U.S. patent application number 09/844450 was filed with the patent office on 2002-03-07 for methods and compositions for enhancing cellular function through protection of tissue components.
Invention is credited to Fawcett, John Randall, Frey, William H. II.
Application Number | 20020028786 09/844450 |
Document ID | / |
Family ID | 27394213 |
Filed Date | 2002-03-07 |
United States Patent
Application |
20020028786 |
Kind Code |
A1 |
Frey, William H. II ; et
al. |
March 7, 2002 |
Methods and compositions for enhancing cellular function through
protection of tissue components
Abstract
Methods and compositions for enhancing cellular function through
protection of a tissue components such receptors, proteins, lipids,
nucleic acids, carbohydrates, hormones, vitamins, and cofactors, by
administering pyrophosphate analogs or related compounds.
Inventors: |
Frey, William H. II; (White
Bear Lake, MN) ; Fawcett, John Randall; (St. Paul,
MN) |
Correspondence
Address: |
GRAY, PLANT, MOOTY, MOOTY & BENNETT, P.A.
P.O. BOX 2906
MINNEAPOLIS
MN
55402-0906
US
|
Family ID: |
27394213 |
Appl. No.: |
09/844450 |
Filed: |
April 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60200843 |
May 1, 2000 |
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60233263 |
Sep 18, 2000 |
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60233025 |
Sep 15, 2000 |
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Current U.S.
Class: |
514/48 ; 514/102;
514/51 |
Current CPC
Class: |
A61K 31/7076 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/352 20130101;
A61K 31/7105 20130101; A61P 25/28 20180101; A61P 9/00 20180101;
A61K 31/708 20130101; A61K 31/409 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 31/409 20130101; A61K 2300/00 20130101; A61K 31/409
20130101; A61P 35/00 20180101; A61P 39/06 20180101; A61K 2300/00
20130101; A61K 31/7105 20130101; A61K 31/6615 20130101; A61K 45/06
20130101; A61K 31/66 20130101; A61P 25/00 20180101; A61K 31/662
20130101; A61K 31/7076 20130101; A61K 31/7076 20130101; A61P 33/00
20180101; A61K 31/66 20130101; A61K 31/708 20130101; A61P 39/00
20180101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/661
20130101; A61K 31/708 20130101; A61K 31/352 20130101; A61K 31/7084
20130101; A61K 31/7072 20130101; A61K 31/663 20130101; A61K 31/7084
20130101 |
Class at
Publication: |
514/48 ; 514/51;
514/102 |
International
Class: |
A61K 031/7105; A61K
031/66 |
Claims
We claim:
1. A method of protecting a tissue component in a subject in need
thereof, comprising administering to the subject at least one
pyrophosphate analog comprising the structure: 5where each X is
independently O, CH.sub.2, NH, or S; R.sup.1 is H, a small alkyl
group, guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol,
serine, threonine, tyrosine, arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2) and m is 1-3; R.sup.2 is H, a
small alkyl group, guanyl, adenylyl, glycerol, acyl glycerol,
diacyl glycerol, serine, threonine, tyrosine, or arachidonyl; and n
is 1-900; or the structure: 6where n=2-4; X is O; RCR.sup.1; CR; C
(n=4), CH (n=3), or CH.sub.2 (n=2); NH; N; S; and R and/or R.sup.1
is H, OH, a small alkyl group, or (CH.sub.2).sub.mNH.sub.2 where
m=1-6; or a dinucleoside-5-5'-pyrophosphate, a cyclopyrophosphate
of purine, a pyrimidine acyclonucleoside, an inositol diphosphate,
an inositol triphosphate, an inositol tetraphosphate, an inositol
pentaphosphate, an inositol hexaphosphate; or a pharmaceutically
acceptable salt thereof.
2. The method of claim 1, wherein the tissue component is at least
one of a receptor, a protein, a lipid, a nucleic acid, a
carbohydrate, a hormone, a vitamin, and a cofactor.
3. The method of claim 2, in which the tissue component is a
receptor for a neurotransmitter, a neuropeptide, a neurotrophin, a
growth factor, a steroid, a histamine, a purine, a benzodiazepine,
arachidonic acid, nitric oxide, carbon monoxide, an odorant, or an
ion channel.
4. A method of protecting tissue from oxidative stress, comprising
administering at least one pyrophosphate analog comprising the
structure: 7where each X is independently O, CH.sub.2, NH, or S;
R.sup.1 is H, a small alkyl group, guanyl, adenylyl, glycerol, acyl
glycerol, diacyl glycerol, serine, threonine, tyrosine,
arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2) and m is 1-3; R.sup.2 is H, a
small alkyl group, guanyl, adenylyl, glycerol, acyl glycerol,
diacyl glycerol, serine, threonine, tyrosine, or arachidonyl; and n
is 1-900; or the structure: 8where n=2-4; X is O; RCR.sup.1; CR; C
(n=4), CH (n=3), or CH.sub.2 (n=2); NH; N; S; and R and/or R.sup.1
is H, OH, a small alkyl group, or (CH.sub.2).sub.mNH.sub.2 where
m=1-6; or a dinucleoside-5-5'-pyrophosphate, a cyclopyrophosphate
of purine, a pyrimidine acyclonucleoside, an inositol diphosphate,
an inositol triphosphate, an inositol tetraphosphate, an inositol
pentaphosphate, an inositol hexaphosphate; or a pharmaceutically
acceptable salt thereof.
5. A method of increasing the efficacy of an agent that directly or
indirectly affects a receptor in a subject in need thereof,
comprising administering the agent, and administering at least one
pyrophosphate analog comprising the structure: 9where each X is
independently O, CH.sub.2, NH, or S; R.sup.1 is H, a small alkyl
group, guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol,
serine, threonine, tyrosine, arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2) and m is 1-3; R.sup.2 is H, a
small alkyl group, guanyl, adenylyl, glycerol, acyl glycerol,
diacyl glycerol, serine, threonine, tyrosine, or arachidonyl; and n
is 1-900; or the structure: 10where n=2-4; X is O; RCR.sup.1; CR; C
(n=4), CH (n=3), or CH.sub.2 (n=2); NH; N; S; and R and/or R.sup.1
is H, OH, a small alkyl group, or (CH.sub.2).sub.mNH.sub.2 where
m=1-6; or a dinucleoside-5-5'-pyrophosphate, a cyclopyrophosphate
of purine, a pyrimidine acyclonucleoside, an inositol diphosphate,
an inositol triphosphate, an inositol tetraphosphate, an inositol
pentaphosphate, an inositol hexaphosphate; or a pharmaceutically
acceptable salt thereof.
6. The method of claim 5, in which the receptor is a receptor for a
neurotransmitter, a neuropeptide, a neurotrophin, a growth factor,
a steroid, a histamine, a purine, a benzodiazepine, arachidonic
acid, nitric oxide, carbon monoxide, an odorant, or an ion
channel.
7. The method of claim 6, in which the receptor is one of
muscarinic acetylcholine, nicotinic acetylcholine, an opiate, a
catecholamine, serotonin, glutamate, aspartate, cannabinoid, gamma
aminobutyric acid, or glycine.
8. The method of claim 6, in which the agent directly or indirectly
affects a mAChR.
9. The method of claim 8, wherein the agent that directly or
indirectly affects a mAChR comprises an anticholinesterase agent, a
neurologic agent, or a muscarinic receptor agonist.
10. The method of claim 8, in which the agent that directly or
indirectly affects a mAChR comprises Xanomeline.
11. The method of claim 8, wherein the agent that directly or
indirectly affects a mAChR comprises donepezil, rivastigmine,
galanthamine, metrifonate, or a combination thereof.
12. A method of protecting a subject from at least one carcinogenic
metal, comprising administering to the subject at least one
pyrophosphate analog comprising the structure: 11where each X is
independently O, CH.sub.2, NH, or S; R.sup.1 is H, a small alkyl
group, guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol,
serine, threonine, tyrosine, arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2) and m is 1-3; R.sup.2 is H, a
small alkyl group, guanyl, adenylyl, glycerol, acyl glycerol,
diacyl glycerol, serine, threonine, tyrosine, or arachidonyl; and n
is 1-900; 12or the structure: where n=2-4; X is O; RCR.sup.1; CR; C
(n=4), CH (n=3), or CH.sub.2 (n=2); NH; N; S; and R and/or R.sup.1
is H, OH, a small alkyl group, or (CH.sub.2).sub.mNH.sub.2 where
m=1-6; or a dinucleoside-5-5'-pyrophosphate, a cyclopyrophosphate
of purine, a pyrimidine acyclonucleoside, an inositol diphosphate,
an inositol triphosphate, an inositol tetraphosphate, an inositol
pentaphosphate, an inositol hexaphosphate; or a pharmaceutically
acceptable salt thereof.
13. The method of claim 12, in which the carcinogenic metal is
selected from the group consisting of arsenic, cadmium, cobalt,
nickel, lead, and chromium.
14. A method of reducing poisoning of a subject by at least one
metal, comprising administering to the subject at least one
pyrophosphate analog comprising the structure: 13where each X is
independently O, CH.sub.2, NH, or S; R.sup.1 is H, a small alkyl
group, guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol,
serine, threonine, tyrosine, arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2) and m is 1-3; R.sup.2 is H, a
small alkyl group, guanyl, adenylyl, glycerol, acyl glycerol,
diacyl glycerol, serine, threonine, tyrosine, or arachidonyl; and n
is 1-900; or the structure: 14where n=2-4; X is O; RCR.sup.1; CR; C
(n=4), CH (n=3), or CH.sub.2 (n=2); NH; N; S; and R and/or R.sup.1
is H, OH, a small alkyl group, or (CH.sub.2).sub.mNH.sub.2 where
m=1-6; or a dinucleoside-5-5'-pyrophosphate, a cyclopyrophosphate
of purine, a pyrimidine acyclonucleoside, an inositol diphosphate,
an inositol triphosphate, an inositol tetraphosphate, an inositol
pentaphosphate, an inositol hexaphosphate; or a pharmaceutically
acceptable salt thereof.
15. The method of claim 14, in which the metal is selected from the
group consisting of iron, copper, mercury, lead, cadmium, vanadium
and their alloys.
16. A method of treating bacterial, fungal, algo, or algae
infections in a subject in need thereof, comprising administering
to the subject at least one pyrophosphate analog comprising the
structure: 15where each X is independently O, CH.sub.2, NH, or S;
R.sup.1 is H, a small alkyl group, guanyl, adenylyl, glycerol, acyl
glycerol, diacyl glycerol, serine, threonine, tyrosine,
arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2) and m is 1-3; R.sup.2 is H, a
small alkyl group, guanyl, adenylyl, glycerol, acyl glycerol,
diacyl glycerol, serine, threonine, tyrosine, or arachidonyl; and n
is 1-900; or the structure: 16where n=2-4; X is O; RCR.sup.1; CR; C
(n=4), CH (n=3), or CH.sub.2 (n=2); NH; N; S; and R and/or R.sup.1
is H, OH, a small alkyl group, or (CH.sub.2).sub.mNH.sub.2 where
m=1-6; or a dinucleoside-5-5'-pyrophosphate, a cyclopyrophosphate
of purine, a pyrimidine acyclonucleoside, an inositol diphosphate,
an inositol triphosphate, an inositol tetraphosphate, an inositol
pentaphosphate, an inositol hexaphosphate; or a pharmaceutically
acceptable salt thereof.
17. The method of claim 16, in which the subject is a plant.
18. The method of claim 17, in which the pyrophosphate analog
comprises imidodiphosphate.
19. The method of claim 16, in which the subject is an animal or
mammal.
20. A method of reducing toxic actions of metal ions in a subject
in need thereof, comprising administering to the subject at least
one pyrophosphate analog comprising the structure: 17where each X
is independently O, CH.sub.2, NH, or S; R.sup.1 is H, a small alkyl
group, guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol,
serine, threonine, tyrosine, arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2) and m is 1-3; R.sup.2 is H, a
small alkyl group, guanyl, adenylyl, glycerol, acyl glycerol,
diacyl glycerol, serine, threonine, tyrosine, or arachidonyl; and n
is 1-900; or the structure: 18where n=2-4; X is O; RCR.sup.1; CR; C
(n=4), CH (n=3), or CH.sub.2 (n=2); NH; N; S; and R and/or R.sup.1
is H, OH, a small alkyl group, or (CH.sub.2).sub.mNH.sub.2 where
m=1-6; or a dinucleoside-5-5'-pyrophosphate, a cyclopyrophosphate
of purine, a pyrimidine acyclonucleoside, an inositol diphosphate,
an inositol triphosphate, an inositol tetraphosphate, an inositol
pentaphosphate, an inositol hexaphosphate; or a pharmaceutically
acceptable salt thereof.
21. The method of claim 20, in which the metal ion is selected from
the group consisting of Fe.sup.++, Hg.sup.++, Cd.sup.++, Cu.sup.++,
As.sup.+++, and Pb.sup.++.
22. A method of protecting a pharmacological agent in a
formulation, comprising combining the agent with at least one
pyrophosphate analog comprising the structure: 19where each X is
independently O, CH.sub.2, NH, or S; R.sup.1 is H, a small alkyl
group, guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol,
serine, threonine, tyrosine, arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2) and m is 1-3; R.sup.2 is H, a
small alkyl group, guanyl, adenylyl, glycerol, acyl glycerol,
diacyl glycerol, serine, threonine, tyrosine, or arachidonyl; and n
is 1-900; or the structure: 20where n=2-4; X is O; RCR.sup.1; CR; C
(n=4), CH (n=3), or CH.sub.2 (n=2); NH; N; S; and R and/or R.sup.1
is H, OH, a small alkyl group, or (CH.sub.2).sub.mNH.sub.2 where
m=1-6; or a dinucleoside-5-5'-pyrophosphate, a cyclopyrophosphate
of purine, a pyrimidine acyclonucleoside, an inositol diphosphate,
an inositol triphosphate, an inositol tetraphosphate, an inositol
pentaphosphate, an inositol hexaphosphate; or a pharmaceutically
acceptable salt thereof.
23. The method of claim 22, in which the pharmacological agent is a
therapeutic agent.
24. The method of claim 22, in which the pharmacological agent is a
diagnostic agent.
25. A method of increasing efficacy of a neurologic agent in a
subject in need thereof, comprising administering to the subject
the neurologic agent, and administering at least one pyrophosphate
analog comprising the structure: 21where each X is independently O,
CH.sub.2, NH, or S; R.sup.1 is H, a small alkyl group, guanyl,
adenylyl, glycerol, acyl glycerol, diacyl glycerol, serine,
threonine, tyrosine, arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2) and m is 1-3; R.sup.2 is H, a
small alkyl group, guanyl, adenylyl, glycerol, acyl glycerol,
diacyl glycerol, serine, threonine, tyrosine, or arachidonyl; and n
is 1-900; or the structure: 22where n=2-4; X is O; RCR.sup.1; CR; C
(n=4), CH (n=3), or CH.sub.2 (n=2); NH; N; S; and R and/or R.sup.1
is H, OH, a small alkyl group, or (CH.sub.2).sub.mNH.sub.2 where
m=1-6; or a dinucleoside-5-5'-pyrophosphate, a cyclopyrophosphate
of purine, a pyrimidine acyclonucleoside, an inositol diphosphate,
an inositol triphosphate, an inositol tetraphosphate, an inositol
pentaphosphate, an inositol hexaphosphate; or a pharmaceutically
acceptable salt thereof.
26. The method of claim 25, wherein the neurologic agent comprises
a ganglioside, a phosphatidylserine, a nerve growth factor, a
neurotrophin, a brain-derived neurotrophic factor, a fibroblast
growth factor, an insulin, an insulin-like growth factor, a
transforming growth factor, an epidermal growth factor, a
platelet-derived growth factor, a neurokine, activity-dependent
neurotrophic factor, a ciliary neurotrophic factor, a glia-derived
neurotrophic factor, a glia-derived nexin, a cholinergic enhancing
factor, an antisense oligonucleotide, a DNA or RNA vector or
plasmid that encodes one or more protein neurologic agents or nerve
growth promoting factors, or a combination thereof.
27. The method of claim 26, wherein the ganglioside comprises GM-1
ganglioside.
28. The method of claim 26, wherein the neurotrophin comprises
neurotrophin 3, neurotrophin 4, neurotrophin 5, or a combination
thereof.
29. The method of claim 26, wherein the fibroblast growth factor
comprises basic fibroblast growth factor, or acidic fibroblast
growth factor.
30. The method of claim 26, wherein the insulin-like growth factor
comprises insulin-like growth factor-I, insulin-like growth
factor-2, or a combination thereof.
31. The method of claim 26, wherein the cholinergic enhancing
factor comprises ethanolamine, thyroid hormone T.3, gallamine or a
combination thereof.
32. The method of any of claims 1, 4, 5, 12, 14, 16, 20, 22, or 25,
wherein the pyrophosphate analog comprises pyrophosphate,
imidodiphosphate, guanylimidodiphosphate, adenylylimidodiphosphate,
or a bisphosphonate.
33. The method of claim 32, wherein the pyrophosphate analog
comprises etidronic acid, pamidronic acid, or a combination
thereof.
34. The method of any of claims 1, 4, 5, 12, 14, 16, 20, 22, or 25,
wherein the subject suffers from at least one of cancer;
neuropathies, diseases, or disorders of the heart, smooth muscles,
blood, blood vessels, glands, or bones; hypertension; myocardial
infarction, ischemic heart disease, or congestive heart failure;
irritable bowel syndrome; diverticular disease; urinary
incontinence; esophageal achalasia; chronic obstructive airways
disease; cardiac arrhythmia; xerostomia; diabetes mellitus;
Sjogren's syndrome; Paget's disease; hereditary hematochromatosis;
or a non-CNS disorder resulting from normal aging.
35. The method of any of claims 1, 4, 5, 12, 14, 16, 20, 22, or 25,
wherein the subject suffers from at least one of a neurologic
disorder and a psychiatric disorder
36. The method of claim 35, wherein the subject suffers from at
least one of Alzheimer's disease, Parkinson's disease, Lewy body
dementia, multiple sclerosis, cerebellar ataxia, progressive
supranuclear palsy, amyotrophic lateral sclerosis, an affective
disorder, an anxiety disorder, schizophrenia, cell damage, nerve
damage, a CNS infection, a tumor of the brain, a tumor of the
spinal cord, a stroke in the brain, a stroke in the spinal cord, a
prion disease, a CNS disorder resulting from ordinary aging, a
brain injury, a spinal cord injury; or a non-CNS disorder resulting
from normal aging.
37. The method of any of claims 1, 4, 5, 12, 14, 16, 20, 22, or 25,
further comprising combining any pyrophosphate analog with at least
one of a bilirubin, biliverdin, carnosol, quercetin, myricetin, a
bioflavinoid, a combination thereof, or a pharmaceutically
acceptable salt thereof.
38. The method of claim 37, further comprising administering heme
oxygenase, a vector encoding a heme oxygenase, heme oxygenase-1, a
vector encoding a heme oxygenase-1, heme oxygenase-2, a vector
encoding a heme oxygenase-2, a biliverdin reductase, a vector
encoding a biliverdin reductase, a catalase, a vector encoding a
catalase, a peroxidase, a vector encoding a peroxidase, or a
combination thereof.
39. The method of claim 37, further comprising administering a heme
binding protein.
40. The method of claim 39, wherein the heme binding protein
comprises hemopexin, a lipoprotein, or a combination thereof.
41. The method of any of claims 1, 4, 5, 12, 14, 16, 20, 22, or 25,
further comprising administering heme oxygenase, a vector encoding
a heme oxygenase, heme oxygenase-1, a vector encoding a heme
oxygenase-1, heme oxygenase-2, a vector encoding a heme
oxygenase-2, a biliverdin reductase, a vector encoding a biliverdin
reductase, a catalase, a vector encoding a catalase, a peroxidase,
a vector encoding a peroxidase, or a combination thereof.
42. The method of any of claims 1, 4, 5, 12, 14, 16, 20, 22, or 25,
further comprising administering a heme binding protein.
43. The method of claim 42, wherein the heme binding protein
comprises hemopexin, a lipoprotein, or a combination thereof.
44. The method of any of claims 1, 4, 5, 12, 14, 16, 20, 22, or 25,
in which the compound is: 23and n is 1-6.
45. A method of protecting a tissue component in a subject in need
thereof, comprising administering to the subject a bilirubin,
biliverdin, carnosol, quercetin, myricetin, a bioflavinoid, a
combination thereof, or a pharmaceutically acceptable salt
thereof.
46. The method of claim 45, wherein the tissue component is at
least one of a receptor, a protein, a lipid, a nucleic acid, a
carbohydrate, a hormone, a vitamin, and a cofactor.
47. The method of claim 46, in which the tissue component is a
receptor for a neurotransmitter, a neuropeptide, a neurotrophin, a
growth factor, a steroid, a histamine, a purine, a benzodiazepine,
arachidonic acid, nitric oxide, carbon monoxide, an odorant, or an
ion channel.
48. A method of protecting tissue from oxidative stress, comprising
administering a bilirubin, biliverdin, carnosol, quercetin,
myricetin, a bioflavinoid, a combination thereof, or a
pharmaceutically acceptable salt thereof.
49. A method of increasing the efficacy of an agent that directly
or indirectly affects a receptor in a subject in need thereof,
comprising administering the agent, and administering a bilirubin,
biliverdin, carnosol, quercetin, myricetin, a bioflavinoid, a
combination thereof, or a pharmaceutically acceptable salt
thereof.
50. The method of claim 49, in which the receptor is a receptor for
a neurotransmitter, a neuropeptide, a neurotrophin, a growth
factor, a steroid, a histamine, a purine, a benzodiazepine,
arachidonic acid, nitric oxide, carbon monoxide, an odorant, or an
ion channel.
51. The method of claim 50, in which the receptor is one of
muscarinic acetylcholine, nicotinic acetylcholine, an opiate, a
catecholamine, serotonin, glutamate, aspartate, cannabinoid, gamma
aminobutyric acid, or glycine.
52. The method of claim 49, in which the agent directly or
indirectly affects a mAChR.
53. The method of claim 52, wherein the agent that directly or
indirectly affects a mAChR comprises an anticholinesterase agent, a
neurologic agent, or a muscarinic receptor agonist.
54. The method of claim 52, in which the agent that directly or
indirectly affects a mAChR comprises Xanomeline.
55. The method of claim 52, wherein the agent that directly or
indirectly affects a mAChR comprises donepezil, rivastigmine,
galanthamine, metrifonate, or a combination thereof.
56. A method of protecting a subject from at least one carcinogenic
metal, comprising administering to the subject a bilirubin,
biliverdin, carnosol, quercetin, myricetin, a bioflavinoid, a
combination thereof, or a pharmaceutically acceptable salt
thereof.
57. The method of claim 56, in which the carcinogenic metal is
selected from the group consisting of arsenic, cadmium, cobalt,
nickel, lead, and chromium.
58. A method of reducing poisoning of a subject by at least one
metal, comprising administering to the subject a bilirubin,
biliverdin, carnosol, quercetin, myricetin, a bioflavinoid, a
combination thereof, or a pharmaceutically acceptable salt
thereof.
59. The method of claim 56, in which the metal is selected from the
group consisting of iron, copper, mercury, lead, cadmium, and their
alloys.
60. A method of treating bacterial, fungal, algo, or algae
infections in a subject in need thereof, comprising administering
to the subject a bilirubin, biliverdin, carnosol, quercetin,
myricetin, a bioflavinoid, a combination thereof, or a
pharmaceutically acceptable salt thereof.
61. The method of claim 60, in which the subject is a plant.
62. The method of claim 60, in which the subject is an animal or
mammal.
63. A method of reducing toxic actions of metal ions in a subject
in need thereof, comprising administering to the subject a
bilirubin, biliverdin, carnosol, quercetin, myricetin, a
bioflavinoid, a combination thereof, or a pharmaceutically
acceptable salt thereof.
64. The method of claim 63, in which the metal ion is selected from
the group consisting of Fe.sup.++, Hg.sup.++, Cd.sup.++, Cu.sup.++,
As.sup.+++, and Pb.sup.++.
65. A method of protecting a pharmacological agent in a
formulation, comprising combining the agent with at least one of a
bilirubin, biliverdin, carnosol, quercetin, myricetin, a
bioflavinoid, a combination thereof, or a pharmaceutically
acceptable salt thereof.
66. The method of claim 65, in which the pharmacological agent is a
therapeutic agent.
67. The method of claim 65, in which the pharmacological agent is a
diagnostic agent.
68. A method of increasing efficacy of a neurologic agent in a
subject in need thereof, comprising administering to the subject
the neurologic agent, and administering a bilirubin, biliverdin,
carnosol, quercetin, myricetin, a bioflavinoid, a combination
thereof, or a pharmaceutically acceptable salt thereof.
69. The method of claim 68, wherein the neurologic agent comprises
a ganglioside, a phosphatidylserine, a nerve growth factor, a
neurotrophin, a brain-derived neurotrophic factor, a fibroblast
growth factor, an insulin, an insulin-like growth factor, a
transforming growth factor, an epidermal growth factor, a
platelet-derived growth factor, a neurokine, activity-dependent
neurotrophic factor, a ciliary neurotrophic factor, a glia-derived
neurotrophic factor, a glia-derived nexin, a cholinergic enhancing
factor, an antisense oligonucleotide, a DNA or RNA vector or
plasmid that encodes one or more protein neurologic agents or nerve
growth promoting factors, or a combination thereof.
70. The method of claim 69, wherein the ganglioside comprises GM-1
ganglioside.
71. The method of claim 69, wherein the neurotrophin comprises
neurotrophin 3, neurotrophin 4, neurotrophin 5, or a combination
thereof.
72. The method of claim 69, wherein the fibroblast growth factor
comprises basic fibroblast growth factor, or acidic fibroblast
growth factor.
73. The method of claim 69, wherein the insulin-like growth factor
comprises insulin-like growth factor-I, insulin-like growth
factor-2, or a combination thereof.
74. The method of claim 69, wherein the cholinergic enhancing
factor comprises ethanolamine, thyroid hormone T.3, gallamine or a
combination thereof.
75. The method of any of claims 45, 47, 48, 49, 56, 58, 60, 63, 65,
or 68, wherein the subject suffers from at least one of cancer;
neuropathies, diseases, or disorders of the heart, smooth muscles,
blood, blood vessels, glands, or bones; hypertension; myocardial
infarction, ischemic heart disease, or congestive heart failure;
irritable bowel syndrome; diverticular disease; urinary
incontinence; esophageal achalasia; chronic obstructive airways
disease; cardiac arrhythmia; xerostomia; diabetes mellitus;
Sjogren's syndrome; Paget's disease; hereditary hematochromatosis;
or a non-CNS disorder resulting from normal aging.
76. The method of any of claims 45, 47, 48, 49, 56, 58, 60, 63, 65,
or 68, wherein the subject suffers from at least one of a
neurologic disorder and a psychiatric disorder.
77. The method of claim 76, wherein the subject suffers from at
least one of Alzheimer's disease, Parkinson's disease, Lewy body
dementia, multiple sclerosis, cerebellar ataxia, progressive
supranuclear palsy, amyotrophic lateral sclerosis, an affective
disorder, an anxiety disorder, schizophrenia, cell damage, nerve
damage, a CNS infection, a tumor of the brain, a tumor of the
spinal cord, a stroke in the brain, a stroke in the spinal cord, a
prion disease, a CNS disorder resulting from ordinary aging, a
brain injury, a spinal cord injury, or a combination thereof.
78. The method of any of claims 45, 47, 48, 49, 56, 58, 60, 63, 65,
or 68, further comprising administering heme oxygenase, a vector
encoding a heme oxygenase, heme oxygenase-1, a vector encoding a
heme oxygenase-1, heme oxygenase-2, a vector encoding a heme
oxygenase-2, a biliverdin reductase, a vector encoding a biliverdin
reductase, a catalase, a vector encoding a catalase, a peroxidase,
a vector encoding a peroxidase, or a combination thereof.
79. The method of any of claims 45, 47, 48, 49, 56, 58, 60, 63, 65,
or 68, further comprising administering a heme binding protein.
80. The method of claim 79, wherein the heme binding protein
comprises hemopexin, a lipoprotein, or a combination thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of provisional
application No. 60/200,843 filed May 1, 2000; provisional
application No. 60/233,263 filed Sep. 6, 2000; and provisional
application No. 60/233,025 filed Sep. 15, 2000; each entitled
"Methods and Compositions for Enhancing Cellular Function and
Protecting Receptor."
BACKGROUND OF THE INVENTION
[0002] Cellular function depends on the maintenance of intact
cellular components including: receptors, proteins, lipids, nucleic
acids, carbohydrates, hormones and cofactors. Cellular receptors,
including cell surface receptors, mediate communication within and
between cells, tissues and organs within a living system. Cellular
receptors also provide a means to signal a living system, tissues,
organs, cells, and subcellular compartments. Receptors are
molecules or macromolecules that bind or interact with agents to
alter or enhance their function. Many receptors are membrane bound
proteins, which require not only that their protein structure be
intact but also that the membrane lipids and carbohydrates be
intact and functional. Through various signaling mechanisms, the
messages sent by the receptor, either in the presence or absence of
an interacting or bound agent, can be transmitted. Following
receptor activation, signalling also requires intact cellular
proteins, lipids, nucleic acids and carbohydrates in order for the
message to be properly received.
[0003] Often as a result of damage, the ability of cellular
receptors to interact with or bind various agents is decreased,
resulting in an impairment of vital intrinsic and extrinsic
communication. Damage to cellular receptors and other cellular
components diminishes the ability of a receptor to bind agents and
elicit a communication or signaling event. This can result in
damage or death to cells, resulting in damage or diseases of
tissues, organs and living systems. Accordingly, there is a need
for a means to protect receptors and other cellular components from
damage and to increase the efficacy of agents that exert their
effects through cellular receptors.
SUMMARY OF THE INVENTION
[0004] The invention provides methods for enhancing cellular
function through protection of tissue components and/or increasing
the efficacy of a therapeutic agent in a subject in need thereof.
The method includes administering a composition, such as a
pharmaceutical composition, of a pyrophosphate analog. In a second
embodiment, the method includes administering a composition, such
as a pharmaceutical composition, of a protective agent.
[0005] Preferably, the invention provides a method for protecting a
muscarinic acetylcholine receptor (mAChR) and/or increasing the
efficacy of an agent that directly or indirectly affects a mAChR in
a subject in need thereof. Suitable agents that directly or
indirectly affect a muscarinic receptor include anticholinesterase
agents, muscarinic agonists, allosteric regulators of a muscarinic
receptor, muscarinic antagonists, and neurotrophic and neuritogenic
factors that are similar to naturally occurring nerve growth
promoting substances. In one embodiment, the invention provides a
method to protect a mAChR and/or increase the efficacy of agents
that directly or indirectly affect a mAChR in the central nervous
system (CNS) of a subject in need thereof. Preferably, a muscarinic
receptor is protected from an endogenous low molecular weight
inhibitor from Alzheimer's brain tissue, a metal, or oxidative
stress. In another embodiment, the invention provides a method to
protect a mAChR and/or increase the efficacy of agents that
directly or indirectly affect a mAChR not in the CNS of a subject
in a need thereof. In a first embodiment, the method includes
administering a pyrophosphate analog. In a second embodiment, the
method includes administering a protective agent.
[0006] The invention also provides a method for increasing the
efficacy of a therapeutic agent, preferably a neurologic agent, in
a subject in need thereof. In a first embodiment, this method
includes administering a pyrophosphate analog. In this embodiment,
the increased efficacy of the neurologic agent preferably results
from protection of a muscarinic receptor caused or induced by the
pyrophosphate analog. In a second embodiment, this method includes
administering a protective agent. In this second embodiment, the
increased efficacy of the neurologic agent preferably results from
protection of a muscarinic receptor caused or induced by the
protective agent. In each embodiment, the subject preferably is
concurrently receiving, has recently received, or will soon receive
a neurologic agent such as nerve growth factor (NGF), insulin
growth factor (IGF-1), brain derived neurotrophic factor (BDNF),
fibroblast growth factor (FGF), and the like; certain other known
neurotrophins and neuroprotectants; and medications used for
stroke, Alzheimer's disease, Parkinson's disease, ALS, traumatic
brain or spinal cord injury, cancer, diabetes, neuropathies,
hypertension, irritable bowel syndrome; diseases or disorders of
the heart and smooth muscles, blood, blood vessels, glands or bones
and other disorders. Preferably, the therapeutic agent directly or
indirectly affects a mAChR. Such agents include an
anticholinesterase agent, a muscarinic agonist, and a muscarinic
antagonist.
[0007] Pyrophosphate analogs that can be employed in the
appropriate embodiment of the method of the invention include
compounds of Formula I: 1
[0008] where each X is independently O, CH.sub.2, NH, or S; R.sup.1
is H, a small alkyl group, guanyl, adenylyl, glycerol, acyl
glycerol, diacyl glycerol, serine, threonine, tyrosine,
arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2), and m is 1-3; R.sup.2 is H, a
small alkyl group, guanyl, adenylyl, glycerol, acyl glycerol,
diacyl glycerol, serine, threonine, tyrosine, or arachidonyl; and n
is 1-900. Compounds of Formula I in which R.sup.1 is a small alkyl
group, guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol,
serine, threonine, arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2); or R.sup.2 is a small alkyl
group, guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol,
serine, threonine, tyrosine, or arachidonyl can be referred to as
substituted pyrophosphate analogs. Compounds of Formula I can also
include substituted pyrophosphate analogs such as a
dinucleoside-5-5'-pyrophosphate, a cyclopyrophosphate of purine,
and a pyrimidine acyclonucleoside. The compound of Formula I can be
any pharmaceutically acceptable salt or basic addition salt.
Preferably, X is O, CH.sub.2, NH, or S; R.sup.1 is H; R.sup.2 is H;
and n is 1-6. More preferably the pyrophosphate analog is
pyrophosphate or imidodiphosphate.
[0009] Additional pyrophosphate analogs include compounds of
Formula II: 2
[0010] where n=2-4; X is O, RCR.sup.1; CR; C (n=4), CH (n=3), or
CH.sub.2 (n=2); NH; N; S; and R and/or R.sup.1 is H, OH, a small
alkyl group, such as CH.sub.3 or (CH.sub.2).sub.mNH.sub.2 where
m=1-6. Further included are bisphosphonic acids, which are also
known as bisphosphonates, where X is preferably RCR.sup.1, where R
and R.sup.1 groups are chosen independently from OH,
H.sub.2N(CH.sub.2).sub.2, or CH.sub.3. For example, RCR.sup.1 can
be H.sub.2N(CH.sub.2).sub.2C(OH) or CH.sub.3COH. More specifically,
the bisphosphonates include etidronic acid
((1-Hydroxyethylidene)bisphosp- honic acid) and pamidronic acid
((3-Amino-1-hydroxypropylidene)bisphosphon- ic acid) where
preferably n=2.
[0011] Yet more additional pyrophosphate analogs include
substituted pyrophosphate analogs such as an inositol diphosphate,
an inositol triphosphate, an inositol tetraphosphate, an inositol
pentaphosphate, and an inositol hexaphosphate.
[0012] Suitable protective agents that can be employed in an
embodiment of the method of the invention include a bilirubin,
biliverdin, carnosol, quercetin, myricetin, a bioflavinoid, a
combination thereof, or a pharmaceutically acceptable salt thereof;
a heme binding compound, such as hemopexin, lipopexin, a
lipoprotein, or ApoE-2; and a heme oxygenase, such as heme
oxygenase-1 or heme oxygenase-2, biliverdin reductase, a catalase,
a peroxidase, a vector encoding a biliverdin reductase, a vector
encoding a heme oxygenase (e.g. a vector encoding a heme
oxygenase-1 or a vector encoding a heme oxygenase-2), a vector
encoding a catalase, a vector encoding a peroxidase, or a
combination thereof Biliverdin reductase can be administered alone
or in combination with a heme oxygenase. Heme oxygenases include
recombinant heme oxygenase. Preferably, a heme oxygenase is a human
heme oxygenase.
[0013] The method of the invention can treat or prevent a CNS
disorder. Preferably, the method of the invention can treat or
prevent neurodegeneration, can improve memory and cognition, can
treat or prevent brain deterioration or cognitive and memory loss
associated with aging, or can treat or prevent Alzheimer's Disease,
Parkinson's disease, Lewy body dementia, multiple sclerosis,
cerebellar ataxia, progressive supranuclear palsy, amyotrophic
lateral sclerosis, affective disorders, anxiety disorders, and/or
schizophrenia; nerve damage from cerebrovascular disorders such as
stroke or atherosclerosis in the brain or spinal cord, from CNS
infections including meningitis and HIV, from tumors of the brain
and spinal cord, prion diseases, and CNS disorders resulting from
ordinary aging (e.g., anosmia), brain injury, or spinal cord
injury.
[0014] In another embodiment, the method of the invention can treat
or prevent a disease or disorder not of the CNS. Preferably, the
method of the invention can treat or prevent cancer, or
neuropathies or diseases or disorders of the heart, smooth muscles,
blood, blood vessels, glands, or bones. Such diseases or disorders
include hypertension, myocardial infarction, ischemic heart
disease, congestive heart failure, cardiac arrhythmias, cancer,
irritable bowel syndrome, diverticular disease, urinary
incontinence, esophageal achalasia, chronic obstructive airways
disease, xerostomia, diabetes mellitus, Sjogren's syndrome or dry
eye syndrome which involves decreased secretion of tears by, for
example, the lacrimal glands, Paget's disease, hereditary
hematochromatosis or a non-CNS disorder resulting from normal
aging.
[0015] In another embodiment, the method of the invention treats
infections, including (without limitation) bacterial, fungal, algo,
or algae infections. Such infections can occur in plants (for which
a preferred embodiment of the invention employs imidodiphosphate as
the pyrophosphate analog), animals, or mammals.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 illustrates protection of a mAChR by pyrophosphate.
Pyrophosphate protected the mAChR from inactivation by the
endogenous low molecular weight inhibitor. Pyrophosphate protected
the receptor from loss of antagonist (.sup.3H-QNB (quinulidinyl
benzilate)) binding.
[0017] FIG. 2 illustrates protection of a mAChR by pyrophosphate.
Pyrophosphate protected the mAChR from inactivation by heme and
peroxide. Pyrophosphate protected the receptor from loss of
antagonist (.sup.3H-QNB) binding.
[0018] FIG. 3 illustrates protection of a mAChR by pyrophosphate.
Pyrophosphate protected the mAChR from inactivation by the
endogenous low molecular weight inhibitor. Pyrophosphate protected
the receptor from loss of agonist (oxotremorine) binding.
[0019] FIG. 4 illustrates protection of a mAChR by
imidodiphosphate. Imidodiphosphate protected the mAChR from
inactivation by the endogenous low molecular weight inhibitor.
Imidodiphosphate protected the receptor from loss of antagonist
(.sup.3H-QNB) binding.
[0020] FIG. 5 illustrates protection of a mAChR by
guanylimidodiphosphate. Guanylimidodiphosphate protected the mAChR
from inactivation by the endogenous low molecular weight inhibitor.
Guanylimidodiphosphate protected the receptor from loss of
antagonist (.sup.3H-QNB) binding.
[0021] FIG. 6 illustrates protection of a mAChR by
adenylylimidodiphosphat- e. Adenylylimidodiphosphate protected the
mAChR from inactivation by the endogenous low molecular weight
inhibitor. Adenylylimidodiphosphate protected the receptor from
loss of antagonist (.sup.3H-QNB) binding.
[0022] FIG. 7 illustrates protection of a mAChR by
tripolyphosphate. Tripolyphosphate protected the mAChR from
inactivation by the endogenous low molecular weight inhibitor.
Tripolyphosphate protected the receptor from loss of antagonist
(.sup.3H-QNB) binding.
[0023] FIG. 8 illustrates protection of a mAChR by bilirubin.
Bilirubin protected the mAChR from inactivation by the endogenous
low molecular weight inhibitor. Bilirubin protected the receptor
from loss of antagonist (.sup.3H-QNB) binding.
[0024] FIG. 9 illustrates protection of a mAChR by bilirubin.
Bilirubin protected the mAChR from inactivation by heme and
peroxide. Bilirubin protected the receptor from loss of antagonist
(.sup.3H-QNB) binding.
[0025] FIG. 10 illustrates protection of a mAChR by bilirubin.
Bilirubin protected the mAChR from inactivation by the endogenous
low molecular weight inhibitor. Bilirubin protected the receptor
from loss of agonist (oxotremorine) binding.
[0026] FIG. 11 illustrates protection of a mAChR by biliverdin.
Biliverdin protected the mAChR from inactivation by the endogenous
low molecular weight inhibitor. Biliverdin protected the receptor
from loss of antagonist (.sup.3H-QNB) binding.
[0027] FIG. 12 illustrates protection of a mAChR by carnosol,
Carnosol protected the mAChR from inactivation by the endogenous
low molecular weight inhibitor. Carnosol protected the receptor
from loss of antagonist (.sup.3H-QNB) binding.
[0028] FIG. 13 illustrates protection of a mAChR by quercetin.
Quercetin protected the mAChR from inactivation by the endogenous
low molecular weight inhibitor. Quercetin protected the receptor
from loss of antagonist (.sup.3H-QNB) binding.
[0029] FIG. 14 illustrates protection of a mAChR by myricetin.
Myricetin protected the mAChR from inactivation by the endogenous
low molecular weight inhibitor. Myricetin protected the receptor
from loss of antagonist (.sup.3H-QNB) binding.
[0030] FIG. 15 illustrates protection of a mAChR by catalase.
Catalase protected the mAChR from inactivation by the endogenous
low molecular weight inhibitor. Catalase protected the receptor
from loss of antagonist (.sup.3H-QNB) binding.
[0031] FIG. 16 illustrates protection of a mAChR by catalase.
Catalase protected the mAChR from inactivation by heme and
peroxide. Catalase protected the receptor from loss of antagonist
(.sup.3H-QNB) binding.
[0032] FIG. 17 illustrates protection of a mAChR by a peroxidase.
The peroxidase protected the mAChR from inactivation by the
endogenous low molecular weight inhibitor. The peroxidase protected
the receptor from loss of antagonist (.sup.3H-QNB) binding.
[0033] FIG. 18 illustrates protection of a mAChR by a peroxidase.
The peroxidase protected the mAChR from inactivation by heme and
peroxide. The peroxidase protected the receptor from loss of
antagonist (.sup.3H-QNB) binding.
[0034] FIG. 19 illustrates protection of mAChR by pamidronate.
Pamidronate protected the mAChR from inactivation by the endogenous
low molecular weight inhibitor.
[0035] FIG. 20 illustrates protection of a mAChR by pyrophosphate.
Pyrophosphate protected the mAChR from damage by the metal lead in
the form of PbCl.sub.2. Pyrophosphate protected the receptor from
loss of antagonist (.sup.3H-QNB (quinulidinyl benzilate))
binding.
DETAILED DESCRIPTION OF THE INVENTION
[0036] Definitions
[0037] As used herein, "cholinesterase" refers to an enzyme capable
of hydrolyzing acetylcholine and includes acetylcholinesterase.
[0038] As used herein, "agonist" refers to an agent that binds to
or interacts with a receptor and elicits a response transduced
through the receptor. Agonist includes full agonists, partial
agonists, and inverse agonists. A full agonist is an agent that can
elicit a maximal response from a receptor. A partial agonist is an
agent that can elicit, at best, a less than maximal response from a
receptor. An inverse agonist is an agent that produces a response
that is opposite that of a full or partial agonist. For example, if
agonist binding or interaction with a receptor results in increased
concentration of cAMP within a cell, then inverse agonist binding
or interaction with the same receptor will result in a decreased
concentration of cAMP within the cell.
[0039] As used herein, "antagonist" refers to an agent that is
capable of partially or completely inhibiting, or reversing, the
effect of an agonist on a receptor.
[0040] As used herein, "allosteric modifier" refers to an agent
that binds or interacts with a site other than the agonist binding
site of a receptor and modifies the ability of an agonist or an
antagonist to elicit or inhibit, respectively, a response
transduced through a receptor, without itself eliciting a
response.
[0041] As used herein, "tissue component" includes receptors,
proteins, lipids, nucleic acids, carbohydrates, hormones, vitamins,
and cofactors.
[0042] As used herein, "receptor" refers to any molecule or
macromolecule within or on a cell that interacts with another
molecule or macromolecule to confer a response or transduce a
signal and includes nuclear receptors, mitochondrial receptors,
cytoplasmic receptors, and cell surface receptors. Receptors
include receptors for neurotrophins (including, without limitation,
nerve growth factor, neurotrophins 3, 4, and/or 5 (NT-3, NT-4
and/or NT-5) and brain derived growth factors); neurotransmitters;
hormones; steroids; local mediators such as nitric oxide, carbon
monoxide, histamine, and growth factors like insulin, insulin-like
growth factor-I, fibroblast growth factors, cilliary neurotrophic
factor, glia-derived neurotrophic factor, glia-derived nexin,
cholinergic enhancing factor, transforming growth factors, activity
dependent neurotrophic factor, neurokines, gangliosides,
phosphatidylserine, PDGF (platelet derived growth factor) and EGF
(epidermal growth factor); benzodiazepines; arachidonic acid;
purines (including, without limitation, adenosine and ATP);
nucleotides and cyclic nucleotides; calcium and other divalent
cations; odorants; antisense oligonucleotides; opiates;
cannabinoids; glutamate; melatonin; angiotensin II; secretin;
vasoactive intestinal peptide; cholecystokinin; ACTH; vasopressin;
thrombin; ion channels; and the like. Receptors also include but
are not limited to G-protein-coupled receptors, ion-channel-linked
receptors and enzyme-linked receptors.
[0043] As used herein, "protecting a receptor" refers to protecting
the physical integrity of a receptor and/or the function of a
receptor, such as enhancing the function of a receptor; or
maintaining the ability of the receptor to respond to agonists, to
respond to antagonists, to transmit a message to the interior of a
cell, or to send a signal within a cell, cell nucleus, or
mitochondria.
[0044] As used herein, "central nervous system" (CNS) refers to the
brain and spinal cord and associated tissues.
[0045] As used herein, "disorders and diseases of the CNS" refers
to brain diseases such as Alzheimer's disease, Parkinson's disease,
Lewy body dementia, multiple sclerosis, cerebellar ataxia,
progressive supranuclear palsy, amyotrophic lateral sclerosis,
affective disorders, anxiety disorders, and/or schizophrenia; cell
damage; nerve damage from cerebrovascular disorders such as stroke
in the brain or spinal cord, from CNS infections including
meningitis and HIV, from tumors of the brain and spinal cord, prion
diseases, and CNS disorders resulting from ordinary aging (e.g.,
anosmia), brain injury, or spinal cord injury.
[0046] As used herein, a disease or disorder that relates to or is
caused at least in part by dysfunction, alteration, or loss of one
or more G-protein coupled receptors refers to Alzheimer's disease;
Parkinson's disease; drug addiction, such as opiate addiction or
cannabinoid abuse; pain; Sjogren's or dry eye syndrome; heart
diseases including congestive heart failure, myocardial infarction,
cardiac arrhythmia; diseases of smooth muscle organs or glands such
as irritable bowel syndrome, colitis, hypertension, erectile
dysfunction, diabetes, obesity, blood coagulation disorders; and
the like.
[0047] An "effective amount" of agent is an amount sufficient to
prevent, treat, reduce and/or ameliorate the symptoms and/or
underlying causes of any of the above disorders or diseases. In
some instances, an "effective amount" is sufficient to eliminate
the symptoms of those diseases and, perhaps, overcome the disease
itself. Preferably, an effective amount of an agent yields a tissue
concentration in the range of about 10.sup.-7 molar to about
10.sup.-5 molar, but the concentrations may be greater provided
that toxicity is avoided.
[0048] In the context of the present invention, the terms "treat"
and "therapy" and the like refer to alleviate, slow the
progression, prophylaxis, attenuation or cure of existing disease.
Prevent, as used herein, refers to putting off, delaying, slowing,
inhibiting, or otherwise stopping, reducing or ameliorating the
onset of such diseases or disorders. It is preferred that a large
enough quantity of the agent be applied in non-toxic levels in
order to provide an effective level of activity against the
disease. The method of the present invention may be used with any
animal, such as a mammal or a bird (avian), more preferably a
mammal. Poultry are a preferred bird. Exemplary mammals include,
but are not limited to rats, cats, dogs, horses, cows, sheep, pigs,
and more preferably humans.
[0049] Protecting a Tissue Component
[0050] The invention provides a method for protecting any
biomolecule or tissue component including a protein, a lipid, a
nucleic acid, a carbohydrate, a hormone and the like. The invention
is best illustrated, but is not limited to, the example of the
protection of a receptor, preferably a muscarinic receptor,
preferably a mAChR. In a first embodiment, the method includes
administering a pyrophosphate analog. In a second embodiment, the
method includes administering a protective agent. Protecting a
receptor includes protecting the physical integrity of a receptor
and/or the function of a receptor, such as maintaining the ability
of the receptor to respond to agonists, to respond to antagonists,
to transmit a message to the interior of a cell, or to send a
signal within a cell, cell nucleus, or mitochondria.
[0051] An embodiment of the invention provides a method for
protecting a receptor from free radical damage. Free radicals and
other reactive oxygen species (e.g., H.sub.2O.sub.2, HOCl, and
radicals such as O.sub.2.sup.-, sulfur cation, nitric oxide
radical, ferryl, peroxyl, peroxynitrite, thiyl, thiylperoxyl, and
alkoxyl) are highly reactive, and many free radical reactions are
highly damaging to cellular components. Free radical reactions can
crosslink proteins, mutagenize DNA, and peroxidize lipids. Such
reactions can have deleterious effects on cellular receptors.
Preferably, the method of the invention includes protection of a
receptor, such as a mAChR, or of DNA, RNA, lipids, and proteins
necessary for receptor function from deleterious effects.
[0052] In another embodiment, the invention provides a method for
reducing or eliminating deleterious effects of an endogenous
inhibitor found in elevated levels in the brains of Alzheimer's
disease patients. This endogenous, low molecular weight inhibitor,
as it is known, inhibits agonist and antagonist binding to mAChRs.
This inhibitor has a molecular weight of less than 3500 Da and is
believed to generate free radicals, in the presence of glutathione
or other sulthydryl compounds, that irreversibly inhibit or
inactivate the mAChR The inhibitor also contains free heme, which
can generate free radicals, including superoxide radicals, peroxyl
radicals, and thiyl radicals, and can cause neurotoxicity. Heme has
been shown to damage protein and lipid components of membranes by
Vincent (Oxidative Effects of Heme and Porphyrins on Proteins and
Lipids, Seminars in Hematology 26(2): 105-113, 1989). Membrane
lipid defects have been demonstrated in Alzheimer's disease by
Ginsberg et al. (Evidence for a Membrane Lipid Defect in
Alzheimer's Disease, Mol. and Chem. Neuropathol. 19: 37-46, 1993).
In addition, heme has been proposed to contribute to
atherosclerosis by Jacob (Newly recognized causes of
atherosclerosis: The role of microorganisms and of vascular iron
overload, J. Lab. Clin. Med. 123: 808-816, 1994).
[0053] In one embodiment, the method of the invention includes
increasing the efficacy of an agent that directly or indirectly
affects a mAChR. By way of example, administration of a
pyrophosphate analog can increase the efficacy of a muscarinic
agonist in the presence of the inhibitor. In another embodiment,
the method of the invention includes reducing or eliminating
deleterious effects the low molecular weight inhibitor or heme by
decreasing or preventing the generation of free radicals or
trapping radicals once formed.
[0054] Receptors
[0055] The invention provides a method for protecting a receptor
and/or increasing the efficacy of agents that directly or
indirectly affect a receptor. Such receptors include
G-protein-coupled receptors, ion-channel-linked receptors and
enzyme-linked receptors. Examples include receptors for
neurotrophins; neurotransmitters; hormones; steroids; local
mediators such as nitric oxide, histamine, and growth factors like
PDGF (platelet derived growth factor) and EGF (epithelial growth
factor); nucleotides and cyclic nucleotides; calcium and other
divalent cations; odorants; antisense oligonucleotides; and the
like. Preferably the receptor is a muscarinic receptor. Examples of
G-protein coupled receptors include receptors that respond to
odorants, opiates, cannabinoids, glutamate, melatonin, angiotensin
II, secretin, vasoactive intestinal peptide (VIP), cholecystokinin
(CCK), adrenaline (adrenergic receptors), acetylcholine (muscarinic
receptors), ACTH, vasopressin, thrombin, and the like.
[0056] In one embodiment the invention provides a method for
protecting a receptor and/or increasing the efficacy of agents that
directly or indirectly affect a receptor in the CNS. In another
embodiment, the invention provides a method for protecting a
receptor and/or increasing the efficacy of agents that directly or
indirectly affect a receptor not in the CNS. Agents whose efficacy
are increased by the method of the invention include receptor
agonists, allosteric modifiers of receptors, and receptor
antagonists.
[0057] An embodiment of the invention provides a method for
treating or preventing a disease or disorder that relates to or is
caused at least in part by dysfunction, alteration, or loss of one
or more G-protein coupled receptors. These diseases and disorders
include Alzheimer's disease; Parkinson's disease; stroke; multiple
sclerosis; ALS; drug addiction, such as opiate addiction or
cannabinoid abuse; pain; Sjogren's or dry eye syndrome; heart
diseases including congestive heart failure, myocardial infarction,
cardiac arrhythmia; cancer; diseases of smooth muscle organs or
glands such as irritable bowel syndrome, colitis, hypertension,
erectile dysfunction, diabetes, obesity, blood coagulation
disorders; and the like.
[0058] Muscarinic Receptors
[0059] The invention provides a method for protecting a mAChR
and/or increasing the efficacy of agents that directly or
indirectly affect a mAChR. There are at least five pharmacological
classes of mAChRs, including the M1, M2, and M3 muscarinic
receptors, and several genetic subclasses including m1, m2, m3, m4,
and m5. These muscarinic receptors are G-protein coupled receptors.
Each receptor subtype has its own unique pattern of expression
throughout various tissues. As such, dysfunction of each receptor
subclass, or combinations thereof, may have deleterious effects
leading to a variety of diseases or disorders. The method of the
invention can provide protection to a muscarinic receptor in any or
several mAChR subclasses, and therefore, can be of benefit to those
at risk or suffering from diseases associated with dysfunction of
one or more muscarinic receptor subtype. Preferably, the method of
the invention provides protection to M1 and M2 muscarinic
receptors.
[0060] Muscarinic receptors mediate numerous of the inhibitory and
excitatory effects of the neurotransmitter acetylcholine in the
heart, smooth muscle, blood vessels, glands and in neurons (both
presynaptic and postsynaptic) in the autonomic and the central
nervous system. Dysfunction of mAChRs thus can contribute to a
variety of diseases and/or disorders. Through protection of a mAChR
and/or through increasing the efficacy of agents that directly or
indirectly affect a mAChR, the method of the invention can provide
benefit to subjects suffering from or at risk of a disease or
disorder associated with mAChR dysfunction.
[0061] An embodiment of the invention provides a method for
protecting a mAChR and/or increasing the efficacy of agents that
directly or indirectly affect a mAChR in the nervous system of a
subject, and therefore, can be of benefit to subjects suffering
from or at risk of central nervous system or peripheral nervous
system disorders. For example, mAChRs and other receptors are
involved in the regulation of the function of cells throughout the
CNS. Accordingly, the method of the invention can provide benefit
to subjects suffering from or at risk of CNS disorders such as
Alzheimer's disease, Parkinson's disease, Lewy body dementia,
multiple sclerosis, cerebellar ataxia, progressive supranuclear
palsy, amyotrophic lateral sclerosis, affective disorders, anxiety
disorders, and/or schizophrenia; nerve damage from cerebrovascular
disorders such as stroke, from CNS infections including meningitis
and HIV, from tumors of the brain and spinal cord, prion diseases,
and CNS disorders resulting from ordinary aging, brain injury, or
spinal cord injury. mAChRs are also involved in the regulation of
the function of cells in the peripheral nervous system, including
the autonomic nervous system. Accordingly, the method of the
invention can provide benefit to subjects suffering from or at risk
of peripheral nervous system disorders, such as peripheral
neuropathy, including that associated with diabetes. For example,
in diabetic patients nerves can deteriorate as blood vessels that
contain muscarinic or other receptors are lost. Preferably, the
method of the invention can benefit a subject suffering from or at
risk of Alzheimer's disease.
[0062] Another embodiment of the invention provides a method for
protecting a mAChR and/or increasing the efficacy of agents that
directly or indirectly affect a mAChR not within the nervous system
of a subject, and therefore, can be of benefit to subjects
suffering from or at risk of disease or disorder outside the
nervous system. For example, mAChRs are involved in the regulation
(e.g., stimulation or inhibition) of smooth muscle contraction, the
regulation of heart rate and cardiac contractility, the regulation
of secretion of enzymes or hormones, including the release of
amylase from the parotid gland and the release of digestive enzymes
and insulin from the pancreas, the regulation of bone growth, and
the regulation of iron metabolism. Accordingly, the method of the
invention can provide benefit to subjects suffering from or at risk
of smooth muscle related disorders such as irritable bowel
syndrome, diverticular disease, urinary incontinence, esophageal
achalasia, diseases or disorders of the blood vessels (e.g.
hypertension), or chronic obstructive airways disease; heart muscle
related disorders such as pathologic bradycardia or tachycardia,
arrhythmia, flutter or fibrillation; blood related disorders such
as hereditary hematochromatosis; bone disorders such as Paget's
disease; cancer; and gland related disorders such as xerostomia,
diabetes mellitus, or Sjogren's syndrome or dry eye syndrome which
involves decreased secretion of tears by, for example, the lacrimal
glands. For example, tear secretion is known to require muscarinic
cholinergic stimulation and intact muscarinic receptors.
Accordingly, protection of a mAChR and/or increasing the efficacy
of an agent that directly or indirectly affects a mAChR can be of
benefit to a subject suffering from Sjogren's syndrome or dry eye
syndrome.
[0063] Protecting a muscarinic receptor can result in enhancing the
effectiveness of agents that directly or indirectly affect a mAChR.
Useful agents that affect a mAChR include, but are not limited to,
anticholinesterase agents, muscarinic agonists, muscarinic
antagonists, and other agents useful for treatment of diseases
associated with dysfunction of muscarinic receptors, including
neurodegenerative and other CNS disorders.
[0064] The method of the invention also provides enhanced efficacy
of agents that do not act directly, or indirectly, with a mAChR.
Such enhanced efficacy can be achieved, for example, through
protection of a receptor, preferably a mAChR. Protecting a
muscarinic receptor can result in enhancing the efficacy of agents
that do not exert their action directly or indirectly on the
muscarinic receptor. Such enhanced efficacy can be achieved through
desirable effects on cells to which protection of muscarinic
receptors provides benefit. Typically, cells that derive benefit
from protection of a muscarinic receptor are cells that contain
muscarinic receptors. Examples of cells that contain a mAChR
include particular neurons, smooth muscle cells, and gland
cells.
[0065] Cells that lack a muscarinic receptor but which interact
with enzymes, hormones, and/or other compounds released from cells
with a muscarinic receptor can derive benefit from protecting a
muscarinic receptor. Examples of cells lacking a mAChR that can
derive benefit from protecting a mAChR include cells that are
presynaptic or postsynaptic relative to cells that contain a mAChR
and cells that can interact with enzymes, hormones, and/or other
compounds released from cells that contain a mAChR.
[0066] Examples of this phenomenon include: Stimulation of m.sub.2
receptors on presynaptic membranes increases the release of
acetylcholine which can then stimulate nicotinic receptors on other
post synaptic cells. Stimulation of mAChR releases arachidonic
acid, which can then affect a variety of other nearby brain cells.
Arachidonic acid also increases secretion of amyloid precursor
protein. Emmerling, M. R. et al. (1996) Ann. N.Y. Acad. Sci.
777:310-315. Activation of m1 and m3 mAChR attenuates release of
amyloid B protein. Hung, A. Y. et al. (1993) J. Biol. Chem.
268:22959-22962. Stimulation of mAChR is required for memory and
learning which also involve the proper function of noncholinergic
cells. Stimulation of mAChR can increase the nitric oxide-cyclic
GMP signaling system in neurons (Bauer, M. B. (1994) Neuroscience
62:351-359) and nitric oxide can travel from one cell to another to
produce its effects. Stimulation of mAChRs markedly increase
hippocampal BDNF and NGF in RNA levels. Once produced, these
neurotrophins can produce important effects on other nerve cells in
the brain. M. da Penha Berzaghi (1993) J. Neuroscience 13(9)
3818-3826.
[0067] By way of further example, some cells containing a mAChR in
the pancreas can release insulin. Released insulin can then
interact with cells in close proximity to, or at relatively great
distances from, the cell from which it was released. Protection of
a mAChR on a cell in the pancreas that releases insulin can have
beneficial effects on a cell that interacts with insulin. Thus,
cells lacking a mAChR can benefit from protection of a mAChR.
Similarly, the efficacy of agents acting on cells lacking a mAChR
can be enhanced by the method of the invention.
[0068] By way of yet further example, stimulation of mAChR in
certain brain cells increases potassium ion evoked release of the
neurotransmitter dopamine which then goes on to affect other brain
cells having dopamine receptors. Joseph, J. A. et al. (1995) Brain
Res. 673:195-193. Thus, CNS cells lacking a mAChR can benefit from
protection of a mAChR. Similarly, the efficacy of agents acting on
CNS cells lacking a mAChR can be enhanced by the method of the
invention.
[0069] Pyrophosphate Analogs
[0070] In one embodiment, the method of the invention provides
protection to a receptor and/or increases the efficacy of agents by
administering to a subject a pyrophosphate analog. Useful
pyrophosphate analogs include compounds of Formula I: 3
[0071] where each X is independently O, CH.sub.2, NH, or S; R.sup.1
is H, a small alkyl group, guanyl, adenylyl, glycerol, acyl
glycerol, diacyl glycerol, serine, threonine, tyrosine,
arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2), and m is 1-3; R.sup.2 is H, a
small alkyl group, guanyl, adenylyl, glycerol, acyl glycerol,
diacyl glycerol, serine, threonine, tyrosine, or arachidonyl; and n
is 1-900. Compounds of Formula I in which R.sup.1 is a small alkyl
group, guanyl, adenylyl, glycerol, acyl glycerol, diacyl glycerol,
serine, threonine, arachidonyl, --PO(OH)(OR.sup.2), or
--(PO(OH)O).sub.m--PO(OH)(OR.sup.2); or R.sup.2 is H, guanyl,
adenylyl, glycerol, acyl glycerol, diacyl glycerol, serine,
threonine, tyrosine, or arachidonyl can be referred to as
substituted pyrophosphate analogs. Compounds of Formula I can also
include substituted pyrophosphate analogs such as
dinucleoside-5-5'-pyrop- hosphates, cyclophosphates of purine and
pyrimidine acyclonucleosides. The compound of Formula I can be any
pharmaceutically acceptable salt or basic addition salt.
Preferably, X is O, CH.sub.2, NH, or S; R.sup.1 is H; and n is 2-6.
More preferably the pyrophosphate analog is pyrophosphate or
imidodiphosphate.
[0072] Additional preferred compounds of Formula I include
pyrophosphate, glycerol pyrophosphate, arachidonylpyrophosphate,
imidodiphosphate, serine phosphate, serine imidophosphate,
threonine phosphate, threonine imidophosphate,
guanylimidodiphosphate and adenylylimidodiphosphate. More
preferably compounds of Formula I include pyrophosphate,
imidodiphosphate, guanylimidodiphosphate and
adenylylimidodiphosphate.
[0073] Additional pyrophosphate analogs include compounds of
formula II: 4
[0074] where n=2-4; X is O; RCR.sup.1; CR; C (n=4), CH (n=3), or
CH.sub.2 (n=3); NH; N; S; and R and/or R.sup.1 is H, OH, a small
alkyl group (such as CH.sub.3), or (CH.sub.2).sub.mNH.sub.2 where
m=1-6. Further included are bisphosphonic acids, which are also
known as bisphosphonates, where X is preferably RCR.sup.1 and R and
R.sup.1 groups are chosen independently from OH,
H.sub.2N(CH.sub.2).sub.2, or CH.sub.3. For example, RCR.sup.1 can
be H.sub.2N(CH.sub.2).sub.2C(OH) or CH.sub.3COH. More specifically,
the bisphosphonates include etidronic acid
((1-Hydroxyethylidene)bisphosp- honic acid) and pamidronic acid
((3-Amino-1-hydroxypropylidene)bisphosphon- ic acid) where
preferably n=2.
[0075] Yet more additional pyrophosphate analogs include
substituted pyrophosphate analogs such as inositol diphosphate,
inositol triphosphate, inositol tetraphosphate, inositol
pentaphosphate, and inositol hexaphosphate.
[0076] Such pyrophosphate and imidodiphosphate compounds and the
like can be prepared as basic addition salts, such as sodium,
potassium, or magnesium salts. It is believed that the use of a
basic addition salt, such as a magnesium salt, will reduce the
charge and allow for freer movement of the compound throughout the
body. Pyrophosphate compounds, imidopyrophosphate compounds, and
the like can be covalently bound to other phosphates creating
polyphosphates or polyimidophosphates. One or more pyrophosphate
analogs can be administered in combination. In another embodiment,
the pyrophosphate analog can be administered with a protective
agent. In another embodiment, the pyrophosphate analog can be
administered with a neurologic agent, and optionally with a
protective agent.
[0077] Protective Agents
[0078] In another embodiment, the invention provides a method for
protecting receptors and/or increasing the efficacy of agents by
administering to a subject a protective agent. Protective agents
useful in an embodiment of the method of the invention include a
bilirubin, biliverdin, carnosol, quercetin, myricetin, a
bioflavinoid; a heme binding compound, such as hemopexin,
lipopexin, a lipoprotein, or ApoE-2; and a heme oxygenase, such as
heme oxygenase-1 or heme oxygenase-2, or biliverdin reductase, a
catalase, a peroxidase, a DNA or RNA vector encoding a biliverdin
reductase, a DNA or RNA vector encoding a heme oxygenase (e.g. a
DNA or RNA vector encoding a heme oxygenase-1 or a DNA or RNA
vector encoding a heme oxygenase-2), a DNA or RNA vector encoding a
catalase, a DNA or RNA vector encoding a peroxidase, or a
combination thereof Biliverdin reductase is preferably administered
with bilirubin, because biliverdin reductase can regenerate
bilirubin from biliverdin after bilirubin has been oxidized while
functioning as a protective agent. Biliverdin reductase is also
preferably administered in combination with a heme oxygenase. Heme
oxygenases include recombinant heme oxygenase. Preferably, a heme
oxygenase is a human heme oxygenase.
[0079] One or more protective agents can be administered in
combination. In another embodiment, one or more protective agents
can be administered in combination with one or more pyrophosphate
analogs. In another embodiment, one or more protective agents can
be administered in with one or more neurologic agents, and
optionally with one or more pyrophosphate analogs.
[0080] Agents that Directly or Indirectly Affect a mAChR
[0081] The invention also provides a method for enhancing the
efficacy of one or more agents that directly or indirectly affect a
mAChR. Agents that directly or indirectly affect a mAChR include
agents that (1) bind to or interact with a mAChR to either elicit a
response transduced through a mAChR or reduce or prevent an agent
from binding to or interacting with a mAChR and/or eliciting a
signal transduced through a mAChR, (2) alter the concentration of
agents that bind to or interact with a mAChR to either elicit a
response transduced through a mAChR or reduce or prevent an agent
from binding to or interacting with a mAChR and/or eliciting a
signal transduced through a mAChR, or (3) modify the ability of
agents that bind to or interact with a mAChR to either elicit a
response transduced through a mAChR or reduce or prevent an agent
from binding to or interacting with a mAChR and/or eliciting a
signal transduced through a mAChR. Such agents include
anticholinesterase agents, muscarinic agonists, muscarinic
antagonists, and allosteric modifiers of muscarinic receptors.
Preferably, the invention provides a method for enhancing the
efficacy of agents that either directly or indirectly elicit a
response through a mAChR. Most preferably, the method of the
invention enhances the efficacy of a muscarinic agonist or an
anticholinesterase agent.
[0082] Muscarinic receptor agonists directly elicit a response
through a mAChR by binding to and transducing a signal through a
mAChR. Preferred muscarinic agonists include acetylcholine,
Xanomeline, and the like.
[0083] A muscarinic antagonist is an agent that is capable of
partially or completely inhibiting, or reversing, the effect of a
muscarinic agonist on a mAChR. Examples of muscarinic antagonists
include atropine, N-methyl-scopolamine, quinuclidinyl benzilate,
pirenzepine, and the like.
[0084] Cholinesterase hydrolyzes the neurotransmitter acetylcholine
and provides one of the mechanisms responsible for rapid depletion
of acetylcholine from the synaptic cleft. Anticholinesterase agents
inhibit cholinesterase activity and as a result increase the
concentration of acetylcholine in the synaptic cleft and prolong
the duration of which acetylcholine remains in the synaptic cleft.
Anticholinesterase agents can thus indirectly affect a mAChR by
increasing concentrations of and prolonging the effective duration
of acetylcholine in the synaptic cleft. Anticholinesterase agents
can also interact directly with cholinergic receptors, including
mAChRs; with sodium and potassium ion channels; and effect the
uptake, synthesis and release of neurotransmitters. Preferred
anticholinesterase agents include Aricept, Exelon, Metrifonate, and
the like.
[0085] An allosteric modifier of a mAChR binds or interacts with a
site other than the agonist binding site of a mAChR and modifies
the ability of an agonist or an antagonist to elicit or inhibit,
respectively, a response transduced through a muscarinic receptor,
without itself eliciting a response. Suitable allosteric modifiers
of a muscarinic agonist include gallamine and dynorphin. Preferred
allosteric modifiers of a mAChR include gallamine and
dynorphin.
[0086] Neurologic Agents
[0087] The invention also provides a method for increasing the
efficacy of a neurologic agent in a subject in need thereof In a
first embodiment, this method includes administering a
pyrophosphate analog. In this embodiment, the increased efficacy of
the neurologic agent preferably results from protection of a
muscarinic receptor caused or induced by the pyrophosphate analog.
In a second embodiment, this method includes administering a
protective agent. In this second embodiment, the increased efficacy
of the neurologic agent preferably results from protection of a
muscarinic receptor caused or induced by the protective agent. In
each embodiment, the subject preferably is concurrently receiving,
has recently received, or will soon receive a neurologic agent.
[0088] A neurologic agent promotes nerve cell growth and survival
or augments the activity of functioning cells. Among those agents
that are preferred are cholinergic agonists, allosteric modifiers
of a mAChR, cholinesterase inhibitors, or neurotrophic and
neuritogenic factors that are similar to naturally occurring nerve
growth promoting substances. Among the preferred neurologic agents
are gangliosides (such as GM-I ganglioside), phosphatidylserine
(PS), nerve growth factor (NGF), neurotrophins 3, 4, and/or 5(NT-3,
NT-4 and/or NT-5) brain-derived neurotrophic factor (BDNF),
fibroblast growth factors (FGFs, e.g., basic fibroblast growth
factor), insulin, insulin-like growth factors (IGF-1 and/or IGF-2),
ciliary neurotrophic factor (CNTF), transforming growth factors,
epidermal growth factors, activity-dependent growth factor,
platelet derived growth factor, neurokine, glia-derived
neurotrophic factor (GDNF), glia-derived nexin, and cholinergic
enhancing factors such as phosphoethanolamine and thyroid hormone
T.3, and DNA or RNA vectors or plasmids that encode one or more
protein neurologic agents or nerve growth promoting factors.
Plasmids and vectors for delivery of a coding sequence to a
mammalian tissue are known to those of skill in the art.
[0089] Metal Diseases
[0090] The method of the invention can treat or prevent diseases or
disorders caused or induced by metals, such as cancer and
poisoning. Such metals can include As, Co, Cr, Ni, Hg, Pb, Fe, Cu,
V, and Cd. That is, the method of the invention can treat or
prevent poisoning by (for example) lead or mercury and also
excessive iron toxicity. In one embodiment, the method of the
invention can treat or prevent CNS diseases or disorders caused or
induced by metals. In another embodiment, the method of the
invention can treat or prevent diseases or disorders not of the CNS
but caused or induced by metals, such as heart disease, blood
vessel disease, and gland disease. In another embodiment, the
method of the invention reduces poisoning of a subject by at least
one metal. In another embodiment, the method of the invention
protects a subject from at least one carcinogenic metal. In another
embodiment, the method of the invention reduces toxic actions of
metal ions in a subject, particularly toxic actions due to
Fe.sup.++, Hg.sup.++, Cd.sup.++, Cu.sup.++, As.sup.+++, and
Pb.sup.++ ions.
[0091] Administering Agents
[0092] Administering compounds according to the method of the
invention can include formulating the compounds or compositions as
pharmaceutical compositions and administering the pharmaceutical
compositions to a mammalian host, including a human patient, in a
variety of forms adapted to the chosen route of administration. The
compounds are preferably administered in combination with a
pharmaceutically acceptable carrier. The compounds can be
administered at one of a variety of doses sufficient to provide an
effective amount at the desired point of action of the agent. Doses
for humans and other mammals can range from about 0.001 mg/kg to
about 100 mg/kg, preferably from about 0.01 mg/kg to about 10
mg/kg, preferably from about 0.1 mg/kg to about 1-10 mg/kg.
[0093] A related use of the methods of the invention is to protect
pharmacological agents in formulation. The pharmacological agents
may be for therapeutic, diagnostic, or other purposes.
[0094] The compounds can be administered by known techniques, such
as orally, intranasally, parentally (including subcutaneous
injection, intravenous, intramuscular, intrasternal or infusion
techniques), by inhalation spray, dermally, transdermally,
intrathecal, intracerebroventricular, buccal, sublingual,
topically, by absorption through a mucous membrane or through the
skin, or rectally, in dosage unit formulations containing
conventional non-toxic pharmaceutically acceptable carriers,
adjuvants or vehicles. Pharmaceutical compositions of the invention
can be in the form of suspensions or tablets suitable for oral
administration, nasal sprays, eye drops, nose drops, creams,
sterile injectable preparations, such as sterile injectable aqueous
or oleagenous suspensions or suppositories.
[0095] Controlled or sustained release systems can also be
employed. For example, compositions can include a polymer or other
substance that enhances controlled or sustained release. Controlled
or sustained release systems can include a polymer disk, such as
evac disks, microspheres, and copolymers. Preferred controlled
release polymers are poly(lactide:glycolide) and
poly(ethylene-co-vinyl acetate).
[0096] For oral administration as a suspension, the compositions
can be prepared according to techniques well known in the art of
pharmaceutical formulation. The compositions can contain
microcrystalline cellulose for imparting bulk, alginic acid or
sodium alginate as a suspending agent, methylcellulose as a
viscosity enhancer, and sweeteners or flavoring agents. As
immediate release tablets, the compositions can contain
microcrystalline cellulose, starch, magnesium stearate and lactose
or other excipients, binders, extenders, disintegrants, diluents
and lubricants known in the art.
[0097] In addition to the typical pharmacological methods for oral
administration, the agents employed in the methods of the invention
can be administered as a component of a nutritional or food
supplement. The nutritional or food supplement can also include any
other ingredients typical of a nutritional or food supplement, such
as flavorings, stabilizers, and the like.
[0098] For administration by inhalation or aerosol, the
compositions can be prepared according to techniques well known in
the art of pharmaceutical formulation. The compositions can be
prepared as solutions in saline, using benzyl alcohol or other
suitable preservatives, absorption promoters to enhance
bioavailability, fluorocarbons or other solubilizing or dispersing
agents known in the art.
[0099] For administration as injectable solutions or suspensions,
the compositions can be formulated according to techniques
well-known in the art, using suitable dispersing or wetting and
suspending agents, such as sterile oils, including synthetic mono-
or diglycerides, and fatty acids, including oleic acid.
[0100] For intranasal administration, the compositions can be
formulated according to techniques well known in the art. The means
of applying a pharmaceutical composition intranasally can be in a
variety of forms such as a powder, spray or nose drops.
[0101] For transdermal administration, the compositions can be
formulated according to techniques well known in the art. Delivery
of the composition through the skin can be accomplished by delivery
means well known in the art, including transdermal patch, an
ointment, an iontophoretic patch or device, and the like.
[0102] For rectal administration as suppositories, the compositions
can be prepared by mixing with a suitable non-irritating excipient,
such as cocoa butter, synthetic glyceride esters or polyethylene
glycols, which are solid at ambient temperatures, but liquefy or
dissolve in the rectal cavity to release the drug.
[0103] Preferred administration routes include orally,
parenterally, as well as intravenous, intramuscular or subcutaneous
routes.
[0104] Intraocular administration through the use of an ointment or
eye drops is preferred for treatment of a glandular disease or
disorder of the eye, such as a lachrymal gland disease disorder
like Sjogren's Syndrome or dry eye syndrome.
[0105] Solutions or suspensions of the compounds can be prepared in
water, isotonic saline (PBS) and optionally mixed with a nontoxic
surfactant. Dispersions may also be prepared in glycerol, liquid
polyethylene, glycols, DNA, vegetable oils, triacetin and mixtures
thereof Under ordinary conditions of storage and use, these
preparations may contain a preservative to prevent the growth of
microorganisms.
[0106] The pharmaceutical dosage form suitable for injection or
infusion use can include sterile, aqueous solutions or dispersions
or sterile powders including an active ingredient which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions. In all cases, the ultimate
dosage form should be sterile, fluid and stable under the
conditions of manufacture and storage. The liquid carrier or
vehicle can be a solvent or liquid dispersion medium including, for
example, water, ethanol, a polyol such as glycerol, propylene
glycol, or liquid polyethylene glycols and the like, vegetable
oils, nontoxic glyceryl esters, and suitable mixtures thereof. The
proper fluidity can be maintained, for example, by the formation of
liposomes, by the maintenance of the required particle size, in the
case of dispersion, or by the use of nontoxic surfactants. The
prevention of the action of microorganisms can be accomplished by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be desirable to include isotonic agents, for
example, sugars, buffers, or sodium chloride. Prolonged absorption
of the injectable compositions can be brought about by the
inclusion in the composition of agents delaying absorption--for
example, aluminum monostearate hydrogels and gelatin.
[0107] Sterile injectable solutions are prepared by incorporating
the compounds in the required amount in the appropriate solvent
with various other ingredients as enumerated above and, as
required, followed by filter sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying techniques, which yield a powder of the active
ingredient plus any additional desired ingredient present in the
previously sterile-filtered solutions.
[0108] Administering Agents to the Brain
[0109] Administering agents, e.g. a protective agent, a
pyrophosphate analog, an agent that directly or indirectly affects
a mAChR, and/or a neurologic agent, according to the method of the
invention includes administering agents to a mammalian host in a
manner that allows the agents to exert their effect in the CNS.
Many agents useful for the method of the invention can be absorbed
into the blood stream and readily cross the blood brain
barrier.
[0110] However, some agents useful for the method of the invention
cannot pass, or have difficulty passing, the blood brain barrier.
Such agents can be administered as "prodrugs" which can cross the
blood brain barrier, and upon or after entry into the CNS, the
prodrug is converted to the active agent. Agents that can cross the
blood brain barrier without difficulty can also be administered as
prodrugs.
[0111] To deliver the agent to the CNS, the agent alone or in
combination with other substances as a pharmaceutical composition
may be administered to the spinal cord and to the cerebral vesicles
according to intrathecal and intracerebrovascular administration
methods known in the art. Such pharmaceutical compositions can also
be administered to the nasal cavity, under the tongue, or onto the
eye. The composition may be dispensed intranasally, sublingually,
or conjunctivally as a powdered or liquid nasal spray, nose drops,
a gel or ointment, through a tube or catheter, by syringe, by
packtail, by pledget, or by submucosal infusion. The agent may be
combined with a polymer or other substance that enhances controlled
or sustained release of the agent. In particular, agents can be
delivered to the brain by intranasal administration as described in
X.-Q. Chen et al. (1998) J. Alzheimer's Disease 1:35-44 and W. H.
Frey II et al. (1997) Drug Delivery 4:87-92; the disclosures of
which are incorporated herein by reference.
[0112] The optimal concentration of the active agent will
necessarily depend upon the specific agent used, the
characteristics of the patient and the nature of the disease or
condition for which the treatment is to be used.
[0113] The carrier of the composition may be any material which is
otherwise pharmaceutically-acceptable and compatible with the
active ingredients of the composition. Where the carrier is a
liquid, it is preferred that the carrier is hypotonic or isotonic
with nasal, oral, or conjunctival fluids and have a pH within the
range of 4.5-7.5. Where the carrier is in powdered form, it is
preferred that the carrier is also within an acceptable non-toxic
pH range.
[0114] The pharmaceutical composition may be formulated as a
powder, granules, solution, ointment, cream, aerosol, powder,
drops, or a controlled or sustained release composition such as a
polymer disk. The solution may be sterile, isotonic or hypotonic,
and otherwise suitable for administration by injection or other
means. In addition to the agent, the solution may contain
appropriate adjuvants, buffers, preservatives and salts. The powder
or granular forms of the pharmaceutical composition may be combined
with a solution and with diluting, dispersing or surface active
agents. Solutions such as nose or eye drops may contain an
antioxidant, a buffer, and the like. Further controlled release
polymers may be used to regulate the delivery of the agent.
[0115] The present invention may be better understood with
reference to the following examples. These examples are intended to
be representative of specific embodiments of the invention, and are
not intended as limiting the scope of the invention.
EXAMPLES
Example 1
Protection of Muscarinic Acetylcholine Receptor (mAChR) in Cell
Free Systems
[0116] Materials and Methods
[0117] Membrane mAChR Preparation
[0118] Membranes rich in mAChRs were prepared by a modification of
the method used by Marks and Collins (Characterization of nicotine
binding in mouse brain and comparison with the binding of
a-bungarotoxin and quinuclindinyl benzilate, Mol. Pharmacol.
22:544-564, 1982). Gray matter from nondemented adult human frontal
cortex was homogenized in 9 vol of 50 mM Tris-HCl, pH 7.4, using 5
passes of a glass/Teflon motor-driven homogenizer. The homogenate
was centrifuged at 27 000.times.g for 20 mm at 4.degree. C., and
the subsequent pellet resuspended in 9 vol of cold deionized water
with 5 passes of the homogenizer. The resuspension was incubated at
37.degree. C. for 5 min, then was centrifuged as before. The
resulting pellet was resuspended, incubated and centrifuged again
as above. The final pellet was weighed, resuspended at 15% w/v in
50 mM Tris-HCl buffer, aliquoted in small portions, flash-frozen in
liquid nitrogen and stored at -70.degree. C. for subsequent assays
to determine content and binding capacity. Before use in binding
assays, the thawed membrane preparation was briefly rehomogenized
with 10 passes in a glass/glass homogenizer. A typical mAChR
membrane preparation bound 300 pmol [.sup.3H] quinuclidinyl
benzilate ([.sup.3H]QNB)/g protein.
[0119] Inhibitor Preparation
[0120] Gray matter obtained from the frontal cortex of cases with
AD was homogenized in 9 vol of 1% trifluoroacetic acid (TFA) for 40
s at 4.degree. C. in a Waring blender, then centrifuged at
1200.times.g for 10 min at 4.degree. C. The resulting supernatant
fraction was centrifuged at 11 000.times.g for 100 min at 4.degree.
C. The 11 000.times.g supernatant fraction was centrifuged at 100
000.times.g for 100 min at 4.degree. C., then the 100 000.times.g
supernatant fraction was concentrated using a SpeedVac and
resuspended in 0.1% TFA to half the original tissue volume. The 100
000.times.g supernatant fraction was transferred to a Spectra/Por 3
dialysis membrane bag (3500 dalton cutoff), and dialyzed against 20
vol of 0.1% TFA at 4.degree. C. for 24 h with gentle stirring. The
resulting <3500 Da fraction (dialysate) was concentrated by
SpeedVac to half the original tissue volume. The <3500 Da
fraction (endogenous inhibitor) was then frozen in liquid nitrogen
and stored at --70.degree. C. for subsequent assays to determine
protein count and inhibitor activity. Protein activity was measured
using the bicinchononic acid (BCA) protein assay method,
essentially as described by Smith et al. (Measurement of protein
binding using bicinchonic acid, Ann. Biochem. 150: 76-85, 1985). A
typical inhibitor preparation contained about 4 mg/ml protein and
approximately twice the concentration of inhibitor found in the
original tissue.
[0121] Inhibitor Activity Assay
[0122] Inhibitor activity was measured using a modification of the
method of Fields et al. (Cardiac muscarinic receptors, J. Biol.
Chem. 253:3251-3258, 1978) to assess the binding of [.sup.3H]QNB, a
mAChR antagonist, or [.sup.3H]-oxotremorine M, a mAChR agonist. In
general, binding conditions consisted of 50 mM Tris-HCl, pH 7.4 at
37.degree. C., 10 mM reduced glutathione (GSH), 75 .mu./ml membrane
and 2.times.10.sup.-10 M [.sup.3H]QNB or 3 nM
[.sup.3H]-oxotremorine M, with and without addition of inhibitor.
To control for non-specific binding, 12.5 .mu.M atropine sulfate (a
mAChR antagonist) was added to several tubes. Subtracting
nonspecific binding from total binding yielded specific
binding.
[0123] Pyrophosphate, imidodiphosphate, adenylylimidodiphosphate,
guanylimidodiphosphate, and tripolyphosphate were dissolved in
distilled water. Bilirubin, biliverdin, and heme were dissolved in
DMSO. Carnosol, myricetin, and quercetin were dissolved in ethanol.
Catalase and peroxidase were dissolved in an aqueous, preferably
buffered, solution.
[0124] Enough water was added to all other reaction components in
each tube to make 4 ml total. The binding reaction was initiated by
adding [.sup.3H]QNB or [.sup.3H]-oxotremorine-M, mixing the tubes
briefly, and then incubating the tubes at 37.degree. C. for
[.sup.3H]QNB or at room temperature for [.sup.3H]-oxotremorine-M.
The reaction time for [.sup.3H]QNB was one hour in most
experiments. In some experiments, the mAChR was preincubated with
either the endogenous LMW inhibitor or heme plus peroxide in the
presence or absence of the therapeutic agent being tested. The
effect of the therapeutic agent on receptor function was then
assessed in a binding assay, which for [.sup.3H]QNB was conducted
at 37.degree. C. for 40 min, and for [.sup.3H]-oxotremorine-M was
conducted at room temperature for 20 min. After 60 min, the binding
reaction was terminated by adding 5 ml of cold 50 mM Tris buffer,
pH 7.4, to each tube and chilling the tubes in an ice bath. The
tube contents and one 15 ml rinse of cold 50 mM Tris buffer, pH
7.4, were filtered through Whatman GF/B glass fiber filters using a
Brandel harvester. The filters were placed in Optiflour
scintillation flour and counted in a Beckman LS-6500 scintillation
counter set for tritium detection.
[0125] Results
[0126] The data resulting from the methods presented above and the
results presented in FIGS. 1-20 are discussed in more detail
below.
[0127] Pyrophosphate:
[0128] Pyrophosphate protects the mAChR from inactivation by the
LMW inhibitor or by the combination of heme and peroxide.
Pyrophosphate protected the receptor from both loss of antagonist
(.sup.3H-QNB) binding (FIGS. 1 and 2) and agonist
(.sup.3H-Oxotremorine M) binding (FIG. 3). Approximately 1 .mu.M
pyrophosphate provides 50% protection. Pyrophosphate also protects
the mAChR from damage by PbCl.sub.2. Approximately 57 .mu.M
pyrophosphate provides 50% protection (FIG. 20).
[0129] Imidodiphosphates:
[0130] Imidodiphosphate (FIG. 4), guanylimidodiphosphate (FIG. 5),
and adenylylimidodiphosphate (FIG. 6) all protect the mAChR from
inactivation by the LMW inhibitor.
[0131] Polyphosphates:
[0132] Polyphosphates, such as tripolyphosphate (FIG. 7), protect
the mAChR from inactivation by the LMW inhibitor.
[0133] Bisphosphonates:
[0134] Bisphosphonates, such as pamidronate (FIG. 19), protect the
mAChR from inactivation by the LMW inhibitor.
[0135] Bilirubin and Biliverdin:
[0136] Bilirubin protects the mAChR from inactivation by the LMW
inhibitor or by the combination of heme and peroxide. Approximately
0.7 .mu.M bilirubin provides 50% protection of the receptor from
loss of antagonist (.sup.3H-QNB) binding (FIGS. 8 and 9) and 1.9
.mu.M provides 50% protection from loss of agonist
(.sup.3H-Oxotremorine M) binding (FIG. 10).
[0137] biliverdin at 3 .mu.M provides 50% protection of the mAChR
(FIG. 11).
[0138] Carnosol, Quercetin, and Myricetin:
[0139] Carnosol (FIG. 12), quercetin (FIG. 13), and myricetin (FIG.
14) all protected the mAChR from inactivation. Carnosol provided
100% protection at 1 .mu.M. while quercetin and myricetin provided
50% protection at 0.24 .mu.M and 0.4 .mu.M respectively.
[0140] Catalase and Peroxidase
[0141] Catalase protected the mAChR from inactivation by the LMW
inhibitor or by the combination of heme and peroxide (FIGS. 15 and
16, respectively). As little as 0.34 units/mL of catalase provided
50% protection from inactivation by heme and peroxide.
[0142] A peroxidase, specifically glutathione peroxidase, protected
the mAChR from inactivation by the LMW inhibitor or by the
combination of heme and peroxide (FIGS. 17 and 18, respectively).
Glutathione peroxidase at 0.5 units/mL provided 71% protection from
inactivation by heme and peroxide.
[0143] Conclusion
[0144] The results indicate that pyrophosphate, imidodiphosphates,
polyphosphates, bisphosphonates, bilirubin, biliverdin, carnosol,
quercetin and myricetin protect a receptor and increase the ability
of agents to bind a receptor. Particularly, the results demonstrate
the ability of these agents to protect a muscarinic receptor from
the effects of endogenous LMW inhibitor, heme and metals and
increase the ability of muscarinic agonists and antagonists to bind
a mAChR, suggesting that these agents can be used effectively to
protect other receptors and increase the efficacy of other
agents.
[0145] Because the mAChR is essential for memory and learning, the
specific demonstration that these agents can protect the human
brain mAChR from inactivation and increase agonist binding
indicates that these agents have therapeutic potential for the
treatment of cognitive and memory disorders including those
associated with aging, such as Alzheimer's disease.
Example 2
Protection of the mAChR in Cell Culture
[0146] Various systems for determining the protection of a mAChR in
cell culture are known in the art. Such cell culture systems can be
used to determine if mAChR is protected from a damaging agent or
condition according to the method of the invention. For example, by
administering a mAChR antagonist or agonist alone or in combination
with one or more protective agent and/or one or more pyrophosphate
analog, one of skill in the art can determine if a mAChR is
protected by the one or more protective agent and/or one or more
pyrophosphate analog.
Example 3
Protection of the mAChR Receptor in Animals
[0147] Various systems for determining the protection of a mAChR in
animals are known in the art. Such animal systems can be used to
determine if mAChR is protected according to the method of the
invention. For example, by administering a mAChR antagonist or
agonist alone or in combination with one or more protective agents
and/or one or more pyrophosphate analogs, one of skill in the art
can determine if a mAChR is protected by the one or more protective
agents and/or one or more pyrophosphate analogs.
Example 4
Increased Efficacy of Neurologic Agents in Model Systems
[0148] Various models systems for determining the efficacy of
neurologic agents are known in the art. Such model systems can be
used to determine if the efficacy of a neurologic agent is
increased by the method of the invention. For example, by
administering one or more neurologic agents alone or in combination
with one or more protective agents and/or one or more pyrophosphate
analogs, one of skill in the art can determine if the efficacy of
the one or more neurologic agents is increased when administered
with one or more protective agents and/or one or more pyrophosphate
analogs within the parameters of the model system according to
techniques known in the art.
[0149] It should be noted that, as used in this specification and
the appended claims, the singular forms "a," "an," and "the"
include plural referents unless the content clearly dictates
otherwise. Thus, for example, reference to a composition containing
"a compound" includes a mixture of two or more compounds. It should
also be noted that the term "or" is generally employed in its sense
including "and/or" unless the content clearly dictates
otherwise.
[0150] All publications and patent applications in this
specification are indicative of the level of ordinary skill in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated by reference.
[0151] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the
invention.
* * * * *