvum, Dinoflagellate neurotoxin (Paralytic
Shellfish Toxin), Drancunculus medianensis (guinea worm), Ebola
virus, Entamoeba histolytica (amoebiasis), Epsilon toxin of
Clostridium perfringens, Escherichia coli, Flavivirus Yellow Fever
virus (e.g., West Nile virus, Dengue), Francisella tularensis
(tularemia), Giardia lamblia (giardiasis), Hantavirus, Henipavirus
Nipah virus (Nipa encephalitis), HIV and AIDS, Influenza,
Leishmania donovane, Marburg virus, Methicillin-resistant
staphylococcus aureus (MRSA), Mycobacterium leprea (leprosy),
Mycobacterium ulcerans (Burulu ulcer), Nairo virus Crimean-Congo
hemorrhagic fever virus, Necator Americanus/Ancylostoma duodenale
(hookworm), Onchocerca volvulus (river blindness), Orthopox virus,
Pathogenic Haemophilus, Pathogenic Salmonella, Pathogenic Shigella,
Pathogenic Streptococcus, Phlebovirus Rift Valley fever virus,
Plasmodium falciparum, P. ovale, P. vivax, P. malariae (malaria),
Ricin toxin (castor bean oil), Rickettsia rickettsii (Rocky
Mountain Spotted Fever), Rickettsia typhi (typhus), Salmonella
typhi (typhoid fever), Schistosoma mansoni, S. haematobium, S.
japonicum, Shigella dysenteriae, Smallpox, Staphylococcus
enterotoxin B, Tickborne encephalitis virus, Tickborne hemorrhagic
fever viruses, Toxoplasma gondii, Treponema, Trichothecene
Mycotoxins, Trichuris trichiura (whipworm), Trypanosoma brucei, T.
gambiense or T. rhodesiense, Vibrio species (cholera), Wuchereria
bancrofti and Brugia malayi, Yersinia pestis (black death).
[0098] Other Threats: Blister agents, including Lewisite, nitrogen
and sulfur mustards; Blood agents, including hydrogen cyanide and
cyanogens chloride; Exotic agents, including hybrid organisms,
genetically modified organisms, antibiotic-induced toxins,
autoimmune peptides, immune mimicry agents, binary bioweapons,
stealth viruses and bioregulators and biomodulators; Heavy metals,
including arsenic, lead and mercury; incapacitating agents,
including BZ; nerve agents, including Tabun, Sarin, Soman, G F, VX,
V-gas, third generation nerve agents, organophosphate pesticides
and carbamate insecticides; nuclear and radiological materials,
pulmonary agents, including phosgene and chorine vinyl chloride;
volatile toxins, including benzene, chloroform and trihalomethanes.
In accordance with the present invention, NAC amide or derivatives
thereof can serve as an innovative treatment for known and emerging
natural infectious disease threats, as well as trauma, e.g.,
excessive bleeding and other events, associated with and/or
resulting from an act of bioterrorism.
[0099] Illustratively, Rickettsia, which causes the pathogenesis of
typhus and spotted fever rickettsioses, results in serious adverse
vascular and hemorrhagic conditions, (e.g., increased vascular
permeability and edema) notably in the brain and lung, following
its entry into vascular endothelial cells. R. rickettsii-infected
endothelial cells produce ROS causing peroxidative damage to cell
membranes. (D. J. Silverman et al., 1990, Ann. N.Y. Acad. Sci.,
590:111-117; D. H. Walker et al., 2003, Ann. N.Y. Acad. Sci.,
990:1-11). Because the oxidative-stress mediated damage to R.
rickettsii-infected endothelial cells is associated with the
depletion of host components such as GSH and levels of catalase
that act as host defenses against ROS-induced damage, the
concentration of hydrogen peroxide and ROS increase in the cells to
cause ROS-induced cellular damage. In a similar manner, cells,
e.g., fibroblasts that are infected with Mycoplasma (e.g.,
Mycoplasma pneumoniae) also produce increased intracellular levels
of hydrogen peroxide and decreased levels of catalase, resulting in
oxidative stress that can lead to death of the infected cells. (M.
Almagor et al., 1986, Infect. Immun., 52(1):240-244). To provide an
ameliorating effect of oxidative stress induced in cells by
infecting microorganisms such as Rickettsia, Mycoplasma, etc., NAC
amide or a derivative thereof is provided to an infected host as an
antioxidant therapeutic. NAC amide administration to cells and/or
organisms (e.g., infected host mammals) in accordance with the
present invention, alone or in combination with other agents and/or
antioxidants, can limit the amount and/or extent of oxidative
damage that is induced by microbial infection.
[0100] In another embodiment, the invention encompasses methods and
compositions comprising NAC amide or a derivative thereof for use
in preventing periventricular leukomalacia (PVL). NAC amide or a
derivative thereof may provide neural protection and attenuate the
degeneration of OPCs against LPS evoked inflammatory response in
white matter injury in developing brain. Moreover, NAC amide or a
derivative thereof may be used as a treatment for placental
infection as a means of minimizing the risk of PVL and cerebral
palsy (CP).
[0101] In another embodiment, the invention encompasses methods and
compositions comprising NAC amide or a derivative thereof for the
treatment of osteoporosis. The tumor necrosis factor member RANKL
regulates the differentiation, activation and survival of
osteoclasts through binding of its cognate receptor, RANK. RANK can
interact with several TNF-receptor-associated factors (TRAFs) and
activate signaling molecules including Akt, NF-.kappa.B and MAPKs.
Although the transient elevation of reactive oxygen species by
receptor activation has been shown to act as a cellular secondary
messenger, the involvement of ROS in RANK signal pathways has not
been characterized. RANKL can stimulate ROS generation and
osteoclasts. According to this embodiment, NAC amide can be used to
pretreat or treat osteoclasts so as to achieve a reduction in
RANKL-induced Akt, NF-.kappa.B, and ERK activation. The reduced
NF-.kappa.B activity by NAC amide may be associated with decreased
IKK activity and I.kappa.B.alpha. phosphorylation. Pretreatment
with NAC amide or a derivative thereof can be used to reduce
RANKL-induced actin ring formation required for bone resorbing
activity and osteoclast survival. The methods and compositions
comprising NAC amide or a derivative thereof can be used for the
improvement of osteoporosis through blockage and interference with
osteoclasts, and to lower reactive oxidative stress levels so as to
have beneficial effects on preventing bone loss by reducing
RANKL-induced cellular function.
[0102] In a related embodiment, NAC amide or a derivative thereof
is used in the treatment of osteoporosis by blockage of thiol
thioredoxin-1, which mediates osteoclast stimulation by reactive
oxidation species (ROS), as well as blockage of TNF-.alpha., which
causes loss of bone, particularly in circumstances of estrogen
deficiency.
[0103] In another embodiment, the invention embraces methods and
compositions comprising NAC amide or a derivative thereof are used
for the treatment of polycystic ovary syndrome. NAC amide or a
derivative thereof may also be used as a therapeutic agent to
ameliorate the homocysteine and lipid profiles in PCOS-polycystic
ovary syndrome.
[0104] In another embodiment, the invention encompasses the use of
NAC amide or a derivative thereof in treatments and therapies for
toxin exposure and conditions related thereto, e.g., sulfur mustard
(HD-induced lung injury). Treatment of individuals having been
exposed to toxins or suffering from toxin exposure with NAC amide
or a derivative thereof may reduce neutrophil counts to achieve a
decreased inflammatory response. NAC amide and its derivatives may
be useful as a treatment compound for patients having sulfur
mustard vapor exposure induced lung injury. Administration of NAC
amide or a derivative thereof can be either orally or as a
bronchoalveolar lavage. As an agent having anti-glutamate toxin
activity, NAC amide and its derivatives are useful in methods and
compositions for the blockage of brain and/or lung damage and
cognitive dysfunction in mechanical warfare agents including CW,
vesicants, sulfur mustard, nitrogen mustards, chloroethyl amine,
lewisite, nerve agents O-ethyl S-(2-[di-isopropylamino]ethyl)methyl
phosphorothioate (VX), tabun (GA) and sarin (GB) and soman DG and
the blood agents cuianogenchloride, and in the prevention of
organophosphate induced convulsions and neuropathological
damage.
[0105] In another embodiment, the present invention encompasses
methods and compositions comprising NAC amide for use in the
treatment of burn trauma. NAC amide or a derivative thereof can
block NF-.kappa.B, which has been shown to reduce burn and burn
sepsis. NAC amide or a derivative thereof can be used to protect
microvascular circulation, reduce tissue lipid peroxidation,
improve cardiac output and reduce volume of required fluid
resuscitation. NAC amide or a derivative thereof can be used in the
prevention of burn related cardiac NF-.kappa.B nuclear migration,
and improve cardiomyocyte secretion of TNF-.alpha., IL-1.beta., and
IL-6 and to improve cardiac malfunction. An association between
cellular oxidative stress and burn-mediated injury provides an
avenue for administering NAC amide or a derivative thereof as an
antioxidant that can inhibit free radical formation and/or scavenge
free radicals to protect tissues and organs in patients with burn
injury.
[0106] In another embodiment, the present invention encompasses
methods and compositions comprising NAC amide or a derivative of
NAC amide for use in the prevention of lung injury due to the
adverse effects of air pollution and diesel exhaust particles.
[0107] In another embodiment, the present invention encompasses
methods and compositions comprising NAC amide or a derivative
thereof for use in the treatment and therapy of cardiovascular
disease and conditions. NAC amide and its derivatives can be used
as a blocker of angiotensin-converting enzyme. In acute myocardial
infarction, NAC amide or a derivative thereof can be used to
decrease oxidative stress, and to cause more rapid re-profusion,
better left ventricular preservation, reduced infarct size, better
preservation of global and regional left ventricular function and
modification of QSR complex morphology and ECG. NAC amide or a
derivative thereof can also be used in the treatment of focal
cerebral ischemia with protection of the brain and reduction of
inflammation in experimental stroke. NAC amide can be used in the
treatment of reperfusion injuries, as well as apoptosis of
myocardial endothelial cells and interstitial tissue. As a
nutriceutical, NAC amide or a derivative thereof may assist in the
elevation of nitric oxide levels, play an important role in the
management of cardiovascular disease, reduce chronic inflammation
in cardiovascular disease and prevent restenosis of cardiovascular
stents placed in coronary arteries and carotid arteries. NAC amide
and its derivatives can be used in the prevention of cardiac
failure following MI and cardiomyopathy due to prevention of
oxidative stress and improvement of left ventricular remodeling.
Use of NAC amide or a derivatives of NAC amide in this capacity
supports the involvement of oxidative stress in myocardial vascular
dysfunction and hypertension and provides a role for antioxidant
strategies to preserve the myocardial microvasculature. NAC amide
or a derivative thereof can also be used in the prevention of
oxidized proteins in muscles.
[0108] In another embodiment of the present invention, method and
compositions comprising NAC amide or a derivative thereof can be
used to treat arterial sclerosis and to increase high density
lipoprotein (HDL)-cholesterol serum levels in hyperlipidemic and
normal lipidemic individuals with documented coronary stenosis. NAC
amide or a derivative thereof can also be used to decrease coronary
and alpha-beta stress; to prevent further myocardial infarctions;
and to cause a reduction in body fat thereby improving glucose
tolerance, particularly in overweight or obese individuals. NAC
amide or a derivative thereof be used to improve muscular
performance and decrease levels of tumor necrosis factor in old
age.
[0109] In other embodiments, the present invention is directed to
the use of method and compositions comprising NAC amide or a
derivative thereof in the treatment of thalassemic blood by
ameliorating oxidative stress in platelets. The activation of
platelets causes thromboembolic consequences and produces a
hypercoagulable state that is amenable to treatment by the
antioxidant NAC amide or a derivative thereof. In an embodiment,
NAC amide or a derivative thereof is useful as a wound dressing to
permit enhancement of neutrophil function. In an embodiment, NAC
amide or a derivative thereof is used to block the effects of
leptin, which is a cardiovascular risk factor in diabetic patients.
In an embodiment, NAC amide or a derivative thereof is used in the
treatment of total plasma homocysteine and cysteine levels with
increased urinary excretion, as well as in the treatment for
hyperhomocysteinemic conditions, to improve oxidative stress. It
has been found that elevated levels of homocysteine pose a
significant risk in vascular disease, such as atherosclerosis,
venous thrombosis, heart attack and stroke, as well as neural tube
defects and neoplasia. Homocysteine promotes free radical
reactions. In patients with defective homocysteine metabolism,
relatively high levels of homocysteine are present in the blood.
Thus, in accordance with this invention, NAC amide or a derivative
thereof is administered to patients with elevated homocysteine
levels. In an embodiment, NAC amide or a derivative thereof is used
as a chemoprotectant against bone marrow toxicity after or during
chemotherapy, e.g., alkylators, with or without accompanying
glutathione depletion. In an embodiment, NAC amide or a derivative
thereof is used in the treatment of lithium induced renal failure.
In an embodiment, NAC amide or a derivative thereof is used in the
treatment of prostatic inflammation, which may contribute to
prostatic carcinogenesis and inflammation.
[0110] In another embodiment, NAC amide or a derivative thereof is
used in pulmonary disease medicine, particularly in oxygen-mediated
lung disease. NAC amide or a derivative thereof can improve
oxygenation in cardiopulmonary bypass during coronary artery
surgery and is useful in the treatment of chronic obstructive
pulmonary disease and pulmonary hypertension. In an embodiment, NAC
amide or a derivative thereof is used in the treatment of injury in
the lung due to high-energy impulse noise-blasts, which can induce
antioxidant depletion. Thus, the administration of NAC amide or its
derivatives provide an advantageous antioxidant source. NAC amide
or a derivative thereof is particularly useful if provided as a
supplement prior to noise blast exposure. NAC amide or a derivative
thereof is useful in the treatment of asthma with increased
oxidative stress. NAC amide or a derivative thereof is useful for
the treatment of adult respiratory distress syndrome; in the
treatment of pulmonary fibrosis, in the treatment of idiopathic
pulmonary fibrosis and asbestos exposure; and in the treatment of
chronic lung rejection. Further, NAC amide or a derivative thereof
is contemplated for use in occupational isocyanate exposure and the
development of isocyanate allergy, which is believed to develop by
two processes, namely, isocyanate-protein conjugation and airway
epithelial cell toxicity. More specifically, NAC amide or a
derivative thereof can serve to protect against hexamethylene
diisocyanate (HDI) conjugation to cellular proteins and to reduce
HDI toxicity to human airway epithelial cells following isocyanate
exposure. Thus, NAC amide or a derivative thereof can help to
prevent the development of allergic sensitization and asthma that
are associated with this occupational hazard.
[0111] In another embodiment, the present invention encompasses the
use of NAC amide or a derivative thereof to inhibit HIV replication
in chronically and acutely infected cells. NAC amide can be used in
GSH replacement therapy, as NAC amide and its derivatives may
interfere with the expression of the integrated HIV genome, thus,
attacking the virus in a manner that is different from that of the
currently employed anti-retrovirals, e.g., AZT, ddI, ddC or D4T.
NAC amide or a derivative thereof can also be beneficial in
countering the excess free radical reactions in HIV infection,
which may be attributable to: 1) the hypersecretion of TNF-.alpha.
by B-lymphocytes in HIV infection, and 2) the catalysis of
arachidonic acid metabolism by the gp120 protein of HIV. The
physiologic requirements for antioxidants by key cell types of the
immune system, and the ability of macrophages to take up
intercellular antioxidants, as well as to metabolically interact
with T-lymphocytes to indirectly cause their antioxidant levels to
increase, offer additional reasons that NAC amide or a derivative
thereof is useful for correcting antioxidant deficiency in patients
with HIV/AIDS. NAC amide and its derivatives can serve as a
suppressant of viral and bacterial species in vaginal tissues by
the use of intravaginal placement of gel induced thiol.
[0112] Because HIV is known to start pathologic free radical
reactions which lead to the destruction of antioxidant molecules,
as well as the exhaustion of GSH and destruction of cellular
organelles and macromolecules, NAC amide and its derivatives can be
used to restore antioxidant levels in a mammal in need thereof, to
arrest the replication of the virus at a unique point, and
specifically prevent the production of toxic free radicals,
prostaglandins, TNF-.alpha., interleukins, and a spectrum of
oxidized lipids and proteins that are immunosuppressive and cause
muscle wasting and neurological symptoms. The administration of NAC
amide or a derivative thereof to elevate or replace antioxidant
levels could slow or stop the diseases progression safely and
economically.
[0113] Because certain viral infections, such as infection by HIV,
are associated with reduced antioxidant levels, an aspect of this
invention is to increase intracellular levels of antioxidant in
infected cells, as well as to increase extracellular of
antioxidant, by introducing or administering AD3 so as to interfere
with the replication of HIV and to prevent, delay, reduce or
alleviate the cascade of events that are associated with HIV
infection. Because AIDS may also be associated with reduced GSSG
levels, providing an amount of NAC amide to cells and/or to an
individual in need thereof, can overcome any interference with de
novo synthesis of antioxidant such as GSH, as well as the oxidation
of existing GSH, which may occur in HIV infected cells. In
accordance with the present invention NAC amide or a derivative
thereof is used to inhibit cytokine-stimulated HIV expression and
replication in acutely infected cells, chronically infected cells,
and in normal peripheral blood mononuclear cells. NAC amide or
derivatives thereof can be used to effect concentration-dependent
inhibition of HIV expression induced by TNF-.alpha. or IL-6 in
chronically infected cells. Due to NAC amide's superior ability to
cross cellular membranes and enhanced lipophilic properties, NAC
amide and derivatives thereof can be used at lower concentrations
as compared to NAC or GSH, such as 2-fold, 5-fold, 10-fold,
100-fold, 1000-fold, 10,000-fold or lower, concentrations.
[0114] Further, the depletion of antioxidants by HIV in infected
cells is also associated with a process known as apoptosis, or
programmed cell death. By providing NAC amide or a derivative
thereof to HIV infected individuals and/or cells, the intercellular
processes, which artificially deplete GSH and which may lead to
cell death can be prevented, interrupted, or reduced. Similarly,
the NAC amide thiol can be used as a blocker of bio-replication
from West Nile Virus and protection of cells from the cytopathic
effect after infection of West Nile Virus, as well as other RNA and
DNA virus infections.
[0115] In accordance with the invention, NAC amide or a derivative
thereof may be administered by several routes that are suited to
the treatment or therapy method, as will be appreciated by the
skilled practitioner. Nonlimiting examples of routes and modes of
administration for NAC amide and its derivatives include parenteral
routes of injection, including subcutaneous, intravenous,
intramuscular, and intrasternal. Other modes of administration
include, but are not limited to, oral, inhalation, topical,
intranasal, intrathecal, intracutaneous, opthalmic, vaginal,
rectal, percutaneous, enteral, injection cannula, timed release and
sublingual routes. Administration of NAC amide and its derivatives
may also be achieved through continuous infusion. In one embodiment
of the present invention, administration of NAC amide and its
derivatives may be mediated by endoscopic surgery. For the
treatment of various neurological diseases or disorders that affect
the brain, NAC amide or a derivative thereof can be introduced into
the tissues lining the ventricles of the brain. The ventricular
system of nearly all brain regions permits easier access to
different areas of the brain that are affected by the disease or
disorder. For example, for treatment, a device, such as a cannula
and osmotic pump, can be implanted so as to administer a
therapeutic compound, such as NAC amide, or derivative thereof as a
component of a pharmaceutically acceptable composition. Direct
injection of NAC amide and its derivatives are also encompassed.
For example, the close proximity of the ventricles to many brain
regions is conducive to the diffusion of a secreted or introduced
neurological substance in and around the site of treatment by NAC
amide.
[0116] For administration to a recipient, for example, injectable
administration, a composition or preparation formulated to contain
water-soluble NAC amide or a derivative thereof is typically in a
sterile solution or suspension. Alternatively, NAC amide or a
derivative thereof can be resuspended in pharmaceutically- and
physiologically-acceptable aqueous or oleaginous vehicles, which
may contain preservatives, stabilizers, and material for rendering
the solution or suspension isotonic with body fluids (i.e. blood)
of the recipient. Non-limiting examples of excipients suitable for
use include water, phosphate buffered saline (pH 7.4), 0.15M
aqueous sodium chloride solution, dextrose, glycerol, dilute
ethanol, and the like, and mixtures thereof. Illustrative
stabilizers are polyethylene glycol, proteins, saccharides, amino
acids, inorganic acids, and organic acids, which may be used either
on their own or as admixtures.
[0117] Formulations comprising NAC amide or a derivative thereof
for topical administration may include but are not limited to
lotions, ointments, gels, creams, suppositories, drops, liquids,
sprays and powders. NAC amide or a derivative thereof may be
administered to mucous membranes in the form of a liquid, gel,
cream, and jelly, absorbed into a pad or sponge. Conventional
pharmaceutical carriers, aqueous, powder or oily bases, thickeners
and the like may be necessary or desirable. Compositions comprising
NAC amide or a derivative thereof for oral administration include
powders or granules, suspensions or solutions in water or
non-aqueous media, sachets, capsules or tablets. Thickeners,
diluents, flavorings, dispersing aids, emulsifiers or binders may
be desirable. Formulations for parenteral administration may
include, but are not limited to, sterile solutions, which may also
contain buffers, diluents and other suitable additives.
[0118] The present invention also provides a food additive
comprising NAC amide or a derivative thereof for mammalian,
preferably human, consumption. NAC amide and other cysteine
derivatives have been detected in many different food products,
including but not limited to, garlic, peppers, turmeric, asparagus,
and onions. See, for example, Hsu, C. C., et al, (2004) J. Nutr.
134:149-152 and Demirkol, O. et al, (2004) J. Agric. Food Chem. 52.
The food additive can comprise NAC amide or its derivative in a
liquid or solid material intended to be added to a foodstuff. The
food additives can be added to "food compositions" including any
products--raw, prepared or processed--which are intended for human
consumption in particular by eating or drinking and which may
contain nutrients or stimulants in the form of minerals,
carbohydrates (including sugars), proteins and/or fats, and which
have been modified by the incorporation of a food additive
comprising NAC amide or a derivative of NAC amide as provided
herein. The present modified food compositions can also be
characterized as "functional foodstuffs or food compositions".
"Foodstuffs" can also be understood to mean pure drinking
water.
[0119] The term "food additive" is understood to mean any a liquid
or solid material intended to be added to a foodstuff. This
material can, for example, have a distinct taste and/or flavor,
such as a salt or any other taste or flavor potentiator or
modifier. It is to be noted, however, that the food additive
comprising NAC amide or a NAC amide derivative does not necessarily
have to be an agent having a distinct taste and/or flavor.
[0120] Other food additives that can be added in combination with
NAC amide, or in food additive formulations of NAC amide include,
but are not limited to, acids which are added to make flavours
"sharper", and also act as preservatives and antioxidants, such as
vinegar, citric acid, tartaric acid, malic acid, fumaric acid,
lactic acid, acidity regulators, anti-caking agents, antifoaming
agents, antioxidants such as vitamin C and tocopherols such as
vitamin E, bulking agents, such as starch are additives, food
coloring, color retention agents, emulsifiers flavors, flavor
enhancers, humectants, preservatives, propellants, stabilizers,
thickeners and gelling agents, like agar or pectin, and sweeteners
.
[0121] Doses, amounts or quantities of NAC amide, or derivative
thereof as well as the routes of administration used, are
determined on an individual basis, and correspond to the amounts
used in similar types of applications or indications known to those
having skill in the art. As is appreciated by the skilled
practitioner in the art, dosing is dependent on the severity and
responsiveness of the condition to be treated, but will normally be
one or more doses per day, with course of treatment lasting from
several days to several months, or until a cure is effected or a
diminution of disease state is achieved. Persons ordinarily skilled
in the art can easily determine optimum dosages, dosing
methodologies and repetition rates. For example, a pharmaceutical
formulation for orally administrable dosage form can comprise NAC
amide, or a pharmaceutically acceptable salt, ester, or derivative
thereof in an amount equivalent to at least 25-500 mg per dose, or
in an amount equivalent to at least 50-350 mg per dose, or in an
amount equivalent to at least 50-150 mg per dose, or in an amount
equivalent to at least 25-250 mg per dose, or in an amount
equivalent to at least 50 mg per dose. NAC amide or a derivative
thereof can be administered to both human and non-human mammals. It
therefore has application in both human and veterinary
medicine.
[0122] Examples of suitable esters of NAC amide include alkyl and
aryl esters, selected from the group consisting of methyl ester,
ethyl ester, hydroxyethyl ester, t-butyl ester, cholesteryl ester,
isopropyl ester and glyceryl ester.
[0123] As described herein, a number of conditions, diseases and
pathologies are believed to be associated with reduced
intracellular antioxidant levels, including AIDS, diabetes, macular
degeneration, congestive heart failure, cardiovascular disease and
coronary artery restenosis, lung disease, asthma, virus infections,
e.g., toxic and infectious hepatitis, rabies, HIV; sepsis,
osteoporosis, toxin exposure, radiation exposure, burn trauma,
prion disease, neurological diseases, blood diseases, arterial
disease, muscle disease, tumors and cancers. Many of these diseases
and conditions may be due to insufficient glutathione levels.
Further, exposure to toxins, radiation, medications, etc., may
result in free radical reactions, including types of cancer
chemotherapy. Accordingly, the present invention provides NAC amide
or a derivative thereof as an agent that can treat these diseases
and conditions in a convenient and effective formulation,
particularly for oral administration. The administration of
exogenous NAC amide or a derivative thereof can serve to supplement
or replace the hepatic output of GSH and to assist in the
maintenance of reduced conditions within the organism. The failure
to alleviate free radical reactions allows an undesirable cascade
that can cause serious damage to macromolecules, as well as lipid
peroxidation and the generation of toxic compounds. Maintaining
adequate levels of GSH is necessary to block these free radical
reactions. When natural GSH levels are debilitated or jeopardized,
NAC amide or a derivative thereof is able to provide efficient and
effective remedial action.
[0124] NAC amide can form chelation complexes with copper and lead.
NAC amide may also form circulating complexes with copper in the
plasma. Thus, NAC amide or a derivative thereof can be administered
to treat metal toxicity. NAC amide-metal complexes will be
excreted, thus reducing the metal load. Thus, NAC amide or a
derivative thereof may be administered for the treatment of
toxicity associated with various metals, e.g., iron, copper,
nickel, lead, cadmium, mercury, vanadium, manganese, cobalt,
transuranic metals, such as plutonium, uranium, polonium, and the
like. It is noted that the chelation properties of NAC amide are
independent from its antioxidant properties. However, because some
metal toxicities are free radical mediated, e.g., iron, NAC amide
administration may be particularly advantageous for such
conditions.
[0125] In order to provide high bioavailability, NAC amide or a
derivative thereof can be provided in a relatively high
concentration in proximity to the mucous membrane, e.g., the
duodenum for oral administration. Thus, NAC amide or a derivative
thereof can be administered as a single bolus on an empty stomach.
The preferred dosage is between about 100-10,000 mg NAC amide or
between about 250-3,000 mg NAC amide. Further, the NAC amide or NAC
amide derivative formulation can be stabilized with a reducing
agent, e.g., ascorbic acid, to reduce oxidation both during storage
and in the digestive tract prior to absorption. The use of
crystalline ascorbic acid has the added benefit of providing
improved encapsulation and serving as a lubricant for the
encapsulation apparatus. Capsules, e.g., a two-part gelatin
capsule, are dosage forms that protect NAC amide from air and
moisture, while dissolving quickly in the stomach. The capsule is
preferably a standard two-part hard gelatin capsule of double-O
(OO) size, which may be obtained from a number of sources. After
filling, the capsules are preferably stored under nitrogen to
reduce oxidation during storage. The capsules are preferably filled
according to the method of U.S. Pat. No. 5,204,114, incorporated
herein by reference in its entirety, using crystalline ascorbic
acid as both an antistatic agent and stabilizer. Further, each
capsule preferably contains 500 mg of NAC amide and 250 mg of
crystalline ascorbic acid. A preferred composition includes no
other excipients or fillers; however, other compatible fillers or
excipients may be added. While differing amounts and ratios of NAC
amide and stabilizer may be used, these amounts are preferable
because they fill a standard double-O capsule, and provide an
effective stabilization and high dose. Further, the addition of
calcium carbonate is avoided as it may contain impurities and may
accelerate the degradation of NAC amide in the small intestine due
to its action as a base, which neutralizes stomach acid.
[0126] NAC amide or a derivative thereof is advantageously
administered over extended periods. Therefore, useful combinations
include NAC amide or NAC amide derivatives and drugs intended to
treat chronic conditions. Such drugs are well absorbed on an empty
stomach and do not have adverse interactions or reduced or variable
combined absorption. One particular class of drugs includes central
or peripheral adrenergic or catecholenergic agonists, or reuptake
blockers, which may produce a number of toxic effects, including
neurotoxicity, cardiomyopathy and other organ damage. These drugs
are used, for example, as cardiac, circulatory and pulmonary
medications, anesthetics and psychotropic /antipsychotic agents.
Some of these drugs also have abuse potential, as stimulants,
hallucinogens, and other types of psychomimetics. Other free
radical initiation associated drugs include thorazine, tricyclic
antidepressants, quinolone antibiotics, benzodiazepines,
acetaminophen and alcohol. Accordingly, NAC amide or a derivative
thereof can advantageously be provided in an oral pharmaceutical
formulation in an amount of between about 50-10,000 mg, along with
an effective amount of a pharmacological agent that is capable of
initiating free radical reactions in a mammal. The pharmacological
agent is, for example, an adrenergic, dopaminergic, serotonergic,
histaminergic, cholinergic, gabaergic, psychomimetic, quinone,
quinolone, tricyclic, and/or steroid agent.
[0127] In the following aspects of the invention, formulations of
NAC amide or a derivative thereof provide an advantageous
alternative to GSH administration. NAC amide or a derivative
thereof offers beneficial properties of lipophilicity and
cell-permeability, allowing it to more readily enter cells and
infiltrate the blood-brain barrier more readily than GSH, NAC or
other compounds. The properties of NAC amide or a derivative
thereof may increase its bioavailability following administration
to provide an improved treatment for the various diseases,
disorders, pathologies and conditions as described herein.
[0128] Hepatic glutathione is consumed in the metabolism,
catabolism and/or excretion of a number of agents, including
aminoglycoside antibiotics, acetominophen, morphine and other
opiates. The depletion of hepatic glutathione may result in hepatic
damage or a toxic hepatitis. High dose niacin, used to treat
hypercholesterolemia, has also been associated with a toxic
hepatitis. The present invention therefore encompasses an oral
pharmaceutical formulation comprising NAC amide or a derivative
thereof in an amount between about 50-10,000 mg, administered in
conjunction with an effective amount of a pharmacological agent
that consumes hepatic glutathione reserves.
[0129] A number of pathological conditions result in hepatic
damage. This damage, in turn, reduces the hepatic reserves of
glutathione and the ability of the liver to convert oxidized
glutathione to its reduced form. Other pathological conditions are
associated with impaired glutathione metabolism. These conditions
include both infectious and toxic hepatitis, cirrhosis, hepatic
primary and metastatic carcinomas, traumatic and iatrogenic hepatic
damage or resection. The present invention encompasses a
pharmaceutical formulation comprising NAC amide or a derivative of
NAC amide and an antiviral or antineoplastic agent. The antiviral
or antineoplastic agent is, for example, a nucleoside analog.
[0130] Glutathione is degraded, and cysteine is excreted, possibly
in the urine. Very high doses of glutathione may therefore result
in cysteinuria, which may result in cysteine stones. Other long
term toxicity or adverse actions may result. Therefore, a daily
intake of greater than about 10 gm for extended period should be
medically monitored. On the other hand, individual doses below
about 50 mg are insufficient to raise the concentration of the
duodenal lumen to high levels to produce high levels of absorption,
and to provide clinical benefit. Therefore, the formulations
according to the present invention have an NAC amide or NAC amide
derivative content greater than 50 mg, and are provided in one or
more doses totaling up to about 10,000 mg per day.
[0131] In the treatment of HIV infection, it is believed that the
oral administration of a relatively high dose bolus of glutathione,
i.e., 1-3 grams per day, on an empty stomach, will have two
beneficial effects. First, HIV infection is associated with a
reduction in intracellular glutathione levels in PBMs, lung, and
other tissues. It is further believed that by increasing the
intracellular glutathione levels, the functioning of these cells
may be returned to normal. Therefore, the administration of NAC
amide or a derivative thereof according to the present invention
will treat the effects of HIV infection. Oral administration of NAC
amide, or derivative thereof, optionally in combination with
ascorbic acid and/or with an antiretroviral agent. It is noted that
the transcription mechanisms and control involved in retroviral
infection is believed to be relatively conserved among the
different virus types. Therefore, late stage retroviral suppression
is expected for the various types of human retroviruses and
analogous animal retroviruses. It has also been found in in vitro
tests that by increasing the intracellular levels of glutathione in
infected monocytes to the high end of the normal range, the
production of HIV from these cells may be suppressed for about 35
days. This is believed to be related to the interference in
activation of cellular transcription of cytokines, including
NF-.kappa.B and TNF-.alpha.. Therefore, the infectivity of HIV
infected persons may be reduced, helping to prevent transmission.
This reduction in viral load may also allow the continued existence
of uninfected but susceptible cells in the body.
[0132] NAC amide, or derivative thereof administered according to
the present method, can be use in the treatment of congestive heart
failure (CHF). In CHF, there are believed to be two defects. First,
the heart muscle is weakened, causing enlargement of the heart.
Second, peripheral vasospasm is believed to be present, causing
increased peripheral resistance. NAC amide or a derivative thereof
can be effective in enhancing the effects of nitric oxide, and
therefore can be of benefit to these patients by decreasing
vasoconstriction and peripheral vascular resistance, while
increasing blood flow to the tissues. The present invention thus
encompasses the oral administration of NAC amide or a derivative
thereof in conjunction with a congestive heart failure medication,
for example, digitalis glycosides, dopamine, methyldopa,
phenoxybenzamine, dobutamine, terbutaline, aminone, isoproterenol,
beta blockers, calcium channel blockers, such as verapamil,
propranolol, nadolol, timolol, pindolol, alprenolol, oxprenolol,
sotalol, metoprolol, atenolol, acebutolol, bevantolol, tolamolol,
labetalol, diltiazem, dipyridamole, bretylium, phenyloin,
quinidine, clondine, procainamide, acecamide, amiodarione,
disopyramide, encamide, flecanide, lorcamide, mexiletine, tocamide,
captopril, minoxodil, nifedipine, albuterol, pargyline,
vasodilators, including nitroprusside, nitroglycerin, phentolamine,
phenoxybeizamine, hydrazaline, prazosin, trimazosin, tolazoline,
trimazosin, isosorbide dinitrate, erythrityl tetranitrate, aspirin,
papaverine, cyclandelate, isoxsuprine, niacin, nicotinyl alcohol,
nylidrin, diuretics, including furosemide, ethacrynic acid,
spironolactone, triamterine, amiloride, thiazides, bumetanide,
caffeine, theophylline, nicotine, captopril, salalasin, and
potassium salts.
[0133] In another of its embodiments, the present invention
embraces NAC amide or a derivative thereof to treat hepatitis of
various types by oral administration. For example, both alcohol and
acetaminophen are hepatotoxic and result in reduced hepatocyte
glutathione levels. Therefore, these toxicities may be treated
according to the present invention with the use of NAC amide or a
derivative thereof. NAC amide and its derivatives may also be
effective in the treatment of toxicities to other types of cells or
organs, which result in free radical damage to cells or reduced
glutathione levels.
[0134] Diabetes, especially uncontrolled diabetes, results in
glycosylation of various enzymes and proteins, which may impair
their function or control. In particular, the enzymes which produce
reduced glutathione (e.g., glutathione reductase) become
glycosylated and non-functional. Therefore, diabetes is associated
with reduced glutathione levels, and in fact, many of the secondary
symptoms of diabetes may be attributed to glutathione metabolism
defects. According to this invention, NAC amide or a derivative
thereof can be used to supplement diabetic patients in order to
prevent a major secondary pathology. The present invention also
encompasses an oral pharmaceutical formulation comprising NAC amide
and an antihyperglycemic agent.
[0135] High normal levels of glutathione deactivate opiate
receptors. Thus, the administration of NAC amide or a derivative
thereof may be of benefit for treating obesity and/or eating
disorders, other addictive or compulsive disorders, including
tobacco (nicotine) and opiate additions. This invention also
encompasses administering NAC amide or a derivative thereof in
conjunction with nicotine. The physiologic effects of nicotine are
well known. NAC amide or a derivative thereof may cause
vasodilation and improve cerebral blood flow, thereby resulting in
a synergistic cerebral function-enhancing effect.
[0136] In mammals, the levels of glutathione in the plasma are
relatively low, in the micromolar range, while intracellular levels
are typically in the millimolar range. Therefore, intracellular
cytosol proteins are subjected to vastly higher concentrations of
glutathione than extracellular proteins. The endoplasmic reticulum,
a cellular organelle, is involved in processing proteins for export
from the cell. It has been found that the endoplasmic reticulum
forms a separate cellular compartment from the cytosol, having a
relatively oxidized state as compared to the cytosol, and thereby
promoting the formation of disulfide links in proteins, which are
often necessary for normal activity. In a number of pathological
states, cells may be induced to produce proteins for export from
the cells, and the progression of the pathology is interrupted by
interference with the production and export of these proteins. For
example, many viral infections rely on cellular production of viral
proteins for infectivity. The interruption of the production of
these proteins will interfere with infectivity. Likewise, certain
conditions involve specific cell-surface receptors, which must be
present and functional. In both cases, cells that are induced to
produce these proteins will deplete reduced glutathione in the
endoplasmic reticulum. It is noted that cells that consume
glutathione will tend to absorb glutathione from the plasma, and
may be limited by the amounts present. Therefore, by increasing
plasma glutathione levels, even transiently, the reducing
conditions in the endoplasmic reticulum may be interfered with, and
the protein production blocked. Normal cells may also be subjected
to some interference; however, in viral infected cells, or cells
otherwise abnormally stimulated, the normal regulatory mechanisms
may not be intact, and the redox conditions in the endoplasmic
reticulum will not be controlled by the availability of
extracellular glutathione. The administration of NAC amide or its
derivatives may serve to replenish GSH or the effects of GSH and
provide significant effects for such conditions.
[0137] Reproduction of herpes viruses, which are DNA viruses, is
inhibited or reduced in cell culture by the administration of
extracellular glutathione. Examples of DNA viruses include Herpes
Simplex Virus I, Herpes Simplex Virus II, Herpes zoster,
cytomegalovirus, Epstein Barr virus and others. Therefore,
according to the present invention, DNA virus and herpes virus
infections may be treated by administering NAC amide or a
derivative thereof. In addition, infection by the rabies virus, an
RNA virus, may be treated by the administration of glutathione.
While standard treatments are available, and indeed effective when
timely administered, glutathione may be useful in certain
circumstances. Therefore, rabies virus infection may be treated, at
least in part, by administering NAC amide or a derivative thereof
according to the present invention. One available treatment for
rabies is an immune serum. The present invention encompasses the
parenteral administration of NAC amide, or derivative thereof
separately, or in combination with one or more immunoglobulins.
[0138] Coronary heart disease risk is increased by the consumption
of a high-fat diet and is reduced by the intake of antioxidant
vitamins, including vitamin E and vitamin C, as well as flavonoids.
High fat meals impair the endothelial function through oxidative
stress, resulting in impaired nitric oxide availability. It has
been found that vitamin C and vitamin E restore the
vasoconstriction resulting from nitric oxide production by
endothelium after a high fat meal. According to the present
invention, NAC amide or a derivative thereof may be administered
prophylactically to combat vascular disease.
[0139] There are known to be qualitative differences among several
species of free radicals. Accordingly, their rates of formation
will differ, as will the different types of inciting agents that
may have to be simultaneously controlled. For example, for those
with macular degeneration, continued, unprotected exposure of the
eyes to strong sunlight and to tobacco smoke would limit the
benefits from an antioxidant used as a therapeutic agent for
control of this disease. Therefore, one aspect of the invention
provides synergistic therapies to patients by increasing
antioxidant levels systemically or in specific organs as well as
reducing oxidative, free radical generating and ionizing
influences. In this case, NAC amide therapy would be complemented
with ultraviolet blocking sunglasses, and a tobacco smoking
cessation plan, as necessary. NAC amide or a derivative thereof can
be used in combination with alpha tocopherol succinate, if
necessary. Free radicals occur in different parts or subparts of
tissues and cells, with different inciting agents. For example, in
trauma to the brain or spinal cord, the injurious free radicals are
in the fatty (lipid) coverings that insulate nerve fibers, i.e.,
the myelin sheaths. Extremely high doses of a synthetic
corticosteroid, 5 to 10 grams of methyl prednisolone sodium
succinate (MPSS), given for just 24 hours, rapidly reach the brain
and spinal cord and diffuse rapidly into the myelin, neutralizing
the trauma-induced radicals. The present invention therefore
provides a pharmaceutical composition comprising a combination of
NAC amide or a NAC amide derivative and a glucocorticoid agent.
[0140] According to the present invention, orally administered NAC
amide or a derivative thereof can raise cell levels of glutathione
to inhibit a number of pathologic processes. For example, NAC amide
can be used to curtail the virtually self-perpetuating, powerful
biochemical cycles producing corrosive free radicals and toxic
cytokines that are largely responsible for the signs and symptoms
of AIDS. These biochemical cycles destroy considerable quantities
of glutathione but they can eventually be brought under control,
and normalized with sufficient, ongoing NAC amide therapy. A
typical example is the over production of a substance, 15 HPETE
(15-hydroperoxy eicosatetraenoic acid), from activated macrophages.
15 HPETE is a destructive, immunosuppressing substance and requires
glutathione for conversion into a non-destructive, benign molecule.
The problem is that once macrophages are activated, they are
difficult to normalize. Once inside cells, GSH curtails the
production of free radicals and cytokines, corrects the
dysfunctions of lymphoctyes and of macrophages, reinforces defender
cells in the lungs and other organs and halts HIV replication in
all major infected cell types, by preventing the activation of the
viral DNA by precluding the activation of NF-.kappa.B, inhibiting
the TAT gene product of HIV that drives viral replication and
dismantling the gp120 proteins of the virus coat. NAC amide can be
provided to disrupt the gp120 protein, thereby offering a potential
mode of preventing transmission of virus not only to other cells in
the patient, but perhaps to others.
[0141] Besides classic antiviral or antiretroviral agents (reverse
transcriptase inhibitors, protease inhibitors), a number of other
therapies may be of benefit for AIDS patients, and the present
invention provides combinations of NAC amide or a derivative
thereof with the following drugs: cycloporin A, thalidomide,
pentoxifylline, selenium, desferroxamine, 2L-oxothiazolidine,
2L-oxothiazolidine-4-carboxylate, diethyldithiocarbamate (DDTC),
BHA, nordihydroguairetic acid (NDGA), glucarate, EDTA, R-PIA,
alpha-lipoic acid, quercetin, tannic acid, 2'-hydroxychalcone,
2-hydroxychalcone, flavones, alpha-angelicalactone, fraxetin,
curcurmin, probucol, and arcanut (areca catechul).
[0142] Inflammatory responses are accompanied by large oxidative
bursts, resulting in large numbers of free radicals. Therefore, NAC
amide and its derivatives may have application in the therapy for
inflammatory diseases. NAC amide or a derivative thereof may
advantageously reduce the primary insult, as well as undesired
aspects of the secondary response. According to the present
invention, NAC amide or a derivative thereof may be administered to
patients suffering from an inflammatory disease, such as arthritis
of various types, inflammatory bowel disease, etc. The present
invention also provides combination pharmaceutical therapy
including NAC amide or NAC amide derivative and an analgesic or
anti-inflammatory agent, for example, opiate agonists,
glucocorticoids or non-steroidal anti-inflammatory drugs (NSAIDS),
including opium narcotics, meperidine, propoxyphene, nalbuphine,
pentazocine, buprenorphine, aspirin, indomethacin, diflunisal,
acetominophen, ibuprofen, naproxen, fenoprofen, piroxicam,
sulindac, tolmetin, meclofenamate, zomepirac, penicillamine,
phenylbutazone, oxyphenbutazone, chloroquine, hydroxychloroquine,
azathiaprine, cyclophosphamide, levamisole, prednisone,
prednisolone, betamethasone, triamcinolone, and methylprednisolone.
NAC amide and its derivatives may also be beneficial for the
treatment of parotitis, cervical dysplasia, Alzheimer's disease,
Parkinson's disease, aminoquinoline toxicity, gentamycin toxicity,
puromycin toxicity, aminoglycoside nephrotoxicity, paracetamol,
acetaminophen and phenacetin toxicity.
[0143] NAC amide or a derivative thereof may be added to a
virus-contaminated fluid or potentially contaminated fluid to
inactivate the virus. This occurs, for example, by reduction of
critical viral proteins. According to an embodiment, NAC amide or a
derivative thereof is added to blood or blood components prior to
transfusion. The added NAC amide or derivative of NAC amide is
added in a concentration of between about 100 micromolar to about
500 millimolar or to a solubility limit, whichever is lower, and
more preferably in a concentration of about 10-50 millimolar.
Additionally, the addition of NAC amide or a derivative thereof to
whole blood, packed red blood cells, or other formed blood
components (white blood cells, platelets) may be used to increase
the shelf like and/or quality of the cells or formed
components.
[0144] In another embodiment, the present invention encompasses the
use of NAC amide, or derivative thereof or a pharmaceutically
acceptable salt or ester thereof, in the treatment and/or
prevention of cosmetic conditions and dermatological disorders of
the skin, hair, nails, and mucosal surfaces when applied topically.
In accordance with the invention, compositions for topical
administration are provided that include (a) NAC amide, or
derivative thereof or a suitable salt or ester thereof, or a
physiologically acceptable composition containing NAC amide; and
(b) a topically acceptable vehicle or carrier. The present
invention also provides a method for the treatment and/or
prevention of cosmetic conditions and/or dermatological disorders
that entails topical administration of NAC amide- or NAC
amide-derivative containing compositions to an affected area of a
patient. Such compositions and methods are useful in anti-aging
treatments and therapies, as well as for the treatment of wrinkles,
facial lines and depressions, particularly around the eyes and
mouth, creases in the skin, age spots and discolorations, and the
like.
[0145] In another embodiment, the present invention provides
methods and compositions useful for cancer and pre-cancer therapy
utilizing NAC amide, or derivative thereof or its pharmaceutically
acceptable salts or esters. The present invention particularly
relates to methods and compositions comprising NAC amide or a
derivative thereof in which apoptosis is selectively induced in
cells of cancers or precancers. In another embodiment, the present
invention relates to a method of selectively inducing apoptosis of
precancer cells by administering an effective amount of NAC amide
or a derivative thereof to a subject. In this embodiment, NAC amide
or a derivative thereof can be topically administered to the
subject. In another embodiment, the present invention relates to a
method of selectively inducing apoptosis in cancer cells by
administering an effective amount of NAC amide or a derivative
thereof to a subject. NAC amide or its derivative can be topically
administered to the subject in this embodiment. Selective apoptosis
refers to a situation in which corresponding normal,
non-transformed cells do not undergo NAC amide-induced cell death.
In yet another embodiment, the present invention relates to a
method comprising reducing the number of cancer cells present in a
subject by administering NAC amide or a derivative thereof to the
subject as an adjunct to chemotherapy or radiation therapies such
that the susceptibility of the cancer cells to apoptosis is
enhanced relative to the non-cancer cells of the subject. In a
further embodiment, the present invention relates to a method
comprising administering an effective amount of NAC amide or a
derivative thereof as an adjunct to p53 therapy, including p53 gene
therapy. The cancer or precancer cells in which apoptosis is
induced are generally those which exhibit at least one functional
p53 allele. In certain instances, administration of NAC amide
results in restoration of mutant p53 protein conformation and/or
activity to a functional state. It is to be understood that an
endogenous functional p53 allele is not necessary for methods
comprising p53 therapy, including p53 gene therapy.
[0146] In another embodiment of the invention, methods are provided
which comprise administering NAC amide or a derivative thereof to
selectively induce cells which arise in hyperproliferative or
benign dysproliferative disorders. Another embodiment of the
present invention encompasses the use of NAC amide or a derivative
thereof in methods for selective cell cycle arrest comprising
contacting the cell with an amount of NAC amide or a derivative
thereof to selectively arrest cells at a particular stage of the
cell cycle. For example, administration of NAC amide can lead to
prolonged transition through G1 phase. This cell cycle arrest may
be influenced by an increase in p21 expression. The methods of the
present invention can also be utilized to reduce or inhibit tumor
vascularization, or to induce differentiation in cancer cells.
[0147] In another of its aspects, the present invention is directed
to the use of NAC amide or a derivative thereof to treat cancers
and tumors that may be induced by faulty signals from the
microenvironment that result in loss of tissue organization in
cancerous organs and loss of genomic stability in individual cancer
cells. Loss of tissue structure may lead to certain cancers.
Involved in this process are matrix metalloproteinases (MMPs),
which are enzymes that are important not only during an organism's
development and during wound healing, but also in promoting
tumorigenesis or carcinogenesis. In particular, MMPs contribute
prominently to microenvironmental signals because these proteolytic
enzymes degrade structural components of the basement membrane and
extracellular matrix (ECM) and digest the contacts that bind
epithelial cells into sheets, thereby permitting the invasion of
tumor cells and metastasis. MMPs can also release cell-bound
inactive precursor forms of growth factors; degrade cell-cell and
cell-ECM adhesion molecules; activate precursor zymogen forms of
other MMPs; and inactivate inhibitors of MMPs and other proteases.
Further, these enzymes induce the epithelial-mesenchymal
transition, or EMT, a transition of one cell state to another that
causes epithelial cells to disassociate from their neighbors, break
free and acquire the ability to move through the body. While this
process is essential for normal development in the embryo, in
cancers, such as breast cancer, EMT provides mobility for tumor
cells and assists tumor cells in penetrating barriers, such as wall
of lymph and blood vessels, thus facilitating metastasis.
[0148] MMP-3 is a particular type of metalloproteinase that has
been observed to induce transformation in mammary epithelial cells
in culture and in transgenic mice. MMP-3 has been found to cause
normal cells to express the Rac1b protein, an unusual form of Rho
GTPase that has previously been found only in cancers. Rac1b
dramatically alters the cell skeleton, which facilitates the
separation and movement of epithelial cells from surrounding cells.
(D. C. Radisky et al., 2005, Nature, 436:123-127). Changes in the
cell skeleton induced by Rac1b stimulate the production of highly
reactive oxygen molecules, called reactive oxygen species (ROS),
which can promote cancer by leading to tissue disorganization and
by damaging genomic DNA. The increased amounts of ROS induced by
Rac1b activate major genes that control the EMT, which then begins
a cascade of massive tissue disorganization and stimulates the
development of cancer by directly affecting genomic DNA, for
example, causing deletion or duplication of large regions of the
DNA. By altering the tissue structure, MMPs can also activate
oncogenes and comprising the integrity of the DNA in an organism's
genome.
[0149] For treating cancers, e.g., breast cancer, especially those
involving the above-described mechanisms leading to abnormal cell
structure and function and loss of tissue integrity, NAC amide in
accordance with the present invention can be used to block the
effects of ROS. This can be achieved, for example, by administering
or introducing NAC amide or a derivative thereof to cells, tissues,
and/or the body of a subject in need thereof, to affect or target
molecules in the pathways leading to epithelial-mesenchymal
transition. Accordingly, NAC amide or a derivative thereof can be
used to inhibit MMP-3 and its functions, such as MMP-3-induced
downregulation of epithelial cytokeratins and upregulation of
mesenchymal vimentin, as well as MMP3-induced cell motility,
invasion and morphological alterations. NAC amide or a derivative
thereof can also be used to target ROS indirectly or directly,
and/or the processes by which ROS activate genes that induce the
EMT.
[0150] In another embodiment, the present invention encompasses
compositions and methods comprising NAC amide or a derivative
thereof for the suppression of allograft rejection in recipients of
allografts.
[0151] In another embodiment, the present invention provides a NAC
amide or derivative of NAC amide in a method of supporting or
nurturing the growth of stem cells for stem cell transplants,
particularly stem cells cultured in vitro prior to introduction
into a recipient animal, including humans.
[0152] In another embodiment, the present invention provides
methods of inhibiting, preventing, treating, or both preventing and
treating, central nervous system (CNS) injury or disease,
neurotoxicity or memory deficit in a subject, involving the
administration of a therapeutically effective amount of NAC amide,
or derivative thereof or a pharmaceutically acceptable composition
thereof. Examples of CNS injuries or disease include traumatic
brain injury (TBI), posttraumatic epilepsy (PTE), stroke, cerebral
ischemia, neurodegenerative diseases of the brain such as
Parkinson's disease, Dementia Pugilistica, Huntington's disease,
Alzheimer's disease, brain injuries secondary to seizures which are
induced by radiation, exposure to ionizing or iron plasma, nerve
agents, cyanide, toxic concentrations of oxygen, neurotoxicity due
to CNS malaria or treatment with anti-malaria agents, and other CNS
traumas. In other related embodiments, the present invention
embraces a method of treating a subject suffering from a CNS injury
or disease comprising administering to the subject a composition
comprising a therapeutically effective amount of NAC amide or a
derivative thereof. In another embodiment, the present invention
relates to a method of preventing or inhibiting a CNS injury or
disease in a subject comprising administering to the subject a
composition comprising a therapeutically effective amount of NAC
amide or a derivative thereof. In other embodiments, the present
invention embraces a method of preventing, inhibiting or treating
neurotoxicity or memory deficit in a subject comprising
administering to the subject a composition comprising a
therapeutically effective amount of NAC amide or a derivative
thereof. Where the memory deficit may be induced by
electroconvulsive shock therapy for treating diseases and disorders
such as depression and schizophrenia, the composition may be
administered before the electroconvulsive shock therapy to mitigate
memory loss. In related embodiments, the CNS injury or disease may
be traumatic brain injury (TBI), posttraumatic epilepsy (PTE),
stroke, cerebral ischemia, or a neurodegenerative disease. In
related embodiments, CNS injury may be induced by fluid percussion,
by trauma imparted by a blunt object, for example on the head of
the subject, by trauma imparted by an object which penetrates the
head of the subject, by exposure to radiation, ionizing or iron
plasma, a nerve agent, cyanide, toxic concentrations of oxygen, CNS
malaria, or an anti-malaria agent. In the embodiments of the
present invention, the therapeutically effective amount of NAC
amide or a derivative thereof administered to the subject is the
amount required to obtain the appropriate therapeutic effect, for
example, about 0.001 mg to about 20 mg per kg of the subject,
preferably about 1 mg to about 10 mg per kg of the subject, more
preferably about 3 mg to about 10 mg per kg of the subject. In
additional embodiments, the total daily amount of NAC amide or a
derivative thereof administered to the subject is about 50 mg to
about 1200 mg, or about 100 mg to about 1000 mg, or about 200 mg to
about 800 mg, or about 300 mg to about 600 mg.
[0153] In other embodiments, the invention encompasses a method of
treating a subject (e.g., an animal, including humans) before the
subject is exposed or likely to be exposed to a risk of CNS injury
or damage, or before the subject is exposed to conditions likely to
cause neurotoxicity or memory deficit or both, by administering NAC
amide or a derivative thereof to a subject in a period of time
prior to the exposure of the subject to the risk of CNS injury or
damage, etc. Illustratively, conditions that may cause CNS injury
or damage, neurotoxicity or memory deficit include
electroconvulsive shock therapy, traumatic brain injury (TBI),
posttraumatic epilepsy (PTE), stroke, cerebral ischemia,
neurodegenerative diseases, fluid percussion, a blunt object
impacting the head of the subject, an object penetrating the head
of the subject, radiation, ionizing or iron plasma, nerve agents,
cyanide, toxic concentrations of oxygen, CNS malaria, and
anti-malaria agents. Other conditions that may cause CNS injury or
damage, neurotoxicity or memory deficit include, without
limitation, certain medical procedures or conditions associated
with risk for CNS ischemia, hypoxia or embolism such as brain
tumor, brain surgery, other brain-related disorders, open heart
surgery, carotid endarterectomy, repair of aortic aneurysm, atrial
fibrillation, cardiac arrest, cardiac or other catheterization,
phlebitis, thrombosis, prolonged bed rest, prolonged stasis (such
as during space travel or long trips via airplane, rail, car or
other transportation), CNS injury secondary to air/gas embolism or
decompression sickness. The period of time may be about 72 hours
prior to the time of expected exposure, or about 48 hours prior to
the time of expected exposure, or about 12 hours prior to the time
of expected exposure, or about 4 hours prior to the time of
expected exposure, or about 30 minutes-2 hours prior to the time of
expected exposure. The administration of NAC amide may be
continuous from the initial time of treatment to the end of
treatment. For example, a transdermal patch or a slow-release
formulation may be used to continually administer NAC amide or a
derivative thereof to the subject for a given period of time.
Alternatively, NAC amide or a derivative thereof may be
administered to the subject periodically. For example, NAC amide or
a derivative thereof may first be administered at about 24 hours
before the time of expected exposure and then administered at about
every 2 hours thereafter. For these embodiments of the invention,
the NAC amide- or NAC amide derivative-containing composition may
further comprise a pharmaceutically acceptable excipient and the
composition may be administered intravenously, intradermally,
subcutaneously, orally, transdermally, transmucosally or
rectally.
[0154] In other embodiments, the present invention encompasses a
pharmaceutical composition for treating or preventing CNS injury,
disease or neurotoxicity in a subject comprising a therapeutically
effective amount of NAC amide or a derivative thereof and a
pharmaceutically acceptable excipient. In a further embodiment, the
invention embraces a kit comprising a composition comprising a
therapeutically effective amount of NAC amide or a derivative
thereof. The kit may further comprise a device for administering
the composition to a subject such as an injection needle, an
inhaler, a transdermal patch, as well as instructions for use.
[0155] In another embodiment of the present invention, anti-cancer
treatments involving NAC amide or a derivative thereof are designed
to specifically target cancer and tumor cells. This embodiment is
directed to the use of nano-sized particles for the in vivo and ex
vivo administration of NAC amide or a derivative thereof to cancer
and tumor cells. According to this embodiment, cancer cells, which
display more receptors for the vitamin folic acid (or folate) and
absorb more folic acid than do normal, healthy cells, are able to
be preferentially targeted. To this end, core or shell nanogels, or
nanoparticles, can be functionalized with folic acid or folate
conjugated or linked to NAC amide or a derivative thereof without
disrupting or destroying the folic acid binding site to its cell
receptor. Such functionalized nanoparticles can be introduced into
a subject, particularly a folate-deprived subject, with a cancer,
e.g., epithelial cancer, in whom the cancer cells have excess folic
acid receptors which will preferentially bind the folic acid-NAC
amide (or folic acid-NAC amide derivative) nanoparticles and
endocytose them. Once inside the cancer cell, NAC amide or a
derivative thereof exert its therapeutic effects, for example, by
inhibiting ROS and/or other target molecules that play a role in
initiating, fueling, and/or maintaining cancer cells, and/or
ultimately killing the cancer cells.
[0156] Illustratively, PAMAM dendritic polymers <5 nm in
diameter can be used as carriers of NAC amide, as described in J.
F. Kukowska-Latallo et al., 2005, Cancer Res., June 15;
65(12):5317-24, to target folic acid receptor-expressing
(overexpressing) tumor and cancer cells. Acetylated dendrimers can
be conjugated to folic acid as a targeting agent and then coupled
to NAC amide or a derivative thereof and either fluorescein or
6-carboxytetramethylrhodamine. Alternatively, NAC amide or a
derivative thereof can be coupled to folic acid to form a conjugate
and the conjugate can be coupled to the nanoparticles. These
conjugates can be injected i.v. into a tumor-bearing patient or
mammal, especially those tumors that overexpress the folic acid
receptor. The folate-conjugated nanoparticles can then concentrate
in the tumor and tissue following administration, where the
delivered NAC amide or NAC derivative can interact with ROS in the
cells, and/or target other molecules to kill the cancer or tumor
cells. The tumor tissue localization of the folate-targeted polymer
may be attenuated by prior i.v. injection of free folic acid.
[0157] In a similar embodiment, polymers or nanoparticles can be
functionalized to display glutathione-NAC amide or glutathione-NAC
amide derivative conjugates, which can then be used to deliver NAC
amide or a derivative thereof to cancer cells which display
increased numbers of glutathione receptors on their cell surfaces.
The NAC amide-glutathione nanoparticles can then be targeted to
those cancer cells having glutathione receptors and preferentially
endocytosed by the cells. In these embodiments, the present
invention provides directed delivery of NAC amide or a derivative
thereof to cells, such as cancer cells that express high levels of
receptors for folic acid (folate) or glutathione. In accordance
with these embodiments, NAC amide ("NACA") or a derivative thereof
is coupled to a ligand for a cell surface receptor (e.g., folic
acid or glutathione) to form a conjugate. This NACA-ligand
conjugate is coated or adsorbed onto readily injectable
nanoparticles using procedures known to those skilled in the art.
Accordingly, the nanoparticles containing NAC amide or a derivative
thereof ("nano-NACA particles") may be preferentially taken up by
cancer or tumor cells where the NAC amide will exert its desired
effects.
[0158] In an embodiment, the present invention is drawn to a method
of directed delivery of NAC amide or a derivative thereof to host
cells expressing high levels of surface receptor for a ligand,
comprising: a) conjugating acetylated dendritic nanopolymers to
ligand; b) coupling the conjugated ligand of step (a) to NAC amide
or a derivative thereof to form NAC amide-ligand nanoparticles; and
c) injecting the nanoparticles of (b) into the host. In another
embodiment, the present invention is drawn to a method of directed
delivery of NAC amide or a derivative thereof to host cells
expressing high levels of surface receptor for a ligand,
comprising: a) coupling NAC amide or a derivative thereof to the
surface receptor ligand to form a NAC amide-ligand conjugate; b)
adsorbing the NAC amide-ligand conjugate onto nanoparticles; and c)
injecting the nanoparticles of (b) into the host.
[0159] Another embodiment of the present invention provides a
compound of the formula I:
##STR00004##
[0160] wherein: [0161] R.sub.1 is OH, SH, or S--S-Z; [0162] X is C
or N; [0163] Y is NH.sub.2, OH, CH.sub.3--C.dbd.O, or NH--CH.sub.3;
[0164] R.sub.2 is absent, H, or .dbd.O [0165] R.sub.3 is absent
or
##STR00005##
[0166] wherein: R.sub.4 is NH or O; [0167] R.sub.5 is CF.sub.3,
NH.sub.2, or CH.sub.3
[0168] and wherein: Z is
##STR00006##
with the proviso that if R.sub.1 is S--S-Z, X and X' are the same,
Y and Y' are the same, R.sub.2 and R.sub.6 are the same, and
R.sub.3 and R.sub.7 are the same.
[0169] In one embodiment, R.sub.1 is S, X is C, Y is NH--CH.sub.3,
R.sub.2 is H, R.sub.3 is
##STR00007##
R.sub.4 is O, and R.sub.5 is CH.sub.3. In another embodiment,
R.sub.1 is S, X is N, Y is CH.sub.3--C.dbd.O, R.sub.2 is H, and
R.sub.3 is absent.
[0170] The present invention also provides compounds of the formula
I above, wherein R.sub.1 is S, X is C, Y is NH.sub.2, R.sub.2
is
##STR00008##
.dbd.O, R.sub.3 is R.sub.4 is O, and R.sub.5 is CF.sub.3. Compounds
of the present invention also include compounds of formula I
wherein R.sub.1 is O, X is C, Y is NH.sub.2, R.sub.2 is .dbd.O,
R.sub.3 is
##STR00009##
R.sub.4 is O, and R.sub.5 is CH.sub.3. Also provided by the present
invention are compounds of formula I wherein R.sub.1 is S, X is C,
Y is OH, R.sub.2 is absent, R.sub.3 is
##STR00010##
R.sub.4 is O, and R.sub.5 is CH.sub.3, or wherein R.sub.1 is S, X
is C, Y is NH.sub.2, R.sub.2 is .dbd.O, R.sub.3 is
##STR00011##
R.sub.4 is NH, and R.sub.5 is NH.sub.2. Another embodiment of the
present invention provides compounds of formula I wherein R.sub.1
is O, X is C, Y is OH, R.sub.2 is absent, R.sub.3 is
##STR00012##
R.sub.4 is O, and R.sub.5 is CH.sub.3; or wherein R.sub.1 is S, X
is C, Y is NH.sub.2, R.sub.2 is .dbd.O, R.sub.3 is
##STR00013##
R.sub.4 is O, and R.sub.5 is CH.sub.3. In a further embodiment, the
present invention provides compounds of formula I wherein R.sub.1
is S--S-Z, X is C, Y is NH.sub.2, R.sub.2 is .dbd.O, R.sub.3 is
##STR00014##
R.sub.4 is O and R.sub.5 is CH.sub.3.
[0171] The compounds disclosed herein can be chiral, i.e.,
enantiomers, such as L- and D-isomers, or can be racemic mixtures
of D- and L-isomers. Preferred compounds include, but are not
limited to, the following:
##STR00015## ##STR00016##
[0172] In one embodiment, Compounds I through XVIII comprise NAC
amide or NAC amide derivatives.
[0173] In another embodiment, a process for preparing an L- or
D-isomer of the compounds of the present invention are provided,
comprising adding a base to L- or D-cystine diamide dihydrochloride
to produce a first mixture, and subsequently heating the first
mixture under vacuum; adding a methanolic solution to the heated
first mixture; acidifying the mixture with alcoholic hydrogen
chloride to obtain a first residue; dissolving the first residue in
a first solution comprising methanol saturated with ammonia; adding
a second solution to the dissolved first residue to produce a
second mixture; precipitating and washing the second mixture;
filtering and drying the second mixture to obtain a second residue;
mixing the second residue with liquid ammonia, and an ethanolic
solution of ammonium chloride to produce a third mixture; and
filtering and drying the third mixture, thereby preparing the L- or
D-isomer compound.
[0174] The base can comprise liquid ammonia or methylamine. The
second solution comprises water, an acetate salt, and an anhydride,
wherein the acetate salt can comprise sodium acetate or sodium
trifluoroacetate, and the anhydride can comprise acetic anhydride
or trifluoroacetic anhydride. Alternatively, the second solution
can comprise dichloromethane, triethylamine, and
1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.
[0175] In addition to liquid ammonia and an ethanolic solution of
ammonium chloride, the second residue can be further mixed with
sodium metal.
[0176] In some embodiments, the process further comprises
dissolving the L- or D-isomer compound in ether; adding to the
dissolved L- or D-isomer compound an ethereal solution of lithium
aluminum hydride, ethyl acetate, and water to produce a fourth
mixture; and filtering and drying the fourth mixture, thereby
preparing the L- or D-isomer compound.
[0177] The compounds of formula II and III are prepared by mixing
L- or D-cystine diamide dihydrochloride with liquid ammonia;
warming the mixture to remove volatiles; warming mixture in vacuo
to 50.degree. C.; adding a warm methanolic solution; filtering the
solution; acidifying the filtrate with alcoholic hydrogen chloride
for obtaining a first residue, dissolving the first residue in a
solution of methanol saturated with ammonia; concentrating to
dryness; adding water, sodium acetate and acetic anhydride; raising
the temperature to 50.degree. C.; precipitating the mixture and
washing the mixture with water; filtering the crude solid; drying
the mixture for obtaining a second residue, mixing the second
residue with liquid ammonia; slowly adding sodium metal; removal of
the solvent; slowly adding an ethanolic solution of ammonium
chloride; filtering and separating the inorganic salt;
concentrating and cooling the filtrate to obtain a third residue;
and crystallizing the third residue from isopropanol.
[0178] The compounds of formula IV and V are prepared by mixing L-
or D-cystine diamide dihydrochloride with methylamine; warming the
mixture to remove volatiles; warming mixture in vacuo to 50.degree.
C.; adding a warm methanolic solution; filtering the solution;
acidifying the filtrate with alcoholic hydrogen chloride for
obtaining a first residue, dissolving the first residue in a
solution of methanol saturated with ammonia; concentrating to
dryness; adding water, sodium acetate and acetic anhydride; raising
the temperature to 50.degree. C.; precipitating the mixture and
washing the mixture with water; filtering the crude solid; drying
the mixture for obtaining a second residue, mixing the second
residue with liquid ammonia; slowly adding sodium metal; removal of
the solvent; slowly adding an ethanolic solution of ammonium
chloride; filtering and separating the inorganic salt;
concentrating and cooling the filtrate to obtain a third residue;
and crystallizing the third residue from isopropanol.
[0179] The compounds of formula VII and VIII are prepared by mixing
L- or D-cystine diamide dihydrochloride with ammonia; warming the
mixture to remove volatiles; warming mixture in vacuo to 50.degree.
C.; adding a warm methanolic solution; filtering the solution;
acidifying the filtrate with alcoholic hydrogen chloride for
obtaining a first residue, dissolving the first residue in a
solution of methanol saturated with ammonia; concentrating to
dryness; adding water, sodium trifluoroacetate and trifluoroacetic
anhydride; raising the temperature to 50.degree. C.; precipitating
the mixture and washing the mixture with water; filtering the crude
solid; drying the mixture for obtaining a second residue, mixing
the second residue with liquid ammonia; slowly adding sodium metal;
removal of the solvent; slowly adding an ethanolic solution of
ammonium chloride; filtering and separating the inorganic salt;
concentrating and cooling the filtrate to obtain a third residue;
and crystallizing the third residue from isopropanol.
[0180] The compounds of formula XIII and XIV are prepared by mixing
L- or D-cystine diamide dihydrochloride with ammonia; warming the
mixture to remove volatiles; warming mixture in vacuo to 50.degree.
C.; adding a warm methanolic solution; filtering the solution;
acidifying the filtrate with alcoholic hydrogen chloride for
obtaining a first residue, dissolving the first residue in a
solution of methanol saturated with ammonia; concentrating to
dryness; adding dichloromethane, triethylamine, and
1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea; lowering the
temperature to 0.degree. C.; precipitating the mixture and washing
the mixture with water; filtering the crude solid; drying the
mixture for obtaining a second residue, mixing the second residue
with liquid ammonia; slowly adding sodium metal; removal of the
solvent; slowly adding an ethanolic solution of ammonium chloride;
filtering and separating the inorganic salt; concentrating and
cooling the filtrate to obtain a third residue; and crystallizing
the third residue from isopropanol.
[0181] The compounds of formula XI and XII are prepared by mixing
L- or D-cystine diamide dihydrochloride with liquid ammonia;
warming the mixture to remove volatiles; warming mixture in vacuo
to 50.degree. C.; adding a warm methanolic solution; filtering the
solution; acidifying the filtrate with alcoholic hydrogen chloride
for obtaining a first residue; dissolving the first residue in a
solution of methanol saturated with ammonia; concentrating to
dryness; adding water, sodium acetate and acetic anhydride; raising
the temperature to 50.degree. C.; precipitating the mixture;
washing the mixture with water; filtering the crude solid; drying
the mixture for obtaining a second residue; mixing the second
residue with liquid ammonia; slowly adding sodium metal; removal of
the solvent; slowly adding an ethanolic solution of ammonium
chloride; filtering and separating the inorganic salt;
concentrating and cooling the filtrate to obtain a third residue;
dissolving the third residue in ether; slowly adding an ethereal
solution of lithium aluminum hydride; slowly adding ethyl acetate;
slowly adding water; filtering and separating the inorganic salts;
concentrating and cooling the filtrate to obtain a fourth residue;
and crystallizing the fourth residue from isopropanol.
[0182] The compounds of formula XVII and XVIII are prepared by
mixing L- or D-cystine diamide dihydrochloride with liquid ammonia;
warming the mixture to remove volatiles; warming mixture in vacuo
to 50.degree. C.; adding a warm methanolic solution; filtering the
solution; acidifying the filtrate with alcoholic hydrogen chloride
for obtaining a first residue; dissolving the first residue in a
solution of methanol saturated with ammonia; concentrating to
dryness; adding of water, sodium acetate and acetic anhydride;
raising the temperature to 50.degree. C.; precipitation of the
mixture; washing the mixture with water; filtering the crude solid;
drying the mixture for obtaining a second residue; and
crystallizing the second residue from isopropanol.
[0183] Another embodiment of the invention provides a process for
preparing an L- or D-isomer of the compounds disclosed herein,
comprising mixing S-benzyl-L- or D-cysteine methyl ester
hydrochloride or O-benzyl-L- or D-serine methyl ester hydrochloride
with a base to produce a first mixture; adding ether to the first
mixture; filtering and concentrating the first mixture; repeating
steps (c) and (d), to obtain a first residue; adding ethyl acetate
and a first solution to the first residue to produce a second
mixture; filtering and drying the second mixture to produce a
second residue; mixing the second residue with liquid ammonia,
sodium metal, and an ethanolic solution of ammonium chloride to
produce a third mixture; and filtering and drying the third
mixture, thereby preparing the L- or D-isomer compound.
[0184] The base can comprise liquid ammonia or methylamine. The
second solution comprises water, an acetate salt, and an anhydride,
wherein the acetate salt can comprise sodium acetate or sodium
trifluoroacetate, and the anhydride can comprise acetic anhydride
or trifluoroacetic anhydride. Alternatively, the second solution
can comprise dichloromethane, triethylamine, and
1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea.
[0185] In some embodiments, the process further comprises
dissolving the L- or D-isomer compound in ether; adding to the
dissolved L- or D-isomer compound an ethereal solution of lithium
aluminum hydride, ethyl acetate, and water to produce a fourth
mixture; and filtering and drying the fourth mixture, thereby
preparing the L- or D-isomer compound.
[0186] The compounds of formula II and III are prepared by mixing
S-benzyl-L- or D-cysteine methyl ester hydrochloride with a cold
methanolic solution of ammonia; passing a stream of ammonia over
the mixture; sealing the flask securely; concentrating the mixture;
adding ether; filtering the solution; concentrating the filtrate;
adding ether and filtering again, to obtain a residue; suspending
the residue with ethyl acetate; adding acetic anhydride to this
suspension; adding water, sodium acetate and acetic anhydride;
raising the temperature to 65.degree. C.; cooling the mixture;
filtering the crude solid; washing with ethyl acetate; drying the
precipitate for obtaining a second residue; mixing the second
residue with liquid ammonia; slowly adding sodium metal; removal of
the solvent; slowly adding an ethanolic solution of ammonium
chloride; filtering and separating the inorganic salt;
concentrating and cooling the filtrate to obtain a third residue;
and crystallizing the third residue from isopropanol.
[0187] The compounds of formula IV and V are prepared by mixing
S-benzyl-L- or D-cysteine methyl ester hydrochloride with a cold
methanolic solution of methylamine; passing a stream of methylamine
over the mixture; sealing the flask securely; concentrating the
mixture; adding ether; filtering the solution; concentrating the
filtrate; adding ether and filtering again, to obtain a residue;
suspending the residue with ethyl acetate; adding acetic anhydride
to this suspension; adding water, sodium acetate and acetic
anhydride; raising the temperature to 65.degree. C.; cooling the
mixture; filtering the crude solid; washing with ethyl acetate;
drying the precipitate for obtaining a second residue; mixing the
second residue with liquid ammonia; slowly adding sodium metal;
removal of the solvent; slowly adding an ethanolic solution of
ammonium chloride; filtering and separating the inorganic salt;
concentrating and cooling the filtrate to obtain a third residue;
and crystallizing the third residue from isopropanol.
[0188] The compounds of formula VII and VIII are prepared by mixing
S-benzyl-L- or D-cysteine methyl ester hydrochloride with a cold
methanolic solution of ammonia; passing a stream of methylamine
over the mixture; sealing the flask securely; concentrating the
mixture; adding ether; filtering the solution; concentrating the
filtrate; adding ether and filtering again, to obtain a residue;
suspending the residue with ethyl acetate; adding trifluoroacetic
anhydride to this suspension; adding water, sodium trifluoroacetate
and trifluoroacetic anhydride; raising the temperature to
65.degree. C.; cooling the mixture; filtering the crude solid;
washing with ethyl acetate; drying the precipitate for obtaining a
second residue; mixing the second residue with liquid ammonia;
slowly adding sodium metal; removal of the solvent; slowly adding
an ethanolic solution of ammonium chloride; filtering and
separating the inorganic salt; concentrating and cooling the
filtrate to obtain a third residue; and crystallizing the third
residue from isopropanol.
[0189] The compounds of formula IX and X are prepared by mixing
O-benzyl-L- or D-serine methyl ester hydrochloride with a cold
methanolic solution of ammonia; passing a stream of methylamine
over the mixture; sealing the flask securely; concentrating the
mixture; adding ether; filtering the solution; concentrating the
filtrate; adding ether and filtering again, to obtain a residue;
suspending the residue with ethyl acetate; adding acetic anhydride
to this suspension; adding water, sodium acetate and acetic
anhydride; raising the temperature to 65.degree. C.; cooling the
mixture; filtering the crude solid; washing with ethyl acetate;
drying the precipitate for obtaining a second residue; mixing the
second residue with liquid ammonia; slowly adding sodium metal;
removal of the solvent; slowly adding an ethanolic solution of
ammonium chloride; filtering and separating the inorganic salt;
concentrating and cooling the filtrate to obtain a third residue;
and crystallizing the third residue from isopropanol.
[0190] The compounds of formula XIII and XIV are prepared by mixing
S-benzyl-L- or D-cysteine methyl ester hydrochloride with a cold
methanolic solution of ammonia; passing a stream of ammonia over
the mixture; sealing the flask securely; concentrating the mixture;
adding ether; filtering the solution; concentrating the filtrate;
adding ether and filtering again, to obtain a residue; suspending
the residue with ethyl acetate; adding acetic anhydride to this
suspension; adding dichloromethane, triethylamine, and
1,3-bis(benzyloxycarbonyl)-2-methyl-2-thiopseudourea; lowering the
temperature to 0.degree. C.; precipitating the mixture; washing the
mixture with water; filtering the crude solid; drying the mixture
for obtaining a second residue; mixing the second residue with
liquid ammonia; slowly adding sodium metal; removal of the solvent;
slowly adding an ethanolic solution of ammonium chloride; filtering
and separating the inorganic salt; concentrating and cooling the
filtrate to obtain a third residue; and crystallizing the third
residue from isopropanol.
[0191] The compounds of formula XI and XII are prepared by (a)
mixing S-benzyl-L- or D-cysteine methyl ester hydrochloride with a
cold methanolic solution of ammonia; passing a stream of ammonia
over the mixture; sealing the flask securely; concentrating the
mixture; adding ether; filtering the solution; concentrating the
filtrate; adding ether and filtering again, to obtain a residue;
suspending the residue with ethyl acetate; adding acetic anhydride
to this suspension; adding of water, sodium acetate and acetic
anhydride; raising the temperature to 65.degree. C.; cooling the
mixture; filtering the crude solid; washing with ethyl acetate;
drying the precipitate for obtaining a second residue; mixing the
second residue with liquid ammonia; slowly adding sodium metal;
removal of the solvent; slowly adding an ethanolic solution of
ammonium chloride; filtering and separating the inorganic salt;
concentrating and cooling the filtrate to obtain a third residue;
dissolving the third residue in ether; slowly adding an ethereal
solution of lithium aluminum hydride; slowly adding ethyl acetate;
slowly adding water; filtering and separating the inorganic salts;
concentrating and cooling the filtrate to obtain a fourth residue;
and crystallizing the fourth residue from isopropanol.
[0192] The compounds of formula XV and XVI are prepared by (a)
mixing O-benzyl-L- or D-serine methyl ester hydrochloride with a
cold methanolic solution of ammonia; passing a stream of ammonia
over the mixture; sealing the flask securely; concentrating the
mixture; adding ether; filtering the solution; concentrating the
filtrate; adding ether and filtering again, to obtain a residue;
suspending the residue with ethyl acetate; adding acetic anhydride
to this suspension; adding of water, sodium acetate and acetic
anhydride; raising the temperature to 65.degree. C.; cooling the
mixture; filtering the crude solid; washing with ethyl acetate;
drying the precipitate for obtaining a second residue; mixing the
second residue with liquid ammonia; slowly adding sodium metal;
removal of the solvent; slowly adding an ethanolic solution of
ammonium chloride; filtering and separating the inorganic salt;
concentrating and cooling the filtrate to obtain a third residue;
dissolving the third residue in ether; slowly adding an ethereal
solution of lithium aluminum hydride; slowly adding ethyl acetate;
slowly adding water; filtering and separating the inorganic salts;
concentrating and cooling the filtrate to obtain a fourth residue;
and crystallizing the fourth residue from isopropanol.
[0193] Yet another embodiment of the invention provides a process
for preparing a compound as disclosed herein, comprising mixing
cystamine dihydrochloride with ammonia, water, sodium acetate, and
acetic anhydride to produce a first mixture; allowing the first
mixture to precipitate; filtering and drying the first mixture to
produce a first residue; mixing the second residue with liquid
ammonia, sodium metal, and an ethanolic solution of ammonium
chloride to produce a second mixture; filtering and drying the
second mixture, thereby preparing the compound.
[0194] The compound of formula VI is prepared by mixing cystamine
dihydrochloride with ammonia; adding water, sodium acetate and
acetic anhydride; raising the temperature to 50.degree. C.;
precipitating the mixture; washing the mixture with water;
filtering the crude solid; drying the mixture for obtaining a
second residue; mixing the second residue with liquid ammonia;
slowly adding sodium metal; removal of the solvent; slowly adding
an ethanolic solution of ammonium chloride; filtering and
separating the inorganic salt; concentrating and cooling the
filtrate to obtain a third residue; and crystallizing the third
residue from isopropanol.
EXAMPLES
Example 1
[0195] In this Example, NAC amide was assessed for its protective
effects against oxidative toxicity induced by glutamate in PC12
cells.
[0196] Materials and methods: N-(1-pyrenyl)-maleimide (NPM) was
purchased from Aldrich (Milwaukee, Wis., USA). N-acetylcysteine
amide was obtained from Novia Pharmaceuticals, (Israel).
High-performance liquid chromatography (HPLC)-grade solvents were
purchased from Fisher Scientific (Fair Lawn, N.J.). All other
chemicals were purchased from Sigma (St. Louis, Mo., USA).
[0197] Cell culture and toxicity studies: Stock culture of PC12
cells, purchased from ATCC, were grown in 75 cm.sup.2 tissue
culture flasks in RPMI 1640, supplemented with 10% (v/v)
heat-inactivated horse serum, and 5% (v/v) fetal bovine serum, to
which 1% (v/v) penicillin and streptomycin were added. Cultures
were maintained at 37.degree. C. in a humidified atmosphere
containing 5% CO.sub.2. The cells were passaged twice a week.
Unless specified, all of the experiments were performed using
Dulbecco's modified Eagle's medium (DMEM) as differentiation
medium, supplemented with 0.5% (v/v) fetal bovine serum, 1% (v/v)
penicillin and streptomycin. PC12 cells were plated at a density of
25.times.10.sup.3 cells/well in a 24-well, collagen-coated plate
for morphological assessment. The plate was divided into five
groups in triplicate: 1) control: no glutamate, no NAC amide; 2)
Nerve Growth Factor (NGF) control: NGF (100 ng/ml), no glutamate,
no NAC amide; 3) NAC amide only: NGF (100 ng/ml), no glutamate, NAC
amide (750 .mu.M); 4) glutamate only: NGF (100 ng/ml), glutamate
(10 mM), no NAC amide; and 5) Glu+NAC amide: NGF (100 ng/ml),
glutamate (10 mM), NAC amide (750 .mu.M). All wells received 100
ng/ml NGF every other day, except Group I. After one week, cells
were treated or not (control) with 10 mM glutamate, with or without
NAC amide, for 24 hours. Twenty-four hours later, the cells were
fixed with 0.5% (v/v) glutaraldehyde in PBS and micropictures were
taken.
[0198] LDH assay: For the lactate dehydrogenase (LDH) assay, cells
were plated at a density of 2.5.times.10.sup.5 cells/well in a 24
well collagen-coated culture plate and, after 24 h; the medium was
replaced with fresh DMEM medium containing the desired
concentration of glutamate and NAC amide. After the desired
incubation period, the LDH activity released was determined using
the kit as described below. For the MTS assay, cells were plated at
a density of 10.sup.5 cells/well in a 24 well collagen-coated
plate. At the end of the experiments, cell viability was assayed
using the kit as described. The LDH activity assay was performed
with the CytoTox96.RTM. Non-Radioactive Cytotoxicity Assay kit
(Promega, Madison, Wis., USA), which quantitatively measured the
activity of LDH, a stable cytosolic enzyme that is released upon
cell lysis [Technical Bulletin No. 163, Promega]. LDH in culture
supernatants was measured with a 30-minute coupled enzymatic assay,
which resulted in the conversion of a tetrazolium salt into a red
formazan product. The amount of color formed was proportional to
the degree of damage to the cell membranes. Absorbance data were
collected using a BMG microplate reader (BMG Labtechnologies, Inc.,
Durham, N.C., USA) at 490 nm. LDH leakage was expressed as the
percentage (%) of the maximum LDH release in the cells treated with
glutamate alone (100%), according to the formula:
% LDH released = Experimental LDH release Maximum LDH release
.times. 100 ##EQU00001##
[0199] MTS assay: The MTS assay (Cell Titer 96.RTM. Aqueous One
solution cell proliferation Assay, Promega) is a cell proliferation
assay in which the administered (3-(4,5-dimethyl
thiazol-2-yl)-5-(3-carboxymethoxy
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, MTS) [21] is
bioreduced by viable cells to a colored formazan product that is
soluble in media. Absorbance at 490 nm is proportional to the
number of living cells in the culture.
[0200] GSH measurement: Cellular levels of GSH were determined by
using the method as described in Winters R. A. et al., Anal
Biochem., 227(1):14-21, 1995. Cells were seeded at a density of
80,000 cells/cm.sup.2 on poly-D-lysine coated (0.05 mg/ml) 75
cm.sup.2 flasks (5 ml/flask) for GSH measurement. After 24 hours,
the flasks were incubated with fresh medium containing glutamate
(10 mM), or BSO (0.2 mM) or Glu+BSO+NAC amide (750 .mu.M) at 370 C
for another 24 h. After the incubation period, cells were removed
from the cultures and homogenized in serine borate buffer (100 mM
Tris-HCl, 10 mM boric acid, 5 mM L-serine, 1 mM DETAPAC, pH 7.4).
Twenty (20) .mu.l of the diluted cell homogenate were added to 2301
of serine borate buffer and 750 .mu.l of NPM (1 mM in
acetonitrile). The resulting solutions were incubated at room
temperature for 5 min. The reaction was stopped by the addition of
5 .mu.l of 2N HCl. The samples were then filtered through a 0.2
.mu.m Acrodisc filter and injected onto the HPLC system.
[0201] MDA measurement: To prepare the solution, 350 .mu.l of
straight cell homogenate, 100 .mu.l of 500 ppm BHT (butylated
hydroxytoluene), and 550 .mu.l of 10% TCA (trichloroacetic acid)
were combined and boiled for 30 min. The tubes were cooled on ice
and centrifuged for 10 min at 2500 rpm. Five hundred (500) .mu.l of
the supernatant were removed and 5001 of TBA (thiobarbituric acid)
were added. The tubes were boiled again for 30 min, and then cooled
on ice. From this solution, 500 .mu.l were removed, added to 1.0 ml
of n-butanol, vortexed, and centrifuged for 5 min at 60 g to
facilitate a phase separation. The top layer was then filtered
through 0.45 .mu.m filters and injected onto a 5 .mu.m C18 column
(250.times.4.6 mm) on a reverse phase HPLC system. The mobile phase
consisted of 69.4% 5 mM sodium phosphate buffer (pH=7.0), 30%
acetonitrile, and 0.6% THF (tetrahydrofuran). The excitation
wavelength was 515 nm; the emission wavelength was 550 nm (Draper
H. H. et al., Free Radic Biol Med., 15(4):353-63, 1993).
[0202] Protein determination and statistical analysis: Protein
levels were determined by the Bradford method with Coomassie Blue
(Bio-Rad) (Bradford M. M., Anal Biochem., 72:248-54, 1976). The
data were given as the mean.+-.SD. The one-way analysis of variance
test was used to analyze the significance of the differences
between the control and experimental groups.
[0203] This Example shows that NAC amide protects cells against
glutamate toxicity. Glutamate toxicity was evaluated by 1)
morphological assessment of PC12 cells in the presence of
glutamate; 2) measuring the amount of LDH released in the media 24
h after glutamate exposure; and 3) measuring cell viability using
the MTS assay. As shown in FIGS. 2A-D, cells completely lost the
normal morphology of their neurites in the presence of 10 mM
glutamate, as compared to the control cells. To determine whether
NAC amide could protect the cells from glutamate toxicity, PC12
cells were exposed to 10 mM glutamate for 24 hours in the presence
of 750 .mu.M NAC amide, and cell viability was examined by light
microscopy. The addition of NAC amide protected the PC12 cells from
glutamate toxicity by slightly decreasing the bleb formation on
neurites.
[0204] To quantify the protection provided by NAC amide, PC12 cells
were exposed to 10 mM glutamate in the presence of NAC amide for 24
hours, and then the amount of LDH released was measured using the
LDH assay. As shown in FIG. 3, inclusion of 750 .mu.M NAC amide in
the assay completely protected the cells from cell damage, even in
the presence of 10 mM glutamate (the % LDH released was
28.9.+-.3.7%). Similar results were obtained when cells were
exposed to 10 mM glutamate in the presence of NAC amide for 24
hours, and the cell viability was assessed by the MTS assay.
[0205] The results of Example 1 demonstrate that NAC amide
treatment significantly increased PC12 cell GSH levels. When cells
were exposed to 10 mM glutamate, a significant reduction in GSH
levels was observed (Table 1).
TABLE-US-00001 TABLE 1 Effect of NAC amide on intracellular GSH
levels in the presence of BSO and Glutamate Group GSH Levels (nM/mg
protein) Control 54 .+-. 13.4 GLU (10 mM) * 23 .+-. 4.2 BSO (0.2
mM) ND NAC amide (750 .mu.M) * 112 .+-. 17.8 GLU + NAC amide ** 88
.+-. 11.0 GLU + BSO + NAC amide *** 30 .+-. 4.3
[0206] PC12 cells were seeded and grown for 24 hours, then they
were treated with either GLU (10 mM); NAC amide (750 .mu.M); GLU
(10 mM)+NAC amide (750 .mu.M); GLU (10 mM)+BSO (0.2 mM)+NAC amide
(750 .mu.M); or BSO (0.2 mM). Twenty hours later, cells were
removed and analyzed for GSH levels, as described in the text.
Values represent means.+-.SD. Statistically different values of *
P<0.05 were determined, compared to control. ** P<0.001
compared to glutamate-treated group. *** P<0.05 compared to
glutamate-treated group. At a 750 .mu.M concentration and 24 hour
treatment time, NAC amide increased the PC12 cell GSH level two
fold, compared to the control group. Interestingly, similar results
were obtained when Chinese hamster ovary (CHO) cells were incubated
with NAC amide (data not shown).
[0207] The intracellular levels of GSH were determined in PC12
cells incubated with 10 mM glutamate for 24 hours, and the effects
of NAC amide were analyzed. Treatment of cells with NAC amide
prevented the marked decline of cellular GSH levels that normally
occurs after glutamate treatment (Table 1). Glutamate inhibits
cystine uptake, resulting in the loss of cellular GSH, while
buthionine-sulfoximine (BSO) inhibits .gamma.-GCS activity and
thereby causes the depletion of intracellular GSH. To determine
whether the increase in intracellular GSH by NAC amide was
.gamma.-GCS-dependent, cells were treated with 0.2 mM BSO. The
simultaneous treatment of glutamate and BSO, depleted the cell GSH
to almost undetectable levels (Table 1). Interestingly, in GSH
synthesis-arrested cells, NAC amide treatment was effective and
maintained 56% of the cells' GSH levels. NAC amide further
protected cells against intracellular peroxide accumulation.
Malondialdehyde (MDA) is a by-product of a free radical attack on
lipids. Marked increase in MDA levels was observed in
glutamate-exposed cells, as compared with the corresponding control
cells (Table 2). Treatment with NAC amide completely protected
cells against glutamate toxicity by lowering MDA levels.
TABLE-US-00002 TABLE 2 Effects of NAC amide on MDA levels in
Glutamate-exposed PC12 cells Group MDA Levels (nM/100 mg protein)
Control 54 .+-. 14 GLU (10 mM) 247 .+-. 26 NAC amide (750 .mu.M) 81
.+-. 22 GLU + NAC amide 88 .+-. 11
[0208] Cells were plated and grown for 24 hours, and then they were
exposed to glutamate (10 mM) in the presence or absence of NAC
amide (750 .mu.M). Twenty-four hours later, the cells were
harvested and malondialdehyde levels were measured. Values
represent means.+-.SD. Statistically different values of *
P<0.002 and ** P<0.05 were determined, compared to control.
*** P<0.05 compared to glutamate-treated group.
[0209] In this Example, it was determined that a high concentration
of glutamate-induced oxidative toxicity was characterized by
various potentially detrimental changes in intracellular GSH
levels, MDA levels, and LDH activity, resulting in a reduction of
PC12 cell viability. Treatment with NAC amide increased
intracellular GSH, and reduced MDA levels, thereby attenuating
glutamate-induced cytotoxicity. Evaluation was done by LDH and MTS
assay. Glutamate cytotoxicity has been attributed to either
excitatory action through the activation of glutamate receptors or
inhibition of cystine uptake that leads to the decreased GSH
levels. Although PC12 cells express NMDA receptors, toxicity
exhibited by glutamate does not solely relate to the presence of
these receptors, as NMDA has no effect on PC12 cell death. The
disruption of intracellular redox homeostasis by high
concentrations of glutamate is thought to be a major contributing
mechanism of cellular damage in vivo. Under conditions such as
cerebral ischemia, extracellular glutamate levels increase 800%, as
compared to control, which would decrease brain GSH levels by
blocking cystine uptake. GSH plays an important role in antioxidant
defense, and redox regulation. GSH deficiency has been associated
with various neurodegenerative diseases. Intracellular GSH levels
were determined by the X c- and ASC systems. The X c-system
transports cystine intracellularly in exchange for glutamate,
whereas the ASC system is a Na.sup.+-dependent neutral amino acid
transporter that mediates the cellular transport of cysteine.
Following uptake, cystine is reduced to cysteine for intracellular
glutathione synthesis. However, elevated levels of glutamate
inhibit cystine uptake, and subsequent restriction of cysteine
availability for the cell, leading to GSH depletion.
[0210] In this Example, incubation of PC12 cells with glutamate
resulted in reduction of GSH (Table 1) and cysteine levels (FIG.
4), when compared to the control group. Reduced levels of cysteine
indicate that the presence of excess glutamate inhibited cystine
uptake, which led to decreased GSH levels. NAC amide treatment was
able to increase GSH (Table 1) and cysteine levels (FIG. 5),
compared to the control group, and effectively reversed the
inhibitory action of glutamate. Increases in GSH and cysteine
levels were also observed 30 minutes after NAC amide was
administered to mice. The possible mechanism for NAC amide to
facilitate the supply of cysteine may be by readily reaching the
cell's interior, and becoming deacetylated to form cysteine. To
understand whether NAC amide could restore the GSH levels in GSH
synthesis-arrested cells, PC12 cells were incubated with glutamate
(10 mM) plus BSO (0.2 mM) in the presence of NAC amide (750 .mu.M).
Results showed that NAC amide elevated intracellular GSH levels in
the presence of BSO, suggesting that the effect is
-GCS-independent. Therefore, NAC amide itself may act as a
sulfhydryl group donor for GSH synthesis.
[0211] In summary, Example 1 shows that NAC amide protects PC12
cells against glutamate-induced cytotoxicity by preventing
glutamate-induced loss of cellular GSH and inhibiting lipid
peroxides. These studies also show that the restoration of GSH
synthesis by NAC amide in GSH synthesis-arrested cells is
-GCS-independent. Without wishing to be bound by theory, the
possible mechanisms by which NAC amide can enhance GSH are 1)
supplying the rate-limiting substrate cysteine to the cells and 2)
reducing GSSG to GSH by a nonenzymatic thiol-disulfide exchange.
Considering the protective effects of NAC amide against
glutamate-induced cytotoxicity, in which oxidative stress seems to
be involved, NAC amide can play a role in the treatment of
neurodegenerative disorders such as cerebral ischemia and
Parkinson's disease in which GSH levels are depleted in certain
regions of the brain.
Example 2
[0212] This Example examines the radioprotective effects of NAC
amide. To evaluate the protective effects of NAC amide against
radiation exposure, the radioprotective role of NAC amide was
compared with that of NAC with respect to increasing the levels of
GSH and returning oxidative stress parameters to their control
values.
[0213] Animal studies: The irradiation of rats was performed at the
Radiation Oncology Department of the Phelps County Regional Medical
Center in Rolla, Mo., using a 16 MeV beam generated by a Varian
linear accelerator, model Clinac 1800, and in accordance with the
standards of humane laboratory animal protocols. A 20.times.20 or
25.times.25 cm field was used and output factors were checked once
a week. Twelve animals were divided into 4 groups each containing 3
animals (Control, XRT, NAC amide+XRT and NAC+XRT). The radiation
(XRT) control received whole body irradiation by 6 Gy of 16 MeV
electrons. The NAC amide+XRT group received 500 mg/kg/day NAC amide
immediately before irradiation and for three days after until
sacrifice. The rats were anesthetized and heparinized blood was
collected via cardiopuncture. Following sacrifice, liver, lung,
brain and spleen were removed and stored at -70.degree. C. until
homogenization.
[0214] All experiments were performed using adult Albino SASCO
Sprague Dawley female rats weighing about 250 g, which were
purchased from Charles River Laboratories Inc. (Portage, Mich.).
Twelve rats were shipped in paper crates (4 in each crate). Rats
were delivered with a certificate including serological,
bacteriological, pathological parasitological information. They
were divided into 4 cages (3 rats in each cage) and kept in a
temperature controlled (20.degree. C.) room equipped to maintain a
12 h light-dark cycle. Standard rat chow (Purina rat chow) and tap
water were supplied in individual glass bottle and given ad
libitum. Water was changed daily. Weights of the animal were taken
before giving the NAC amide treatment solution and amount of food
eaten and water consumed was not measured because NAC amide was
given orally but not in the drinking water or food.
[0215] NAC amide was provided by Novetide Ltd (Haifa Bay, Israel)
including certificate of analysis and MSDS (lot# 40233-64). NAC
amide feeding solution was prepared freshly each day right before
the administration by weighing 1.25 g NAC amide solid sample (Type
HR-120 electronic balance, A&D Company limited, Japan. S/N:
12202464) and adding into 10 ml PBS solution and put on ice. One ml
of this solution was administrated (gavaged) per rat orally by
using animal feeding biomedical needles and 3 ml BD Luer-Lok Tip
syringes. Rats received one-dose total-body 6Gy/16 MeV x-ray
radiation and 3 rats in each group were held in a covered bucket
and received radiation at the same time. Each day at the same time,
500 mg/Kg body weight of NAC amide was administrated to the
animals.
[0216] All the results are normalized into values per unit (mg) of
protein content for all the tissue samples.
[0217] Typical standard curves:
GSH:y=8.57544x-425.092,R2=0.9997
CYS:y=7.53294x+184.35,R2=0.9995
[0218] For GSH and CYS levels, 250 .mu.L tissue homogenate was used
to react with 750 .mu.L NPM solution, therefore, the total volume
was 1000 .mu.L.
[0219] As an example:
[0220] The peak area for GSH in the sample is 90860.25. The GSH
concentration (nM) is calculated from the standard curve. After
determining the protein content (mg/ml) of the sample, for example:
16.5 mg/ml, the calculation is as follows:
[(90860.25+425.092)/8.57544 nmol/L]*[1 L/1000 mL]*[1000 .mu.L/250
.mu.L]/(16.5 mg/ml)=2.58 mmol GSH/mg protein
MDA:y=26.6869x+370.488,R2=0.9990
[0221] For MDA levels, 350 .mu.L tissue homogenate was used to
react with 100 .mu.L of 500 ppm BHT solution and 550 .mu.L solution
of 10% TCA solution, therefore, the total volume here was 1000
.mu.L. After boiling the whole solution, 500 .mu.L was taken out
and react with 500 .mu.L TBA and the total volume here was 1000
.mu.L also.
[0222] As an example:
[0223] The peak area for MDA in the sample as 65289.23, The MDA
concentration (nM) is calculated from the standard curve. After
determining the protein content (mg/ml) of the sample, for example:
16.5 mg/ml, the resulting calculation is as follows:
[(65289.23-370.488)/26.6869 nmol/L]*[1 L/1000 mL]*[1000 .mu.L/350
.mu.L]*[1000 .mu.L/500 .mu.L]/(16.5 mg/ml)*100=84.3 nmol MDA/100 mg
protein
[0224] Catalase:
[0225] Calculation for Specific Activity:
[0226] In assay solution,
k(enzyme activity)= 1/60*ln(A0/A60)*(Total Volume of
reaction/volume of sample)
A0--Absorbance at 0 second A60--Absorbance at 60 second In sample,
K(specific activity)=k/protein concentration.
[0227] Oxidative Stress Parameters in Animals: After the blood
samples were drawn, the animals were perfused by a cold antioxidant
buffer first and then liver, brain and kidney samples were
collected aseptically, rinsed in ice-cold saline and placed in
petri dishes maintained on ice. The tissue samples kept at
-70.degree. C. for the GSH, GSSG, and MDA determinations were
made.
[0228] Glutathione (GSH) and Glutathione Disulfide (GSSG)
Determination: Cells or tissue samples were homogenized on ice and
derivatized with N-(1-pyrenyl)-maleimide (NPM). The derivatized
samples were injected onto a 3 .mu.m C18 column (Column
Engineering) in a reverse phase HPLC system with a mobile phase of
35% water, 65% acetonitrile containing 1 mL/L of acetic acid and
o-phosphoric acid (R. Winters, et al., Anal. Biochem., 227:14-21
(1995) and H. H. Draper et al., Free Rad. Biol. Med., 15:353-363
(1993)). Malondialdehyde (MDA) determinations were made as
described in J. Gutteridge, Anal. Biochem., 69: 518-526 (1975).
[0229] Enzyme Activity Assays: Catalase (CAT) activity was
determined spectrophotometrically and was expressed in kunits/mg
protein and kunits/10.sup.6 cells as described by M. Bradford,
Anal. Biochem., 72:248-256 (1976).
[0230] Statistical Analysis: Tabulated values represent
means.+-.standard deviations. InStat.RTM. by GraphPad Software, San
Diego, Calif. will use One-way Analysis of Variance (ANOVA) and the
Student-Newman-Keuls Multiple Comparisons Test to analyze data from
experimental and control groups. The p values <0.05 is
considered significant.
[0231] The results of the studies described in this Example are
provided in the tables below. In these tables, AD4 is synonymous
with NAC amide.
TABLE-US-00003 TABLE 3 GSH and CYS levels in BRAIN after 6Gy
total-body x-ray radiation with AD4 or NAC administration (500
mg/kg orally) GSH (nmol/mg) CYS (nmol/mg) (n = 3) level Mean SD
level Mean SD CTR-1 8.19 7.5 0.7 3.61 4.1 0.5 CTR-2 6.75 3.88 CTR-3
7.59 4.79 XRT-1 6.42 6.6 0.3 3.48 3.8 0.5 XRT-2 6.35 3.76 XRT-3
6.89 4.36 XRT + AD4-1 7.93 7.6** 0.5 4.47 4.4 0.1 XRT + AD4-2 7.84
4.32 XRT + AD4-3 6.98 4.26 XRT + NAC-1 7.32 7.0 0.3 4.16 4.1 0.4
XRT + NAC-2 6.74 3.76 XRT + NAC-3 7.15 4.47
TABLE-US-00004 TABLE 4 GSH and CYS levels in LIVER after 6Gy
total-body x-ray radiation with AD4 or NAC administration (500
mg/kg orally) GSH (nmol/mg) CYS (nmol/mg) (n = 3) level Mean SD
level Mean SD CTR-1 15.70 16.9 1.1 1.64 1.5 0.3 CTR-2 17.99 1.78
CTR-3 16.97 1.17 XRT-1 14.54 14.4* 0.2 1.34 1.4 0.1 XRT-2 14.26
1.39 XRT-3 14.30 1.55 XRT + AD4-1 17.45 17.2** 0.4 1.51 1.5 0.01
XRT + AD4-2 16.73 1.53 XRT + AD4-3 17.50 1.53 XRT + NAC-1 15.23
16.3 1.0 1.25 1.5 0.2 XRT + NAC-2 16.80 1.61 XRT + NAC-3 16.93
1.51
TABLE-US-00005 TABLE 5 GSH and CYS levels in KIDNEY after 6Gy
total-body x-ray radiation with AD4 or NAC administration (500
mg/kg orally) GSH (nmol/mg) CYS (nmol/mg) (n = 3) level Mean SD
level Mean SD CTR-1 4.62 5.5 0.8 10.29 11.1 0.7 CTR-2 6.25 11.56
CTR-3 5.63 11.37 XRT-1 4.91 4.8 0.3 8.13 8.7* 0.5 XRT-2 4.98 9.07
XRT-3 4.38 8.94 XRT + AD4-1 4.39 5.2 0.9 16.91 12.9** 3.4 XRT +
AD4-2 6.22 11.09 XRT + AD4-3 5.02 10.81 XRT + NAC-1 5.95 6.2** 0.3
12.23 11.8** 0.7 XRT + NAC-2 6.44 12.16 XRT + NAC-3 6.33 11.03
TABLE-US-00006 TABLE 6 GSH and CYS levels in LUNG after 6Gy
total-body x-ray radiation with AD4 or NAC administration (500
mg/kg orally) GSH (nmol/mg) CYS (nmol/mg) (n = 3) level Mean SD
level Mean SD CTR-1 7.04 6.2 0.7 1.91 1.7 0.3 CTR-2 5.87 1.85 CTR-3
5.78 1.44 XRT-1 5.24 5.1 0.8 1.26 1.6 0.3 XRT-2 4.25 1.66 XRT-3
5.93 1.83 XRT + AD4-1 5.12 5.6 0.6 1.43 1.3 0.4 XRT + AD4-2 5.27
1.61 XRT + AD4-3 6.28 0.91 XRT + NAC-1 5.19 5.8 1.3 1.16 1.9 0.7
XRT + NAC-2 7.24 2.04 XRT + NAC-3 4.95 2.43
TABLE-US-00007 TABLE 7 GSH and CYS levels in PLASMA after 6Gy
total-body x-ray radiation with AD4 or NAC administration (500
mg/kg orally) GSH (nmol/mg) CYS (nmol/mg) (n = 3) level Mean SD
level Mean SD CTR-1 7.65 7.4 0.4 16.03 15.5 0.4 CTR-2 7.49 15.20
CTR-3 6.92 15.39 XRT-1 5.27 5.3* 0.1 12.68 13.6* 0.9 XRT-2 5.39
13.63 XRT-3 5.31 14.45 XRT + AD4-1 7.10 7.6** 0.4 16.00 15.6** 0.3
XRT + AD4-2 7.44 15.45 XRT + AD4-3 7.94 15.40 XRT + NAC-1 7.08
6.5**/*** 0.5 14.64 14.2*** 0.5 XRT + NAC-2 6.18 13.75 XRT + NAC-3
6.27 14.36 *P < 0.05 compared to the CTR group; **P < 0.005
compared to the XRT only group ***P < 0.05 compared to the XRT +
AD4-treated group
TABLE-US-00008 TABLE 8 MDA levels in BRAIN after 6Gy total-body
x-ray radiation with AD4 or NAC administration (500 mg/kg orally)
MDA (nmol/100 mg) (n = 3) level Mean SD CTR-1 4.93 4.09 0.80 CTR-2
3.33 CTR-3 4.02 XRT-1 5.64 5.99* 0.68 XRT-2 6.76 XRT-3 5.55 XRT +
AD4-1 5.79 5.48 0.33 XRT + AD4-2 5.53 XRT + AD4-3 5.13 XRT + NAC-1
6.42 6.15 0.72 XRT + NAC-2 6.69 XRT + NAC-3 5.33
TABLE-US-00009 TABLE 9 MDA levels in LIVER after 6Gy total-body
x-ray radiation with AD4 or NAC administration (500 mg/kg orally)
MDA (nmol/100 mg) (n = 3) level Mean SD CTR-1 4.36 4.62 0.39 CTR-2
4.44 CTR-3 5.07 XRT-1 8.9 8.36* 0.53 XRT-2 8.35 XRT-3 7.83 XRT +
AD4-1 4.14 4.38** 0.26 XRT + AD4-2 4.65 XRT + AD4-3 4.36 XRT +
NAC-1 5.1 5.07**/*** 0.04 XRT + NAC-2 5.1 XRT + NAC-3 5.02
TABLE-US-00010 TABLE 10 MDA levels in KIDNEY after 6Gy total-body
x-ray radiation with AD4 or NAC administration (500 mg/kg orally)
MDA (nmol/100 mg) (n = 3) level Mean SD CTR-1 1.61 1.69 0.09 CTR-2
1.8 CTR-3 1.68 XRT-1 2.48 2.28* 0.17 XRT-2 2.17 XRT-3 2.18 XRT +
AD4-1 1.5 1.64** 0.28 XRT + AD4-2 1.96 XRT + AD4-3 1.45 XRT + NAC-1
1.76 1.65** 0.21 XRT + NAC-2 1.78 XRT + NAC-3 1.41
TABLE-US-00011 TABLE 11 MDA levels in LUNG after 6Gy total-body
x-ray radiation with AD4 or NAC Administration (500 mg/kg orally)
MDA (nmol/100 mg) (n = 3) level Mean SD CTR-1 1.47 1.54 0.07 CTR-2
1.53 CTR-3 1.61 XRT-1 2.3 2.80* 0.45 XRT-2 2.94 XRT-3 3.17 XRT +
AD4-1 1.72 1.53** 0.22 XRT + AD4-2 1.58 XRT + AD4-3 1.28 XRT +
NAC-1 2.58 2.52** 0.15 XRT + NAC-2 2.34 XRT + NAC-3 2.63 *P <
0.05 compared to the CTR group **P < 0.005 compared to the XRT
only group ***P < 0.05 compared to the XRT + AD4-treated
group
TABLE-US-00012 TABLE 12 Catalase activities in KIDNEY after 6Gy
total-body x-ray radiation with AD4 or NAC administration (500
mg/kg orally): Catalase (mU/mg) (n = 3) level Mean SD CTR-1 2.75
2.34 0.78 CTR-2 2.84 CTR-3 1.44 XRT-1 8.73 8.69* 1.05 XRT-2 7.59
XRT-3 9.66 XRT + AD4-1 3.89 3.97** 0.56 XRT + AD4-2 3.46 XRT +
AD4-3 4.56 XRT + NAC-1 5.85 4.41 1.48 XRT + NAC-2 3.02 XRT + NAC-3
4.36
TABLE-US-00013 TABLE 13 Catalase activities in LUNG after 6Gy
total-body x-ray radiation with AD4 or NAC administration (500
mg/kg orally): Catalase (mU/mg) (n = 3) level Mean SD CTR-1 1.50
1.24 0.33 CTR-2 0.87 CTR-3 1.37 XRT-1 3.53 2.03 1.43 XRT-2 0.72
XRT-3 1.83 XRT + AD4-1 1.02 0.68** 0.29 XRT + ND4-2 0.50 XRT +
AD4-3 0.53 XRT + NAC-1 2.12 1.13 0.89 XRT + NAC-2 0.79 XRT + NAC-3
0.48
TABLE-US-00014 TABLE 14 Catalase activities in LIVER after 6Gy
total-body x-ray radiation with AD4 or NAC administration (500
mg/kg orally). Catalase (mU/mg) (n = 3) level Mean SD CTR-1 49.48
43.03 6.13 CTR-2 42.39 CTR-3 37.23 XRT-1 89.10 77.44* 10.46 XRT-2
69.23 XRT-3 74.00 XRT + AD4-1 69.63 59.28** 9.80 XRT + AD4-2 57.88
XRT + AD4-3 50.33 XRT + NAC-1 75.22 71.11*** 3.56 XRT + NAC-2 69.09
XRT + NAC-3 69.00 *P < 0.05 compared to the CTR group; **P <
0.05 compared to the XRT only group ***P < 0.05 compared to the
XRT + AD4-treated group
[0232] The data presented support the finding that NAC amide
functions as a strong thiol antioxidant in radiation-induced
oxidative stress. NAC does not increase GSH levels in tissues,
presumably because it does not cross the cell membranes. Although
plasma Cys level increased significantly, this was not reflected in
the liver. NAC generally provides GSH only during increased demand
on the GSH pool.
[0233] Upon irradiation, reactive oxygen species are formed through
oxygen's acceptance of electrons, which are involved in free
radical chain reactions and are highly damaging to the cell through
disruption of the cellular pro-oxidant/antioxidant balance. Normal
tissue damage limits the radiation dose and treatment volume in
radiotherapy. Radioprotection of normal tissue by thiols offers one
way in which radiation dosage can be increased. The focus in this
Example was to examine the radioprotective effects of NAC amide
using a whole body radiation dose of 6 Gy, sufficient to insure
that all animals should progress with lethal gastrointestinal and
hematopoietic syndromes. The time point chosen for analyses, 4
days, approximates the time that the animals would begin to succumb
to the gastrointestinal syndrome, but would be expected to show
only early changes in the hematopoietic syndrome.
[0234] GSH, a tripeptide consisting of
y-glutamyl-cysteinyl-glycine, is the principle water-soluble
intracellular free thiol and acts as a radioprotector. Several
distinct mechanisms of radioprotection by GSH can be identified and
include radical scavenging, hydrogen donation to damaged molecules,
reduction of peroxides, and protection of protein thiol oxidative
status. GSH has been shown to decrease in tissues following
irradiation. Since GSH is an endogenous radioprotector,
modification of GSH concentration may be useful as radiation
protection. Cysteine provides the rate-limiting step in GSH
synthesis since its apparent Km value for .gamma.-glutamyl-cysteine
synthetase is close to the intracellular concentration of the amino
acid. However, administration of cysteine is not the ideal way to
increase intracellular GSH, since it auto-oxidizes rapidly and can
lead to the production of hydroxyl and thiyl radicals.
[0235] NAC, a cysteine analogue that is a mucolytic agent and a
treatment for paracetamol intoxication, promotes hepatic GSH
synthesis. It penetrates the cell membrane and is rapidly
deacetylated to L-cysteine, while also stimulating GSSG reductase.
NAC can rapidly increase the hepatic GSH levels and maintain these
levels for at least 6 hours (B. Wong et al., J. Pharm. Sci.,
75:878-880 (1986)). NAC has also been shown to protect Chinese
hamster ovary cells from lead and .delta.-aminolevulinic
acid-induced toxicity through restoration of the oxidative status
of the cells by GSH replenishment. It has been demonstrated that
NAC protects liver and brain of C57BL/6 mice from GSH depletion as
a result of lead poisoning. Radioprotective effects of select
thiols such as indomethacin, WR-2721, cysteamine, and
diethyldithiocarbamate have been reported, though at higher
concentrations these induce cellular toxicity. The radioprotective
effect of NAC has been demonstrated in human
granulocyte/macrophage-colony forming cells. However, it has also
been shown that the more radioresistant SW-1573 human squamous lung
carcinoma cell line was not protected from X-ray induced cell death
by NAC. NAC amide is more lipophilic and able to more easily cross
cell membranes than NAC. In this Example, the radioprotective
function of NAC amide was compared with that of NAC in terms of
increasing GSH levels and returning oxidative stress parameters to
their control values.
[0236] The exposure of membrane lipids to reactive oxygen species
such as the hydroxyl radical can initiate a chain reaction in
polyunsaturated fatty acid moieties, which results in peroxidation
and causes degradation of membrane function. MDA is a degradation
product of the highly unstable lipid peroxides. As observed in this
Example, irradiation of Sprague Dawley rats resulted in increased
MDA levels in liver and lung. Upon treatment with NAC amide
concurrent with irradiation, lung MDA levels were significantly
lowered, while treatment with NAC did not change the MDA levels
significantly.
[0237] It is generally accepted in the field of radiobiology that
the mechanism of individual cell killing by radiation exposure is
due to direct and indirect ionizing effects specifically upon DNA
in the cell nucleus, although it becomes apparent that in a complex
organism there are ROS effects of some potential importance on
membrane lipids and proteins as well as on nucleic acids.
Furthermore, acute whole body irradiation of the intact animal
under conditions modeling the so called "gastrointestinal syndrome"
causes changes in several tissues apart from gastrointestinal
tract, and some of these effects can be ameliorated by the use of
NAC amide. A given syndrome such as the "gastrointestinal syndrome"
can actually involve a complex of changes in multiple tissues and
organs. Radiation pneumonitis can be a serious hazard in the
therapeutic irradiation of patients with lung cancer. NAC amide may
be considered for use as a thiol radioprotectant to protect against
such a complication. Thus, in accordance with the invention, NAC
amide significantly increases thiol levels in plasma and liver and
performs better than NAC as a radioprotecting agent.
Example 3
[0238] This Example describes a treatment regimen suitable for
humans. NAC amide is administered between 1 and three grams per
day, in two divided doses, between meals (on an empty stomach).
Encapsulated NAC amide (a formulation of NAC amide comprising 500
mg NAC amide and optionally, 250 mg USP grade crystalline ascorbic
acid, and not more than 0.9 mg magnesium stearate, NF grade in an
OO-type gelatin capsule) is suitable for administration. The
administration of exogenous NAC amide is expected to provide a dose
response effect in patients, despite the production of large
quantities of glutathione in the human body.
Example 4
[0239] This Example describes a combination pharmaceutical
composition to ameliorate the detrimental effects of acetaminophen,
a drug that consumes glutathione in the liver during metabolism
and, in excess doses, causes liver damage due to oxidative damage.
The composition includes 500 mg NAC amide, 250 mg crystalline
ascorbic acid and 350 mg acetaminophen.
Example 5
[0240] This Example describes a combination pharmaceutical
composition to ameliorate the detrimental effects of
chlorpromazine, a phenothiazine drug that causes side effects,
including tardive dyskinesia, which may be associated with excess
free radical reactions. The composition includes 500 mg NAC amide,
250 mg crystalline ascorbic acid and 200 mg chlorpromazine.
Example 6
[0241] This Example describes a combination pharmaceutical
composition to ameliorate the detrimental effects of aminoglycoside
drugs (antibiotics), nonlimiting examples of which include
neomycin, kanamycin, amikacin, streptomycin, gentamycin, sisomicin,
netilmicin and tobramycin, a drug class which may be associated
with various toxicities. This damage may be related to oxidative
damage or consumption of glutathione during metabolism. The
composition according to the present invention is an intravenous
formulation, including the aminoglycoside in an effective amount,
and NAC amide in an amount of about 10-20 mg/kg. Ascorbic acid in
an amount of 5-10 mg/kg may be added as a stabilizer.
Example 7
[0242] This Example describes a urethral insert comprising NAC
amide. A composition containing 200 mg NAC amide, 50 mg ascorbic
acid per unit dosage is mixed with carageenan and/or agarose and
water in a quick-gelling composition, and permitted to gel in a
cylindrical form having a diameter of about 3 mm and a length of
about 30 mm. The composition is subjected to nitric oxide to cause
between 0.1-10% of the NAC amide to be converted to nitroso-NAC
amide. The gelled agarose is then freeze dried under conditions
that allow shrinkage. The freeze-dried gel is than packaged in a
gas barrier package, such as a foil pouch or foil "bubble-pack".
The freeze-dried gel may then be used as a source of nitroso-NAC
amide for administration transmucosally. The cylindrical
freeze-dried gel may be inserted into the male urethra for
treatment of impotence, or administered sublingually for systemic
vasodilation.
Example 8
[0243] This Example describes an oral formulation for prophylaxis
of vascular disease, e.g., in men over 40. The composition includes
500 mg NAC amide, 250 mg USP grade crystalline ascorbic acid and 50
mg USP acetyl salicylic acid (aspirin) in an OO-type gelatin
capsule. Typical administration is twice per day. The acetyl
salicylic acid may be provided in enteric release pellets within
the capsule to retard release.
Example 9
[0244] This Example describes an oral formulation for prophylaxis
of vascular disease. The composition contains 500 mg NAC amide, 200
mg USP grade crystalline ascorbic acid, and 200 mg arginine in an
OO-type gelatin capsule. Arginine is the normal starting substrate
for the production of nitric oxide. Because arginine is normally in
limited supply, a relative deficiency of arginine may result in
impaired vascular endothelial function.
Example 10
[0245] This Example describes an oral formulation for prophylaxis
of vascular disease. The composition includes 500 mg NAC amide, 200
mg USP grade crystalline ascorbic acid, and 200 mg vitamin E
succinate in an OO-type gelatin capsule. Vitamin E consumption
reduces the risk of heart attack and other vascular disease.
Vitamin E succinate (alpha-tocopherol succinate) is a dry
powder.
Example 11
[0246] This Example describes an oral formulation for prophylaxis
of vascular disease. Nonspecific esterases having broad substrate
specificity are present in the plasma. According to the present
invention, esters are formed between agents that are useful
combination therapies in order to provide for efficient
administration, high bioavailability, and pharmaceutical stability.
Preferred esters include alpha tocopherol-ascorbate, alpha
tocopherol-salicylate, and ascorbyl-salicylate. The tocopherol
ester maintains the molecule in a reduced state, allowing full
antioxidant potential after ester cleavage. These esters may be
administered alone or in combination with other agents, for example
NAC amide. Typically, the esters are administered to deliver an
effective dose of salicylate equivalent of 100 mg per day for
prophylaxis, or 750-1000 mg per dose for treatment of inflammatory
diseases. Tocopherol is administered in an amount of 100-500 IU
equivalent. Ascorbate is administered in an amount of up to 1000 mg
equivalent. In order to enhance availability, a non-specific
esterase may be provided in the formulation to cleave the ester
after dissolution of the capsule. Therefore, a non-specific
esterase, such as a bacterial or saccharomyces (yeast) enzyme, or
an enriched enzyme preparation, may be included in the formulation
as a powder or as pellets in the capsule.
Example 12
[0247] This Example describes an oral formulation for prophylaxis
of vascular disease. The composition includes 500 mg reduced NAC
amide, 200 mg USP grade crystalline ascorbic acid, and 100 mg
nordihydroguaretic acid, in an OO-type gelatin capsule. Typical
administration is twice per day. Nordihydroguaretic acid is a known
lipoxygenase inhibitor. Thus, this composition may be used to treat
inflammatory processes or as prophylaxis against vascular
disease.
Example 13
[0248] This Example describes a study observing the survival of
rats receiving whole body, single-dose irradiation by X-rays (XRT)
in the presence or absence of NAC or NAC amide (TOVA). In this
experiment, thirty-nine female Sprague-Dawley rats ranging from
about 150-200 g were subjected to total body, single-dose X-ray
irradiation (9 Gy, 16 Mev). The same groups were designated to
receive either NAC or TOVA. For the pre-treatment groups (n=6 in
each group), the first treatment of NAC or TOVA was administered 30
minutes to 1 hour before irradiation. For the post-pretreatment
groups (n=6 in each group), the first treatment of NAC or TOVA was
administered 30 minutes to 1 hour after the irradiation. For groups
receiving NAC or TOVA, the same amount (500 mg/kg NAC or TOVA
daily) was administered for 4 or 5 consecutive days.
[0249] Group 1 was a control group (n=3), where rats received the
same amount of saline solution daily for 5 consecutive days without
XRT. Group 2 rats received NAC only (n=3) at an amount of 500 mg/kg
body weight NAC daily for 5 consecutive days without XRT. Group 3
rats received TOVA only (n=3) at an amount of 500 mg/kg body weight
TOVA daily for 5 consecutive days without XRT. Group 4 rats
received radiation (XRT) only (n=6) and received the same amount of
saline solution daily for 5 consecutive days after single dose
total-body XRT irradiation.
[0250] Group 5 rats received one treatment of NAC at 500 mg/kg body
weight before XRT (XRT+NAC pre-treated), which was then followed by
500 mg/kg body weight NAC daily for 4 consecutive days after XRT.
Group 6 rats received XRT, followed by daily doses of NAC at 500
mg/kg body weight for 5 consecutive days after XRT (XRT+NAC
post-treated). Group 7 rats received one treatment of NAC at 500
mg/kg body weight before XRT (XRT+TOVA pre-treated), which was then
followed by 500 mg/kg body weight TOVA daily for 4 consecutive days
after XRT. Group 8 rats received XRT, followed by daily doses of
TOVA at 500 mg/kg body weight for 5 consecutive days after XRT
(XRT+TOVA post-treated). All rats were then given a normal diet
post-treatment.
[0251] The rats were observed twice a day, and the survival status
of rats in each group will be recorded. The mean survival days were
calculated for each group and compared to the survival differences
of the three groups of rats at the end of the experiment. The
radioprotective effects of NAC and TOVA treatment on the survival
of those irradiated rats were then evaluated, as shown in the
following tables.
[0252] Table 15 shows the number of animals that survived under
conditions where NAC or TOVA was administered pre- or post-XRT
treatment.
TABLE-US-00015 # of # of percentage animals animals survival
survival Groups # of animals dead survived rate rate XRT only (n =
6)-1st time 2 4 (4 + 2)/(6 + 6) 50% (n = 6)-2nd time 4 2 XRT + NAC
(pre-treated) (n = 6)-1st time 1 5 (5 + 5)/(6 + 6) 83.3% (n =
6)-2nd time 1 5 XRT + TOVA (pre-treated) (n = 6)-1st time 0 6 (6 +
6)/(6 + 6) 100% (n = 6)-2nd time 0 6 Control (no XRT and any (n =
3)-1st time 0 3 (3 + 3)/(3 + 3) 100% treatment) (n = 3)-2nd time 0
3 NAC only (n = 2)-2nd time 0 2 (2)/(2) 100% TOVA only (n = 3)-2nd
time 0 3 (3)/(3) 100% XRT + NAC (post-treated) (n = 6)-2nd time 4 2
(2)/(6) 33.3% XRT + TOVA (post-treated) (n = 6)-2nd time 2 4
(4)/(6) 66.7%
[0253] Table 16 shows the survival rate percentage of rats
receiving NAC or TOVA pre- or post-XRT treatment.
TABLE-US-00016 Groups percentage survival rate XRT only 50% XRT +
NAC(pre-treated) 83.3% XRT + TOVA(pre-treated) 100% Control (no XRT
and any treatment) 100% NAC only 100% TOVA only 100% XRT +
NAC(post-treated) 33.3% XRT + TOVA(post-treated) 66.7%
[0254] FIG. 6 is a graphical representation comparing the
percentage survival rates as presented in Table 16. These results
show that rats pre-treated with NAC or TOVA before XRT have a
higher survival rate than those receiving XRT alone.
[0255] All patent applications, published applications, patents,
texts, and literature references cited in this specification are
hereby incorporated herein by reference in their entirety.
[0256] As various changes can be made in the above methods and
compositions without departing from the scope and spirit of the
invention as described, it is intended that all subject matter
contained in the above description, shown in the accompanying
drawings, or defined in the appended claims be interpreted as
illustrative, and not in a limiting sense.
* * * * *