U.S. patent application number 16/928967 was filed with the patent office on 2021-03-11 for methods and compositions for prevention and treatment of cardiac hypertrophy.
The applicant listed for this patent is University of Hawaii. Invention is credited to Alexander Stokes.
Application Number | 20210069184 16/928967 |
Document ID | / |
Family ID | 1000005237015 |
Filed Date | 2021-03-11 |
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United States Patent
Application |
20210069184 |
Kind Code |
A1 |
Stokes; Alexander |
March 11, 2021 |
METHODS AND COMPOSITIONS FOR PREVENTION AND TREATMENT OF CARDIAC
HYPERTROPHY
Abstract
Methods are provided of treating cardiac hypertrophy in a
mammalian subject comprising administering to the subject an
anti-hypertrophic effective amount of an ion channel TR-PV1
inhibitor. The methods include treatment of a symptom of cardiac
hypertrophy in the subject comprises cardiac remodeling, cardiac
fibrosis, apoptosis, hypertension, or heart failure
Inventors: |
Stokes; Alexander;
(Honolulu, HI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Hawaii |
Honolulu |
HI |
US |
|
|
Family ID: |
1000005237015 |
Appl. No.: |
16/928967 |
Filed: |
July 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16181204 |
Nov 5, 2018 |
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16928967 |
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15583867 |
May 1, 2017 |
10137123 |
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16181204 |
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14745060 |
Jun 19, 2015 |
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15583867 |
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13824912 |
Mar 18, 2013 |
9084786 |
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PCT/US2011/058967 |
Nov 2, 2011 |
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14745060 |
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61409781 |
Nov 3, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/00 20130101;
A61K 31/497 20130101; A61P 9/04 20180101; A61K 31/496 20130101 |
International
Class: |
A61K 31/496 20060101
A61K031/496; A61K 31/00 20060101 A61K031/00; A61P 9/04 20060101
A61P009/04; A61K 31/497 20060101 A61K031/497 |
Goverment Interests
U.S. GOVERNMENT RIGHTS
[0002] This invention was made with U. S. Government support under
grant NCRR U54RR026136; NAIPI P20RR016467; NIMHD P20MD006084; NCRR
5P20RR016453 from the National Institutes Health. The U.S.
Government may have certain license rights in this invention.
Claims
1-12. (canceled)
13. A method of treating arrhythmias, kidney dysfunction,
hypertension, or heart failure in a mammalian subject comprising
administering to the subject an effective amount of an ion channel
TRPV1 inhibitor.
14. The method according to claim 13, comprising treating
arrhymthmias.
15. The method according to claim 13, comprising treating kidney
dysfunction.
16. The method according to claim 13, comprising treating
hypertension.
17. The method according to claim 13 wherein the inhibitor is
selected from the group consisting of:
N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)-tetrahydropyrazine-1(2-
H)-carboxamide; N-(3-Methoxyphenyl)-4-chlorocinnamide;
1-Isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea;
(2E)-N-(2,3-Dihydro-1,4-benzodioxin-6-yl)-3-[4-(1,1-dimethylethyl)phenyl]-
-2-propenamide;
2-Acetylamino-4-[6'-(4-trifluoromethylphenyl)-pyrimidin-4'-yl-oxy]-benzot-
hiazole;
N-(2-bromophenyl-N'-[((R)-1-(5-trifluoromethyl-2-pyridyl)pyrrolid-
in-3-yl)]urea;
N-(2-bromophenyl)-N'-{2-[ethyl(3-methylphenyl)amino]ethyl}urea;
(R)-(5-tert
-butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)-urea;
N-(Isoquinolin-5-yl)-N'-[spiro-(cyclobutane-1,2'-(3',4'-dihydro-benzopyra-
n-4'-yl))]urea;
(2R)-4-(3-chloro-2-pyridinyl)-2-methyl-N-[4-(trifluoromethyl)phenyl]-1-pi-
perazinecarboxamide;
4-(4'-Trifluoromethyl-anilino)-7-(3'-trifluoromethyl-pyridin-2-yl)-quinaz-
oline;
N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-b-
enzazepine-2-carbothioamide;
(5R*,8R*,6E,9E)-5,8-Dimethyl-4-methylenetetradeca-6,9-dienoic acid;
1-(3-Fluorobenzyl)-2-(N-(1,2-dimethyl-1,3-isoindazol-5-yl)-acetamido)-{py-
ridine-[3,4-b]-pyrrole};
N-(4-chlorobenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(4-tert-butylbenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(3-fluoro-4-(trifluoromethyl)benzyl)-N'-(1-methyl-1H-indazol-4-yl)-urea-
;
N-(4-fluoro-3-(trifluoromethyl)benzyl)-N'-(1-methyl-1H-indazol-4-yl)-ure-
a; N-(3 ,4-dichlorobenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(2,4-dichlorobenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(4-ethylbenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(2-chlorobenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(4-fluorobenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(2-fluorobenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-[1-(bromophenyl)ethyl-N'-(1-methyl-1H-Indazol-4-yl)urea;
N-(1-methyl-1H-indazol-4-yl)-N'-{4-[(trifluoromethyl)thio]benzyl}urea;
1-(2,3-dichlorophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea;
1-[2-(N-ethyl-3-methylanilino)ethyl]-3-naphthalen-1-ylurea;
1-(4-bromophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea;
1-(3-bromophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea;
1-(chlorophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea;
1-[2-(N-ethyl-3-methylanilino)ethyl]-3-(2-fluorophenyl)urea; 1-[2-1
N-ethyl-3-methylanilino)ethyl]-3-(2-methylphenyl)urea;
1-[2-(N-ethyl-3-methylanilino)ethyl]-3-phenylurea;
2-[(2-bromophenyl)carbamoylamino]ethyl-ethylmethyl-(3-methylphenyl)azaniu-
m iodide;
1-(2-bromophenyl)-3-[2-(N-ethyl-3-fluoro-4-methylanilino)ethyl]u-
rea;
1-(2-bromophenyl)-3-[2-(N-ethyl-3,4-difluoroanilino)ethyl]urea;
1-(2-bromophenyl)-3-[2-(N-ethyl-3-fluoroanilino)ethyl]urea;
1-(2-bromophenyl)-3-[2-(N-ethyl-4-methylanilino)ethyl]urea;
1-(2-bromophenyl)-3-[2-(N-ethyl-2-methylanilino)ethyl]urea;
1-(2-bromophenyl)-3-[2-(N-ethylanilino)ethyl]urea;
N-[2-[(2-bromophenyl)carbamoylamino]ethyl]-N-(3-methylphenyl)acetamide;
1-[2-{N-benzyl-3-methylanilino)ethyl]-3-(2-bromophenyl)urea;
1-(2-bromophenyl)-3-[2-(2,3-dimethylanilino)ethyl]urea;
1-(2-bromophenyl)-3-[2-(3-methylaniIino)ethyl]urea;
1-(2,5-dichlorophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea;
4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-
-carboxamide;
4-fluoro-4(pyridin-2-yl)N-[4-trifluoromethylphenyl]piperidine-1-carboxami-
de;
4-fluoro-4(pyridine-2-yl)N-[4-trifluoromethylbenzyl]piperidine-1-carbo-
xamide;
2-{4-fluoro-1-[4-trifluoromethylbenzoyl]piperidin-4-yl}pyridine;
2-(4-fluoro-1-{[4-trifluoromethylphenyl]acetyl
}piperidin-4-yl)pyridine;
2-(4-fluoro-1-{3-[4-trifluoromethylphenyl]propanoyl}piperidin-4-yl)pyridi-
ne;
4-fluoro-4-(1-methyl-1H-imidazol-2-yl)-N-[4-trifluoromethylphenyl]pipe-
ridine-1-carboxamide;
4-methoxy-4-pyridin-2-yl-N-[4-trifluoromethylphenyl]piperidine-1-carboxam-
ide;
4-methoxy-4-pyridin-2-yl-N-[4-trifluoromethylbenzyl]piperidine-1-carb-
oxamide;
4-fluoro-N-(4-isopropylphenyl)-4-(3-methylpyridin-2-yl)piperidine-
-1-carboxamide; 4-fluoro-4-(3-methylpyridin-2-yl)-N-{4-[
1,2,2,2-tetrafluoro-1-trifluoromethylethyl]phenyl}piperidine-1-carboxamid-
e;
N-(4-Tert-butylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-ca-
rboxamide;
4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-(pentafluoro-lambda(sup
6)-sulfanyl)phenyl]piperidine-1-carboxamide;
N-(4-Butylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxami-
de;
N-(4-Benzylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carbo-
xamide;
N-biphenyl-4-yl-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carb-
oxamide;
4-fluoro-4-(3-methylpyridin-2-yl)-N-[5-trifluoromethylpyridin-2-y-
l]piperidine-1-carboxamide;
4-(3-chloropyridin-2-yl)-4-fluoro-N-[4-trifluoromethylphenyl]piperidine-1-
-carboxamide;
4-fluoro-4-(3-fluoropyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-
-carboxamide;
4-fluoro-4-(3-methoxypyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine--
1-carboxamide;
4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-
-carbothioamide;
N'-cyano-4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethylphenyl]pip-
eridine-1-carboximidamide;
4-fluoro-4-(3-methylpyridin-2-yl)-N'-(1-phenylpiperidin-4-yl)-N-[4-triflu-
oromethylphenyl]piperidine-1-carboximidamide;
4-fluoro-4-phenyl-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide;
(+/-)-(syn)-4-fluoro-2-methyl-4-(3-methylpyridin-2-yl)-N-[4-trifluorometh-
ylphenyl]piperidine-1-carboxamide;
4-(fluoromethyl)-4-pyridin-2-yl-N-[4-trifluoromethylphenyl]piperidine-1-c-
arboxamide; syn- and
anti-3-fluoro-3-pyridin-2-yl-N-[4-trifluoromethylphenyl]-8-azabicyclo[3.2-
-.1]octane-8-carboxamide;
3-fluoro-3-pyridin-2-yl-N-[4-trifluoromethylphenyl]-8-azabicyclo[3.2.1]oc-
tane-8-carboxamide;
4-fluoro-4-pyrimidin-2-yl-N-[4-trifluoromethylphenyl]piperidine-1-carboxa-
mide;
4-fluoro-4-(3-phenylpropyl)-N-[4-trifluoromethylphenyl]piperidine-1--
carboxamide;
2-[4-fluoro-4-(3-methylpyridin-2-yl)piperidin-1-yl]-6-trifluoromethyl-1H--
benzimidazole;
2-(4-fluoro-4-pyridin-2-yl)piperidin-1-yl)-6-(trifluoromethyl)-1H-benzimi-
dazole;
4-fluoro-N-[4trifluoromethylphenyl]-4-[3-trifluoromethylpyridin-2--
yl]piperidine-1-carboxamide;
4-fluoro-N-(4-methylphenyl)-4-(3-methylpyridin-2-yl)piperidine-1-carboxam-
ide;
N-(4-ethylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carbo-
xamide;
N-(4-chlorophenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-c-
arboxamide;
4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethoxyphenyl]piperidine--
1-carboxamide;
N-(4-cyanophenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxami-
de;
N-[4-dimethylaminophenyl]-4-fluoro-4-(3-methylpyridin-2-yl)piperidine--
1-carboxamide;
1-(2-(3,3-dimethylbutyl)-4-(trifluoromethyl)benzyl)-3-(1-methyl-1H-indazo-
-1-4-yl)urea; N-acetyl-1-phenylalanyl-1-leucinamide; and
pharmaceutically acceptable salts thereof.
18. The method according to claim 14 wherein the inhibitor is
selected from the group consisting of:
N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)-tetrahydropyrazine-1(2-
H)-carboxamide; N-(3-Methoxyphenyl)-4-chlorocinnamide;
1-lsoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea;
(2E)-N-(2,3-Dihydro-1,4-benzodioxin-6-yl)-3-[4-(1,1-dimethylethyl)phenyl]-
-2-propenamide;
2-Acetylamino-4-[6'-(4-trifluoromethylphenyl)-pyrimidin-4'-yl-oxy]-benzot-
hiazole;
N-(2-bromophenyl-N'-[((R)-1-(5-trifluoromethyl-2-pyridyl)pyrrolid-
in-3-yl)]urea;
N-(2-bromophenyl)-N'-{2-[ethyl(3-methylphenyl)amino]ethyl}urea;
(R)-(5-tert
-butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)-urea;
N-(Isoquinolin-5-yl)-N'-[spiro-(cyclobutane-1,2'-(3',4'-dihydro-benzopyra-
n-4'-yl))]urea;
(2R)-4-(3-chloro-2-pyridinyl)-2-methyl-N-[4-(trifluoromethyl)phenyl]-1-pi-
perazinecarboxamide;
4-(4'-Trifluoromethyl-anilino)-7-(3'-trifluoromethyl-pyridin-2-yl)-quinaz-
oline;
N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-b-
enzazepine-2-carbothioamide;
(5R*,8R*,6E,9E)-5,8-Dimethyl-4-methylenetetradeca-6,9-dienoic acid;
1-(3-Fluorobenzyl)-2-(N-(1,2-dimethyl-1,3-isoindazol-5-yl)-acetamido)-{py-
rdine-[3,4-b]-pyrrole};
N-(4-chlorobenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(4-tert-butylbenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(3-fluoro-4-(trifluoromethyl)benzyl)-N'-(1-methyl-1H-indazol-4-yl)-urea-
;
N-(4-fluoro-3-(trifluoromethyl)benzyl)-N'-(1-methyl-1H-indazol-4-yl)-ure-
a; N-(3,4-dichlorobenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(2,4-dichlorobenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(4-ethylbenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(2-chlorobenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(4-fluorobenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-(2-fluorobenzyl)-N'-(1-methyl-1H-indazol-4-yl)urea;
N-[1-(bromophenyl)ethyl-N'-(1-methyl-1H-Indazol-4-yl)urea;
N-(1-methyl-1H-indazol-4-yl)-N'-{4-[(trifluoromethyl)thio]benzyl}urea;
1-(2,3-dichlorophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea;
1-[2-(N-ethyl-3-methylanilino)ethyl]-3-naphthalen-1-ylurea;
1-(4-bromophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea;
1-(3-bromophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea;
1-(chlorophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea;
1-[2-(N-ethyl-3-methylanilino)ethyl]-3-(2-fluorophenyl)urea;
1-[2-{N-ethyl-3-methylanilino)ethyl]-3-(2-methylphenyl)urea;
1-[2-(N-ethyl-3-methylanilino)ethyl]-3-phenylurea;
2-[(2-bromophenyl)carbamoylamino]ethyl-ethylmethyl-(3-methylphenyl)azaniu-
m iodide;
1-(2-bromophenyl)-3-[2-(N-ethyl-3-fluoro-4-methylanilino)ethyl]u-
rea;
1-(2-bromophenyl)-3-[2-(N-ethyl-3,4-difluoroanilino)ethyl]urea;
1-(2-bromophenyl)-3-[2-(N-ethyl-3-fluoroanilino)ethyl]urea;
1-(2-bromophenyl)-3-[2-(N-ethyl-4-methylanilino)ethyl]urea;
1-(2-bromophenyl)-3-[2-(N-ethyl-2-methylanilino)ethyl]urea;
1-(2-bromophenyl)-3-[2-(N-ethylanilino)ethyl]urea;
N-[2-[(2-bromophenyl)carbamoylamino]ethyl]-N-(3-methylphenyl)acetamide;
1-[2-1N-benzyl-3-methylanilino)ethyl]-3-(2-bromophenyl)urea;
1-(2-bromophenyl)-3-[2-(2,3-dimethylanilino)ethyl]urea;
1-(2-bromophenyl)-3-[2-(3-methylaniIino)ethyl]urea;
1-(2,5-dichlorophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea;
4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-
-carboxamide;
4-fluoro-4(pyridin-2-yl)N-[4-trifluoromethylphenyl]piperidine-1-carboxami-
de;
4-fluoro-4(pyridine-2-yl)N-[4-trifluoromethylbenzyl]piperidine-1-carbo-
xamide;
2-{4-fluoro-1-[4-trifluoromethylbenzoyl]piperidin-4-yl}pyridine;
2-(4-fluoro-1-{[4-trifluoromethylphenyl]acetyl}piperidin-4-yl)pyridine;
2-(4-fluoro-1-{3-[4-trifluoromethylphenyl]propanoyl}piperidin-4-yl)pyridi-
ne;
4-fluoro-4-(1-methyl-1H-imidazol-2-yl)-N-[4-trifluoromethylphenyl]pipe-
ridine-1-carboxamide;
4-methoxy-4-pyridin-2-yl-N-[4-trifluoromethylphenyl]piperidine-1-carboxam-
ide;
4-methoxy-4-pyridin-2-yl-N-[4-trifluoromethylbenzyl]piperidine-1-carb-
oxamide;
4-fluoro-N-(4-isopropylphenyl)-4-(3-methylpyridin-2-yl)piperidine-
-1-carboxamide;
4-fluoro-4-(3-methylpyridin-2-yl)-N-{4-[1,2,2,2-tetrafluoro-1-trifluorome-
thylethyl]phenyl}piperidine-1-carboxamide;
N-(4-Tert-butylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carb-
oxamide;
4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-(pentafluoro-lambda(sup
6)-sulfanyl)phenyl]piperidine-1-carboxamide;
N-(4-Butylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxami-
de;
N-(4-Benzylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carbo-
xamide ;
N-biphenyl-4-yl-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-car-
boxamide ;
4-fluoro-4-(3-methylpyridin-2-yl)-N-[5-trifluoromethylpyridin-2-
-yl]piperidine-1-carboxamide;
4-(3-chloropyridin-2-yl)-4-fluoro-N-[4-trifluoromethylphenyl]piperidine-1-
-carboxamide;
4-fluoro-4-(3-fluoropyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-
-carboxamide;
4-fluoro-4-(3-methoxypyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine--
1-carboxamide;
4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-
-carbothioamide;
N'-cyano-4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethylphenyl]pip-
eridine-1-carboximidamide;
4-fluoro-4-(3-methylpyridin-2-yl)-N'-(1-phenylpiperidin-4-yl)-N-[4-triflu-
oromethylphenyl]piperidine-1-carboximidamide;
4-fluoro-4-phenyl-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide;
(+/-)-(syn)-4-fluoro-2-methyl-4-(3-methylpyridin-2-yl)-N-[4-trifluorometh-
ylphenyl]piperidine-1-carboxamide;
4-(fluoromethyl)-4-pyridin-2-yl-N-[4-trifluoromethylphenyl]piperidine-1-c-
arboxamide; syn- and
anti-3-fluoro-3-pyridin-2-yl-N-[4-trifluoromethylphenyl]-8-azabicyclo[3.2-
-.1]octane-8-carboxamide;
3-fluoro-3-pyridin-2-yl-N-[4-trifluoromethylphenyl]-8-azabicyclo[3.2.1]oc-
tane-8-carboxamide;
4-fluoro-4-pyrimidin-2-yl-N-[4-trifluoromethylphenyl]piperidine-1-carboxa-
mide;
4-fluoro-4-(3-phenylpropyl)-N-[4-trifluoromethylphenyl]piperidine-1--
carboxamide;
2-[4-fluoro-4-(3-methylpyridin-2-yl)piperidin-1-yl]-6-trifluoromethyl-1H--
benzimidazole;
2-(4-fluoro-4-pyridin-2-ylpiperidin-1-yl)-6-(trifluoromethyl)-1H-benzimid-
azole;
4-fluoro-N-[4trifluoromethylphenyl]-4-[3-trifluoromethylpyridin-2-y-
l]piperidine-1-carboxamide;
4-fluoro-N-(4-methylphenyl)-4-(3-methylpyridin-2-yl)piperidine-1-carboxam-
ide;
N-(4-ethylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carbo-
xamide;
N-(4-chlorophenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-c-
arboxamide;
4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethoxyphenyl]piperidine--
1-carboxamide;
N-(4-cyanophenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxami-
de;
N-[4-dimethylaminophenyl]-4-fluoro-4-(3-methylpyridin-2-yl)piperidine--
1-carboxamide;
1-(2-(3,3-dimethylbutyl)-4-(trifluoromethyl)benzyl)-3-(1-methyl-1H-indazo-
-1-4-yl)urea; N-acetyl-1-phenylalanyl-1-leucinamide; and
pharmaceutically acceptable salts thereof.
19. The method according to claim 13 wherein the inhibitor
comprises
N-(4-t-butyl-phenyl)-4-(3-chloropyridin-2-yl)-tetrahydropyrazine-1(2H)-ca-
rboxamide or a pharmaceutically acceptable salt thereof.
20. The method according to claim 14 wherein the inhibitor
comprises
N-(4-t-butyl-phenyl)-4-(3-chloropyridin-2-yl)-tetrahydropyrazine-1(2H)-ca-
rboxamide or a pharmaceutically acceptable salt thereof.
21. The method according to claim 16 wherein the inhibitor
comprises
N-(4-t-butyl-phenyl)-4-(3-chloropyridin-2-yl)-tetrahydropyrazine-1(2H)-ca-
rboxamide or a pharmaceutically acceptable salt thereof.
Description
PRIORITY CLAIM
[0001] Priority is claimed pursuant to 35 USC 119(e) of U.S.
provisional application Ser. No. 61/409,781, filed Nov. 3, 2010,
the disclosure of which is incorporated by reference herein in its
entirety for all purposes.
FIELD OF THE INVENTION
[0003] The present invention relates to the treatment and
prevention cardiac hypertrophy. More specifically, the invention
relates to methods and compositions fir preventing or treating
cardiac hypertrophy, cardiac remodeling, fibrosis, hypertension,
and heart failure in mammals, including humans, through inhibition
of the ion channel TRPV1.
BACKGROUND
[0004] Myocardial hypertrophy is the fundamental response of the
heart to a chronically increased workload, which can result from
conditions such as hypertension or valve disorders. The progression
of myocardial hypertrophy represents a principal risk factor for
the development of heart failure and subsequent cardiac death.
[0005] The focus of this invention is on combating hypertrophy,
apoptosis fibrosis, and heart failure, focuses on regulation of
TRPV1 (transient receptor potential cation channel, subfamily V,
member 1), a complex and remarkable receptor/channel. TRPV1 is
typically classified as a nocioceptive receptor. Published data
indicate that the open probability of TRPV1 is controlled by the
endocannabinoid anandamide, its endogenous ligand, and pathways
modulating anandamide levels also influence TRPV1 activation. The
etiology of hypertrophic regulation by TRPV1 (transient receptor
potential cation channel, subfamily V, member 1) is unknown. There
is only a general understanding of how TRPV1 is regulated, and of
the identity of several cell and tissue types in which TRPV1
resides.
[0006] TRPV1 has been studied in peripheral sensory neurons as a
pain receptor; however TRPV1 is expressed in numerous tissues and
cell types including those of the cardiovascular system. TRPV1
expression is upregulated in the hypertrophic heart, and the
channel is positioned to receive stimulatory signals in the
hypertrophic heart. TRVP1 is a six trans-membrane tetrameric
nonselective cation channel, typically associated with peripheral
sensory neurons involved in nociception. Exogenous activators of
TRPV1 include temperature of greater than 43.degree. C. and
capsaicin. Endogenously, TRPV1 is activated and potentiated by the
endocannabinoids, anandamide and N-arachidonovl-dopamine, low and
phosphorylation by protein kinase C (PKC) and cyclic AMP-dependent
protein kinase (PKA). The nociceptive involvement of TRPV1
activation in peripheral sensory neurons has prompted substantial
study of TRPV1 as a target for inhibition. Consequently a plethora
of effective TRPV1 antagonists has been produced and demonstrated
to be effective analgesics in the management of inflammatory pain
and hyperalgesia.
[0007] In addition to the peripheral sensory neurons, TRPV1 is also
found in other excitable and non-excitable tissues, including those
of the heart and circulatory system. For example, cardiomyocytes,
cardiac blood vessels, perivascular nerves, pulmonary artery smooth
muscle cells, and coronary endothelial cells, skeletal muscle, mast
cells, and dendritic cells express TRPV1.
[0008] Although TRPV1 inhibition has not been studied in the
context of cardiac hypertrophy, TRPV1 activation has been
implicated in protection from myocardial ischemia reperfusion
injury. In addition, the channel's endogenous ligand, anandamide,
has been implicated in multiple cardiac diseases such as
cardiotoxicity and hypertension.
SUMMARY OF EXEMPLARY EMBODIMENTS OF THE INVENTION
[0009] The invention provides methods of treating cardiac
hypertrophy in a mammalian subject comprising administering to the
subject an anti-hypertrophic effective amount of an ion channel
TRPV1 inhibitor. In some embodiments, the invention provides
methods of treatment where a symptom of cardiac hypertrophy in the
subject comprises cardiac remodeling, cardiac fibrosis, apoptosis,
hypertension, or heart failure.
[0010] The invention further provides methods prophylactic
treatment for cardiac hypertrophy in a mammalian subject comprising
administering to the subject an anti-hypertrophic effective amount
of an ion channel TRPV1 inhibitor.
[0011] The invention also provides pharmaceutical compositions
comprising a pharmaceutically effective amount of an ion channel
TRPV1 inhibitor or mixtures of such inhibitors useful for the
methods of the invention.
[0012] The invention also provides pharmaceutical compositions
comprising a pharmaceutically effective amount of the ion channel
TRPV1 inhibitor
(N-(4-t-butyl-phenyl)-4-(3-chloropyridin-2-yl)-tetrahydropyrazine-1(2H)-c-
arboxamide or mixtures of that inhibitor with other ion channel
TRPV1 inhibitors useful for the methods of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIGS. 1A-1D are graphs of results of data obtained by
procedures described in Example 1.
[0014] FIG. 2 is a graph of results of data obtained by the
picosirius staining procedure described in Example 2.
[0015] FIGS. 3A-3B are graph of results of data obtained by the
TGF-beta RNA expression and ANP expression procedures described in
Example 2.
[0016] FIGS. 4A-4B are graphs of results of data obtained by the
procedures described in Example 3.
[0017] FIGS. 5A-5F are graphs of test results obtained by
procedures described in Example 4 pertaining to gravimetric,
structural, and functional analysis of the heart during and after
applied pressure overload cardiac hypertrophy.
[0018] FIGS. 6A-6D are graphs of test results on measurement of
cardiomycyte cross sectional area, and expression levels of ANP and
TGF.beta. by procedures described in Example 4.
[0019] FIGS. 7A-7E are graphs of test results on measurement of
fibrosis, tissue remodeling, and inflammatory markers by procedures
described in Example 4.
[0020] FIGS. 8A-8E arc graphs of test results on measurement of
heart mass, structure and function during pressure overload cardiac
hypertrophy by procedures described in Example 5.
[0021] FIGS. 9A-9B are graphs of measurements from histological
analysis of mice treated with the TRPV1 antagonist BCTC according
to procedures described in Example 6.
DETAILED DESCRIPTION EXEMPLARY EMBODIMENTS OF THE INVENTION
[0022] Cardiac hypertrophy is classically considered to be an
adaptive and compensatory response that increases the work output
of cardiomyoeytes and thus maintains cardiac function despite
increased load. In mice, cardiac hypertrophy is typically modeled
using transverse aortic constriction (TAC) to induce acute pressure
overload. The increased resistance created by aortic constriction
initially compromises left ventricular (LV) function; the
subsequent development of LV hypertrophy begins to restore systolic
function in the two weeks following TAC. Concentric LV hypertrophy
continues during weeks two to eleven post-TAC, potentially doubling
the LV mass compared to controls. A decline in LV function
accompanies LV chamber dilation and myocardial fibrosis, and around
half of TAC treated mice develop pulmonary congestion by week
eleven. Thus, TAC is an effective stimulus for rapidly producing
cardiac hypertrophy in an experimental setting. The TAC model
provides tremendous utility for identifying important therapeutic
targets in heart disease and exploring the effects of molecular or
pharmacological inhibitors.
[0023] This invention shows that TRPV1 function is a new target for
protective therapy in cardiac hypertrophy, fibrosis and heart
failure using TRPV1-directed therapeutics, this invention has the
potential to shift clinical treatment paradigms for cardiac
hypertrophy and heart failure by repurposing existing drugs.
[0024] As shown below in the examples the loss of TRPV1 function in
mice alters the responses of the heart to TAC-induced pressure
overload. TRPV1 contributes to cardiac hypertrophy, fibrosis,
apoptosis, and loss of contractile function in response to pressure
overload. TRPV1 antagonists previously known as anti-hyperalgesics
are unexpectedly provided by the methods of the present invention
as anti-hypertrophic agents.
[0025] As shown in the examples the knockout of Trpvl significantly
suppresses the ventricular enlargement, apoptosis, tissue
remodeling and fibrosis associated with modeled pressure overload
cardiac hypertrophy. This phenotype mirrors some of the most
desirable effects for anti-hypertrophic treatments. By use of a
transverse aortic constriction to model pressure overload cardiac
hypertrophy mice lacking functional TRPV1, compared to wild type,
have improved heart function, and reduced hypertrophic, fibrotic
and apoptotic markers. TRPV 1 plays a role in the progression of
cardiac hypertrophy, and presents a therapeutic target for the
treatment of cardiac hypertrophy and subsequent disease states
including arrhythmias, kidney dysfunction and heart failure;
treatment and alleviation of symptoms leading to cardiac
hypertrophy and heart failure such as high blood pressure, heart
valve disease, weakness of the heart muscle (cardiomyopathy),
abnormal heartbeat, anemia, thyroid disorders, excessive drug use,
muscular dystrophy and Fabry's disease, aortic valve stenosis, side
effects of chemotherapy agents leading to toxic cardiomyopathy,
obesity, diabetes, cigarette smoking, viral myocarditis (an
infection of the heart muscle), infiltrations of the muscle such as
amyloidosis, HIV cardiomyopathy (caused by human immunodeficiency
virus), connective tissue diseases such as systemic lupus
erythematosus, abuse of drugs such as alcohol and cocaine, and side
effects of arrhythmias or pharmaceutical drugs such as
chemotherapeutic agents.
[0026] In addition to the compounds and compositions having
activity herein, other compounds having the requisite activity may
be identified by the following test. Since cardiac hypertrophy is
classically considered to be an adaptive and compensatory response
that increases the work output of cardiomyocytes and thus maintains
cardiac function despite increased load, the following test will
identify a compound as having the activity useful in accordance
with the invention. In mice, cardiac hypertrophy is typically
modeled using transverse aortic constriction (TAC) to induce acute
pressure overload. The increased resistance created by aortic
constriction initially compromises left ventricular (LV) function;
the subsequent development of LV hypertrophy begins to restore
systolic function in the two weeks following TAC. Concentric LV
hypertrophy continues during weeks two to eleven post TAC,
potentially doubling the LV mass compared to controls. A decline in
LV function accompanies LV chamber dilation and myocardial
fibrosis, and around half of TAC treated mice develop pulmonary
congestion by week eleven. Thus, TAC is an effective stimulus for
rapidly producing cardiac hypertrophy in an experimental setting.
Although there are differences between the TAC model and clinical
cardiac hypertrophy, this model mimics the acute onset of
hypertension rather than the gradual onset in clinical cases.
However, the TAC model is useful for identifying important
therapeutic targets in heart disease and exploring the effects of
molecular or pharmacological inhibitors. (Lygate, 2006; Patten and
Hall-Porter, 2009)
[0027] Test Model
[0028] Generation of the Model in the Mouse
[0029] Transverse Aortic Constriction (TAC). Transverse aortic
constriction was performed as described by Rockman, producing left
ventricular hypertrophy by constriction of the aorta (Rockman et
al., 1994; Rockman et al., 1991). The left side of the chest was
depilated with Nair and a baseline 2-D echocardiogram was obtained.
Mice were then deeply anesthetized with a mixture of ketamine and
xylazine. The transverse aorta between the brachiocephalic and left
carotid artery was banded using 6-0 silk ligature around the vessel
and a 26G blunt needle, after which the needle was withdrawn. Sham
surgeries were identical apart from the constriction of the
aorta.
[0030] Checking for Successful Banding
[0031] Doppler echocardiography. Doppler echocardiography was
performed one week post TAC to measure the level of constriction.
Mice were anesthetized lightly with isofluorene gas and shaved.
Doppler was performed using the Visualsonics Vevo 770 system. In
the parasternal short-axis view, the pulsed wave Doppler sample
volume was placed in the transverse aorta just proximal and distal
to the site of banding. Peak velocity was traced using Vevo 770
software, and the pressure gradient was calculated using the
simplified Bernoulli equation.
[0032] Following the Structural Changes in Heart Dimensions during
the Progression of the Modeled Disease
[0033] Transthoracic echocardiography. Baseline and post TAC
transthoracic echocardiography were used to assess changes in mouse
heart dimensions and function. Briefly, after two days of
acclimatization and depilation, unanesthetized transthoracic
echocardiography was performed using a 30-Mhz transducer (Vevo 770,
VisualSonics). High quality two-dimensional images and. M-mode
images of the left ventricle were recorded. Measurements of left
ventricular end-diastolic (LVIDd) and end-systolic (LVIDS) internal
dimensions were performed by the leading edge to leading edge
convention adopted by the American Society of Echocardiography. The
left ventricular ejection fraction (% EF) was calculated as (LV
Vol; d-LV Vol;s/LV Vol; d.times.100) (Visualsonics Inc.).
[0034] Testing the Degree of Cellular Hypertrophy, Fibrosis, and
Apoptosis Post Treatment
[0035] Markers of hypertrophy, fibrosis, tissue remodeling,
inflammation and apoptosis, are assessed by either Western Blot
(W13) analysis, real-time PCR of extracted RNA (RT), or
histological and immunohistological analysis (H).
[0036] Hypertrophic markers can include: ANP.sup.(WB, RT),
BNP.sup.(RT), ACTAI.sup.(RT), .alpha.-MHC.sup.(RT),
.beta.-MHC.sup.(RT), MLC2A.sup.(RT), and Wheat-germ
agglutinin.sup.(H) to generate cardiomyocyte cross sectional
area.
[0037] Tissue remodeling markers can include: Chymase CMA1.sup.(WB,
RT), MMP-2.sup.(RT), MMP-9.sup.(RT), TGF-.beta..sup.(RT), Collagen
III.sup.(RT,H), fibrinogen.sup.(RT,H), Fibroblast proliferation
CD29.sup.(H).
[0038] Apoptosis markers can include: Cleaved
Caspase-3.sup.(WB).
[0039] Immunological, inflammatory and infiltration markers can
include: IL-6.sup.(RT), TNF-.alpha..sup.(RT), NOS3.sup.(RT), CD68
(Macrophages).sup.(WB,RT,H), histidine decarboxylase.sup.(WB) and
Fe R1.alpha..sup.(WB) (Mast cells).sup.(WB,R,H),
CD4.sup.+/CD8.sup.+ T-cell markers.sup.(WB,H), NK cell
CD161.sup.(RT,WB,H)
[0040] Tissue preparation for histology. Eight weeks post TAC, mice
were euthanized by CO.sub.2 asphyxiation, and hearts were collected
for histological and molecular analysis. For histology, hearts were
perfused with phosphate-buffered saline and 10% formalin in situ,
collected immediately, and fixed overnight in 10% formalin at
4.degree. C. Tissues were then cut in a sagittal orientation,
embedded in paraffin, mounted on glass and stored until use.
Paraffin-embedded sections were stained for the following:
[0041] Collagen: Collagen volume fraction was determined by
analysis of picrosirius stained sections. Sections cut to 5urn
thickness were deparaffinized, stained with Weigert's hematoxylin,
then stained with picrosirius red (0.1% Sirius Red in picric acid).
Sections were subsequently washed and dehydrated before image
analysis.
[0042] Cardiomyocyte cross sectional area: Heart sections were
deparaffinized and permeabilized, then stained with wheat
germ-agglutinin conjugated to Alexa488 (WGA-Alexa488, Invitrogen,
W11261) at a concentration of 50 .mu.g/mL to identify sarcolemmal
membranes and measure cardiomyocyte cross sectional area (described
below).
[0043] Image collection and analysis. Fluorescent and bright field
images were collected on an epifluorescence-microscope (Axioscope,
Zeiss). Fibrosis and cross-sectional cardiomyocyte area were
quantified using ImageJ software (NIH). To quantify fibrosis,
collagen fibers were highlighted, and the red-stained pixels were
counted to determine the percentage of pixels in each field that
represented collagen fibers. Perivascular tissue was excluded from
this calculation. Three heart sections from each animal were imaged
at five images per heart. Images were averaged for each animal and
graphed in Prism GraphPad. Cardiomyocytes from WGA stained sections
were randomly selected in a blinded fashion then traced to
determine the cross sectional area of individual myocytes
(n=100).
[0044] All images were captured and analyzed in a single-blind
manner, except for WGA staining, which was analyzed in a
double-blind manner.
[0045] RT-PCR. For RNA extraction, hearts were collected from mice
and total RNA was isolated from homogenized hearts with Trizol
(Molecular Research Center, TR 118) and further purified with an
RNA isolation kit (Mo Bio Laboratories, Inc, 15000-250).
Single-stranded eDNA was synthesized from 1 .mu.g of total RNA
using a eDNA synthesis kit (Qiagen, 205113). The mRNA levels of
chymase (CMA1), atrial natriuretic peptide (ANP), TGF-, collagen
III, matrix metalloproteinase (MMP) 2 and 9 and cyclophilin (CPN)
were quantified by RT-PCR in triplicate with QuantiTect SYBR Green
(Qiagen, 204245) in an Opticon device (MJ Research, Waltham,
Mass.). The following primer pairs were used: ANP, 5'-AGA ACA CAG
AGA GTG GGC AGA G-3' (SEQ ID NO. 1) and 5'-CAA GAC GAG GAA GAA GCC
CAG-3' (SEQ ID NO. 2); TGF.beta., 5'-TGG AGC AAC ATG TGG AAC TC-3'
(SEQ ID NO, 3) and 5'-CAG CAG CCG GTT ACC AAG-3' (SEQ ID NO. 4);
MMP2, 5'-TGG TGT GGC ACC ACC GAG GA-3' (SEQ ID NO. 5) and 5'-GCA
TCG GGG GAG GGC CCA TA-3' (SEQ ID NO. 6); MMP9, 5'-CGG CAC GCC TTG
GTG TAG CA-3' (SEQ ID NO. 7) and 5'-TCG CGT CCA CTC GGG TAG GG-3'
(SEQ ID NO. 8); Collagen III, 5'-GAC CGA TGG ATT CCA GTT CG-3' (SEQ
ID NO. 9) and 5'-TGT GAC TCG TGC AGC CAT CC-3' (SEQ ID NO. 10);
CMA1, 5'-AGC TCA CTG TGC GGG AAG GTC T-3' (SEQ ID NO. 11) and
5'-CTC AGG GAC CAG GCA GGG CTT-3' (SEQ ID NO. 12).
[0046] Western blot analysis. Hearts were collected, and protein
extracts were prepared from homogenized heart tissue using IGEPAL.
Total protein concentrations were determined by the bicinchoninic
acid (BCA) colorimetric assay. Absorbance was measured at 562 nm by
spectrophotometer (Spectra. Max 340), and concentrations determined
using a standard curve based on bovine serum albumin (BSA) protein
standards. Concentrations were normalized to 30 .mu.g, and samples
were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Protein samples were transferred to
polyvinylidene fluoride (PVDF, Millipore, IPFL00010) membrane at
1.4 amps for 3.5 hours. Membranes were probed overnight at
4.degree. C. with antibodies to cleaved Caspase-3 (Cell Signaling,
9661S), CMA1 (Gene Tex, GTX72388), and GAPDH (Calbiochem, CB1001.).
The membranes were visualized with ECL substrate (GE Healthcare,
RPN2132) and film. Western blot band intensity was quantified as
integrated density by densitometry and normalized to the density of
loading control.
[0047] Any suitable TRPV1 inhibitor or combination of TRPV1
inhibitors may be used in the compositions and methods of the
present invention. Inhibitors of TRPV1 family members, as used
herein, are substances that reduce (partially, substantially, or
completely block) the activity of one or more members of the TRPV1
family, that is, Trpv1, among others. The substances may be
compounds (small molecules of less than about 10 kDa, peptides,
nucleic acids, lipids, etc.), complexes of two or more compounds,
and/or mixtures, among others. Furthermore, the substances may
inhibit TRPV1 family members by any suitable mechanism including
competitive, noncompetitive, uncompetitive, mixed inhibition,
and/or by changing a subject's pH, among others. The expression
"TRPV1 inhibitor" may refer to a product which, within the scope of
sound pharmacological judgment, is potentially or actually
pharmaceutically useful as an inhibitor of TRPV1, and includes
reference to substances which comprise a pharmaceutically active
species and are described, promoted, and/or authorized as a TRPV1
inhibitor. The strength of inhibition for a selective inhibitor may
be described by an inhibitor concentration at which inhibition
occurs (e.g., an IC.sub.50 (inhibitor concentration that produces
50% of maximal inhibition) or a K.sub.i value (inhibition constant
or dissociation constant)) relative to different TRPV1 family
members.
[0048] Any suitable TRP' V I inhibitor or combination of inhibitors
may be used in the methods and compositions herein. For example, a
subject may be treated with a TRIVP1 selective inhibitor and a
nonselective TRPV1 inhibitor. [0049] TRPV1 inhibitors include, but
are not limited to:
TABLE-US-00001 [0049] (N-(4-tertiarybutylphenyl)-4-(3- Bio Trend
chloropyridin-2-yl)tetrahydropyrazine- (Switzerland)
1(2H)-carboxamide (BCTC) N-(3-Methoxyphenyl)-4-chlorocinnamide
Neurosci. Lett. (SB-366791) 385:137-142
1-Isoquinolin-5-yl-3-(4-trifluoromethyl- Eur. J. Pharmacol.
benzyl)-urea (A-425619) 596:62-69
(2E)-N-(2,3-Dihydro-1,4-benzodioxin-6- J. Med. Chem.
yl)-3-[4-(1,1-dimethylethyl)phenyl]-2- 50:3515-3527 propenamide
(AMG-9810) (AZD1386) Phase II- AstraZeneca
2-Acetylamino-4-[6'-(4-trifluoro-
methylphenyl)-pyrimidin-4'-yl-oxy]- benzothiazole (AMG517)
N-(2-bromophenyl-N'-[((R)-1-(5- Phase II
trifluoromethyl-2-pyridyl)pyrrolidin-3-yl)] urea (SB705498)
N-(2-bromophenyl)-N'-{2-[ethyl(3- methylphenyl)amino]ethyl}-urea
(SB-452533) ((R)-(5-tert-butyl-2,3-dihydro-1H-inden-
1-yl)-3-(1H-indazol-4-yl)-urea (ABT-102)
N-(Isoquinolin-5-yl)-N'-[spiro-(cyclo- Phase II
butane-1,2'-(3',4'-dihydro- benzopyran-4'-yl))]-urea (GRC-6211)
(2R)-4-(3-chloro-2-pyridinyl)-2-methyl-
N-[4-(trifluoromethyl)phenyl]-1- piperazinecarboxamide
4-(4'-Trifluoromethyl-anilino)-7-(3'- Phase II
trifluoromethyl-pyridin-2-yl)-quinazoline (MK-2295) JYL 1421 Eur.
J. Pharmacol. 517:35-44 N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-
tetrahydro-7,8-dihydroxy-2H-2-benzazepine- 2-carbothioamide
(Capsazapine) (5R*,8R*,6E,9E)-5,8-Dimethyl-4-
methylenetetradeca-6,9-dienoic acid
1-(3-Fluorobenzyl)-2-(N-(1,2-dimethyl-
1,3-isoindazol-5-yl)-acetamido)-{pyridine- [3,4-b]-pyrrole}
(SAR-115740) N-(4-chlorobenzyl)-N'-(1-methyl-1H- Abbott
indazol-4-yl)urea, Laboratories
N-(4-tert-butylbenzyl)-N'-(1-methyl- (20100249203)
1H-indazol-4-yl)urea, N-(3-fluoro-4-(trifluoromethyl)benzyl)-
N'-(1-methyl-1H-indazol-4-yl)-urea,
N-(4-fluoro-3-(trifluoromethyl)-benzyl)-
N'-(1-methyl-1H-indazol-4-yl)-urea,
N-(3,4-dichlorobenzyl)-N'-(1-methyl- 1H-indazol-4-yl)urea,
N-(2,4-dichlorobenzyl)-N'-(1- methyl-1H-indazol-4-yl)urea,
N-(4-ethylbenzyl)-N'-(1-methyl-1H- indazol-4-yl)urea,
N-(2-chlorobenzyl)-N'-(1-methyl-1H- indazol-4-yl)urea,
N-(4-fluorobenzyl)-N'-(1-methyl-1H- indazol-4-yl) urea,
N-(2-fluorobenzyl)-N'-(1-methyl-1H- indazol-4-yl)urea,
N-[1-(bromophenyl)ethyl-N'-(1- methyl-1H-indazol-4-yl)urea.
N-(1-methyl-1H-indazol-4-yl)-N'-{4-
[(trifluoromethyl)thio]benzyl}urea.
1-(2,3-dichlorophenyl)-3-[2-(N-ethyl- 3-methylanilino)ethyl]urea.
1-[2-(N-ethyl-3-methylanilino)ethyl]-3- naphthalen-1-ylurea
1-(4-bromophenyl)-3-[2-(N-ethyl-3- methylanilino)ethyl]urea
1-(3-bromophenyl)-3-[2-(N-ethyl-3- methylanilino)ethyl]urea
1-(chlorophenyl)-3-[2-(N-ethyl-3- methylanilino)ethyl]urea
1-[2-(N-ethyl-3-methylanilino)ethyl]- 3-(2-fluorophenyl)urea
1-[2-{N-ethyl-3-methylanilino)ethyl]- 3-(2-methylphenyl)urea
1[2-(N-ethyl-3-methylanilino)ethyl]- 3-phenylurea
2-[(2-bromophenyl)carbamoylamino)
ethyl]-ethylmethyl-(3-methylphenyl) azanium iodide
1-(2-bromophenyl)-3-[2-(N-ethyl-3-
fluoro-4-methylanilino)ethyl]urea 1-(2-bromophenyl)-3[2-(N-ethyl-
3,4-difluoroanilino)ethyl]urea 1-(2-bromophenyl)-3[2-(N-ethyl-
3-fluoroanilino)ethyl]urea 1-(2-bromophenyl)-3[2-(N-ethyl-4-
methylanilino)ethyl]urea 1-(2-bromophenyl)-3-[2-(N-ethyl-
2-methylanilino)ethyl]urea 1-(2-bromophenyl)-3-[2-(N-
ethylanilino)ethyl]urea N-[2-[(2-bromophenyl)carbamoyl-
amino]ethyl]-N-(3-methylphenyl) acetamide
1-[2-{N-benzyl-3-methylanilino) ethyl]-3-(2-bromophenyl)urea
1-(2-bromophenyl)-3-[2-(2,3-dimethyl- anilino)ethyl]urea
1-(2-bromophenyl)-3-[2-(3-methyl- anilino)ethyl]urea
1-(2,5-dichlorophenyl)-3-[2-(N-ethyl-3- methylanilino)ethyl]urea
2,6-bis-(4-hydroxy-3-methoxybenzylidene) cyclohexanone (BHMC)
4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-
trifluoromethylphenyl]piperidine-1-carboxamide
4-fluoro-4(pyridin-2-yl)N-[4-trifluoro-
methylphenyl]piperidine-1-carboxamide
4-fluoro-4(pyridine-2-yl)N-[4-trifluoro-
methylbenzyl]piperidine-1-carboxamide
2-{4-fluoro-1-[4-trifluoromethylbenzoyl] piperidin-4-yl}pyridine
2-(4-fluoro-1-{[4-trifluoromethyl-
phenyl]acetyl}piperidin-4-yl)pyridine
2-(4-fluoro-1-{3-[4-trifluoromethyl-
phenyl]propanoyl}piperidin-4-yl)pyridine
4-fluoro-4-(1-methyl-1H-imidazol-2-yl)-N-[4-
trifluoromethylphenyl]piperidine-1-carboxamide
4-methoxy-4-pyridin-2-yl-N-[4-trifluoro-
methylphenyl]piperidine-1-carboxamide
4-methoxy-4-pyridin-2-yl-N-[4- trifluoromethylbenzyl]piperidine-1-
carboxamide 4-fluoro-N-(4-isopropylphenyl)-4-(3-methyl-
pyridin-2-yl)piperidine-1-carboxamide
4-fluoro-4-(3-methylpyridin-2-yl-N-{4-
[1,2,2,2-tetrafluoro-1-trifluoromethyl-ethyl]
phenyl}piperidine-1-carboxamide
N-(4-Tert-butylphenyl)-4-fluoro-4-(3-methyl-
pyridin-2-yl)piperidine-1-carboxamide
4-fluoro-4-(3-methylpyridin-2-yl)-N- [4-(pentafluoro-lambda(sup
6)-sulfanyl) phenyl]piperidine-1-carboxamide
N-(4-Butylphenyl)-4-fluoro-4-(3-methylpyridin-
2-yl)piperidine-1-carboxamide
N-(4-Benzylphenyl)-4-fluoro-4-(3-methyl-
pyridin-2-yl)piperidine-1-carboxamide
N-biphenyl-4-yl-4-fluoro-4-(3-methylpyridin-
2-yl)piperidine-1-carboxamide
4-fluoro-4-(3-methylpyridin-2-yl-N-[5-trifluoro-
methylpyridin-2-yl]piperidine-1-carboxamide
4-(3-chloropyridin-2-yl)-4-fluoro-N-[4-trifluoro-
methylphenyl]piperidine-1-carboxamide
4-fluoro-4-(3-fluoropyridin-2-yl)-N-[4-
trifluoromethylphenyl]piperidine-1- carboxamide
4-fluoro-4-(3-methoxypyridin-2-yl)-N-[4-
trifluoromethylphenyl]piperidine-1- carboxamide
4-fluoro-4-(3-methylpyridin-2-yl)-
N-[4-trifluoromethylphenyl]piperidine-1- carbothioamide
N'-cyano-4-fluoro-4-(3- methylpyridin-2-yl)-N-
[4-trifluoromethylphenyl]piperidine-1- carboximidamide
4-fluoro-4-(3-methylpyridin-2-yl)-N'-(1- phenylpiperidin-4-yl)-
N[4-trifluoro-methylphenyl]piperidine- 1-carboximidamide
4-fluoro-4-phenyl-N-[4-trifluoromethylphenyl]
piperidine-1-carboxamide +/-)-(syn)-4-fluoro-2-methyl-4-(3-
methylpyridin-2-yl)-N-[4-trifluoromethyl-
phenyl]piperidine-1-carboxamide
4-(fluoromethyl)-4-pyridin-2-yl-N-[4-
trifluoromethylphenyl]piperidine-1-carboxamide syn- and
anti-3-fluoro-3-pyridin-2-yl-N-[4-
trifluoromethylphenyl]-8-azabicyclo[3.2-.1] octane-8-carboxamide
3-fluoro-3-pyridin-2-yl-N-[4-trifluoromethyl-
phenyl]-8-azabicyclo[3.2.1]octane-8- carboxamide
4-fluoro-4-pyrimidin-2-yl-N-[4-
trifluoromethylphenyl]piperidine-1-carboxamide
4-fluoro-4-(3-phenylpropyl)-N-[4-
trifluoromethylphenyl]piperidine-1-carboxamide
2-[4-fluoro-4-(3-methylpyridin-2-yl)
piperidin-1-yl]-6-trifluoromethyl-1H- benzimidazole
2-(4-fluoro-4-pyridin-2-ylpiperidin-1-yl)-
6-(trifluoromethyl)-1H-benzimidazole
4-fluoro-N-[4-trifluoromethylphenyl]-4-[3-
trifluoromethylpyridin-2-yl]piperidine- 1-carboxamide
4-fluoro-N-(4-methylphenyl)-4-(3-methyl-
pyridin-2-yl)piperidine-1-carboxamide
N-(4-ethylphenyl)-4-fluoro-4-(3-methyl-
pyridin-2-yl)piperidine-1-carboxamide
N-(4-chlorophenyl)-4-fluoro-4-(3-methyl-
pyridin-2-yl)piperidine-1-carboxamide
4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-
trifluoromethoxyphenyl]piperidine- 1-carboxamide
N-(4-cyanophenyl)-4-fluoro- 4-(3-methylpyridin-2-yl)piperidine-1-
carboxamide N-[4-dimethylaminophenyl]-4-fluoro-
4-(3-methylpyridin-2-yl)piperidine-1- carboxamide 1-(2-(3
3-dimethylbutyl)-4-(trifluoromethyl)
benzyl)-3-(1-methyl-1H-indazo-1-4-yl)urea
N-acetyl-1-phenyl-1-leucinamide Nobilamides A-H (Lin 2011) SB366791
(Gunthrope 2004) TRPV1 antagonists (Messeguer 2006) Capsaicin
receptor ligands PCT WO 02/08221
[0050] Pharmaceutically acceptable salts forming part of this
invention include base addition salts such as alkali metal salts
like Li.sup.+, Na.sup.+, and K.sup.+ salts, alkaline earth metal
salts like Ca.sup.2+ and Mg.sup.2+ salts, salts of organic bases
such as lysine, arginine, guanidine, diethanolamine, choline and
the like, ammonium or substituted ammonium salts. Salts may include
acid addition salts which are sulphates, nitrates, phosphates,
perchlorates, borates, hydrohalides, acetates, tartrates, maleates,
citrates, succinates, palmoates, methanesulphonates, benzoates,
salicylates, hydroxynaphthoates, benzenesulfonates, ascorbates,
glycerophosphates, ketoglutarates and the like. The term
pharmaceutically acceptable solvates includes combinations of
solvent molecules with molecules or ions of the solute compound
(the inhibitor). Pharmaceutically acceptable solvates may be
hydrates or comprising other solvents of crystallization such as
alcohols.
[0051] Preferred salts for the list of compounds above are
hydrochloride, hydrobromide, sodium, potassium or magnesium.
[0052] The present invention provides pharmaceutical compositions
containing a TRPV1 inhibitor or mixture of TRPV1 inhibitors. An
inhibitor may be in the form if a pharmaceutically acceptable salt
or a pharmaceutically acceptable solvate in combination with the
usual pharmaceutically employed carriers, diluents and the
like.
[0053] The pharmaceutical composition may be in the forms normally
employed, such as tablets, capsules, powders, syrups, solutions,
suspensions and the like, may contain flavorants, sweeteners etc.
in suitable solid or liquid carriers or diluents, or in suitable
sterile media to form injectable solutions or suspensions. Such
compositions typically contain from 1 to 25%, preferably 1 to 15%
by weight of active compound, the remainder of the composition
being pharmaceutically acceptable carriers, diluents, excipients or
solvents.
[0054] Suitable pharmaceutically acceptable carriers include solid
fillers or diluents and sterile aqueous or organic solutions. The
active compound will be present in such pharmaceutical compositions
in the amounts sufficient to provide the desired dosage in the
range as described above. Thus, for oral administration, the
compounds can be combined with a suitable solid or liquid carrier
or diluent to form capsules, tablets, powders, syrups, solutions,
suspensions and the like. The pharmaceutical compositions, may, if
desired, contain additional components such as flavorants,
sweeteners, excipients and the like. Pharmaceutically acceptable
solutions in sesame or peanut oil, aqueous propylene glycol and the
like can be used, as well as aqueous solutions of water-soluble
pharmaceutically-acceptable acid addition salts or alkali or
alkaline earth metal salts of the compounds. The injectable
solutions prepared in this manner can then be, administered
intravenously, intraperitonally, subcutaneously, or
intramuscularly, with intramuscular administration being preferred
in humans.
[0055] The pharmaceutical compositions of the invention are shown
to be effective by tests in animal models. The pharmaceutical
compositions of the invention are thus effective for treatment of
cardiac hypertrophy in a mammalian subject, including cardiac
remodeling, cardiac fibrosis, apoptosis, hypertension, or heart
failure. The compositions may also be administered for prophylactic
treatment of cardiac hypertrophy in a mammalian subject.
[0056] Generally, the effective dose for treating a particular
condition in a patient may be readily determined and adjusted by
the physician during treatment to alleviate the symptoms or
indications of the condition or disease. Generally, a daily dose of
active compound (inhibitor) in the range of about 0.01 to 1000
mg/kg of body weight is appropriate for administration to obtain
effective results. The daily dose may be administered in a single
dose or divided into several doses. In some cases, depending upon
the individual response, it may be necessary to deviate upwards or
downwards from the initially prescribed daily dose. Typical
pharmaceutical preparations normally contain from about 0.2 to
about 500 mg of active compound of formula I and/or its
pharmaceutically active salts or solvates per dose.
[0057] The term "therapeutically effective amount,"
"pharmaceutically effective amount," or "effective amount" refers
to that amount of a compound or mixture of compounds of Formula I
that is sufficient to effect treatment, as defined below, when
administered alone or in combination with other therapies to a
mammal in need of such treatment. The term "mammal" as used herein
is meant to include all mammals, and in particular humans. Such
mammals are also referred to herein as subjects or patients in need
of treatment. The therapeutically effective amount will vary
depending upon the subject and disease condition being treated, the
weight and age of the subject, the severity of the disease
condition, the dosing regimen to be followed, timing of
administration, the manner of administration and the like, all of
which can readily be determined by one of ordinary skill in the
art.
[0058] The term "treatment" or "treating" means any treatment of a
disease in a mammal, including: [0059] a) preventing the disease or
condition, that is, causing the clinical symptoms of the disease
not to develop; [0060] b) inhibiting the disease, that is, slowing
or arresting the development of clinical symptoms; and/or [0061] c)
relieving the disease, that is, causing the regression of clinical
symptoms.
[0062] The invention is explained in detail in the examples given
below which are provided by way of illustration only and therefore
should not be construed to limit the scope of the invention.
EXAMPLE 1
[0063] Trpv1 Knockout Suppresses Pressure Overload Cardiac
Hypertrophy
[0064] Pressure overload cardiac hypertrophy was modeled by
transverse aortic constriction (TAC) in 8 week old male TRPV1
knockout mice (Caterina, 2000) and wild type controls. Sham control
mice underwent the same procedure except for aortic constriction.
Baseline pressures were assessed proximal; and distal, to the TAC
banding site, as analyzed by Doppler echo. There was no significant
difference between Trpv1.sup.-/- and control animals. Transthoracic
echocardiography (Echo) was performed using a high resolution Vevo
770.TM. Echo system with a 30 MHz transducer (Visual Sonics,
Toronto, Canada) in unanethestized mice, in order to assess heart
dimensions during pressure overload cardiac hypertrophy, as
compared to sham controls. Mice were sacrificed at 6 weeks post
TAC, and hearts were collected for histological sectioning, RNA
extraction, and protein analysis by Western blot and other methods.
Gravimetric analyses of cardiac hypertrophy at 6 weeks after TAC,
indicate that the heart weight/body weight ratio, as well as the
heart weight/tibia length ratio increases more in the control
animals than the Trpv1.sup.-/- hearts, as compared to sham animals.
Referring to FIG. 1A the effects of TAC are shown in banded animals
on left ventricle (LV) internal diameter, diastolic, from baseline
to 6 weeks in C57BU6 WT (n=17), Trpv1.sup.-/- (n=15), compared to
those effects (FIG. 1B) in sham operated animals (n=9) (middle). In
FIG. 1C the effects of TAC are shown on rate of LVID change/day in
C57BU6 WT (n=17) versus Trpv1.sup.-/- (n=15). (P<0.05) WT and
Trpv1.sup.-/- (p<0.05). In FIG. 1D the effects of TAC on treated
animals on LV function (fractional shortening) from baseline to 8
weeks in C57BU6
[0065] WT (n=17), Trpv1.sup.-/- (n=15), and on sham operated
animals C57BU6 WT (n=9), Trpv1.sup.-/- (n=9). Most notably, in FIG.
1C there is shown a significant increase in the rate of left
ventricle internal diameter, in control mice =17) as compared to
Trpv.sup.-/- mice (n=15) (P<O.OS). Sham Trpv.sup.-/- and control
animals did not differ significantly. FIG. 1D indicates that left
ventricular function as measured by percentage fractional
shortening
[0066] (% FS=([LVDd-LVDs]/[LVDd).times.100) appears to be preserved
in Trpy1.sup.-/- mice from zero to four weeks, as compared to
control animals, but declines from four to six weeks.
EXAMPLE 2
[0067] Trpv1 Knockout Alters Hypertrophic Markers in Pressure
Overload Cardiac Hypertrophy
[0068] Multiple hypertrophy markers were analyzed from extracted
heart lysates and sections. Overall, major hypertrophic indicators
like collagen (FIG. 2), atrial natriuretic peptide and TGF.beta.
(FIGS. 3A, 3B) show a significant reduction in expression in
Trpv1.sup.-/- mice modeled with pressure overload cardiac
hypertrophy for six weeks. In FIG. 2 quantification of picrosirius
staining in TRPV1 deficient and C57BU6 WT controls are shown,
performed in Image J, excluding peri vascular tissue.
[0069] Each heart was stained in replicates of 3. Image J was used
to analyze 5 images from each heart (.times.3) and determine the
pixel count in each field as percentage of overall number of pixels
for a ratio of red-stained collagen/fiber:total tissue area. Images
were averaged for each animal and graphed in Prism GraphPad;
p<0.001 between TRPV1 banded and C57BU6 banded. In FIG. 3A are
shown TGF-beta RNA expression in C5781/6 (n=7), and TRPV1-/- (n=4)
(p<0.0328). In FIG. 3B are shown ANP expression in C5781/6
(n=15), and TRPV1. (n=14) (p<0.0431). Total RNA was isolated
from homogenized hearts with Trizol (Molecular Research Center) and
further purified with a RNA isolation kit (Mo Bio Laboratories,
Inc). Single-stranded eDNA was synthesized from 1 .mu.g of total
RNA using a cDNA synthesis kit (Qiagen), The mRNA levels were
quantified by RT-PCR using SYBR green method.
EXAMPLE 3
[0070] Extracellular Matrix Remodeling
[0071] The composition of cardiac tissue changes during the
development of ventricular hypertrophy and leads to structural
remodeling of the inyocardium. One of these changes is related to
the disruption of the equilibrium between the synthesis and
degradation of collagen, which results in an excessive accumulation
of collagen type I and III fibers within the myocardium. As
collagen and other extracellular matrix components accumulate in
the interstitial space, myocardial stiffness increases and
diastolic and systolic dysfunction occurs. Prior data indicates
less interstitial collagen deposition in the Trpvr1.sup.-/- mice
than control mice, with pressure overload cardiac hypertrophy
(Buckley 2011). Similar results were obtained by collagen protein
assay (Sircol.TM., Biocolor, Northern Island), and RealTime-PCR.
Changes are also seen in the enzymes responsible for degradation of
collagen, the matrix metalloproteinases (MMPs). (FIGS. 4A, 4B)
[0072] The RNA expression changes in sham vs. TAC treated mice
shown in FIG. 4A for MMP2 by RT-PCR (p<0.05), C5781/6 (n=15) and
Trpv1.sup.-/- (n=14), and in FIG. 4B for MMP13 by RT-PCR.
Generally, suppression of Mmp and Timp transcription is observed
more in the Trpv1.sup.-/- mice than in control mice 6 weeks post
TAC. However, MMP13 appears upregulated. MMP13 targets collagen
type I, II and III and may serve to protect tissue from fibrosis,
Mast cell chymase (CMA 1) message and protein is expressed less in
Trpv1.sup.-/- mice than in control mice 8 weeks post TAC (FIG. 7D).
CMA1 is a chymotryptic serine proteinase that belongs to the
peptidase family S1. It is described as expressed in mast cells but
appears to be expressed in other tissues and cell types. It
functions in the degradation of the extracellular matrix and the
generation of vasoactive peptides. In the heart and blood vessels,
this protein, rather than angiotensin converting enzyine (ACE), is
largely responsible for converting angiotensin I to the vasoactive
peptide angiotensin II in the renin-angiotensin system. This system
controls blood pressure and is involved in the pathogenesis of
hypertension, cardiac hypertrophy, and heart failure.
EXAMPLE 4
[0073] Involvement of TRPV1 in the Progression of Cardiac
Hypertrophy
[0074] Mice lacking functional TRPV1 and control mice with
wild-type TRPV1 were modeled for pressure overload cardiac
hypertrophy. Heart dimensions and function were measured and
compared over time using unanesthestized transthoracic
echocardiography and hearts were harvested eight weeks later for
molecular, biochemical and histological analysis. Heart dimensions
and function were better preserved in mice lacking functional
TRPV1. Cellular hypertrophy, markers for hypertrophy, fibrosis and
apoptosis were also significantly reduced in these mice, indicating
involvement of TRPV1 in the progression of cardiac hypertrophy.
[0075] Pressure Overload Model
[0076] To test the involvement of TRPV1 in the remodeling
associated with cardiac hypertrophy and heart failure, ten-week-old
male B6.129X1-Trpv1tm1Jul/J mice (TRPV1 KO), (Catering, 2000) and
age/sex matched C57BL/6J (WT) control mice were subjected to acute
pressure overload by transverse aortic constriction (TAC). Sham
operated control mice from both strains underwent an identical
surgical procedure except for actual aortic constriction. TRPV1 KU
TAC mice and WT TAC mice showed no difference in baseline
pressures, assessed immediately distal to the TAC banding site by
Doppler echocardiography.
[0077] Gravimetric Analysis of the Heart, Alter Pressure Overload
Cardiac Hypertrophy
[0078] This analysis reveals that TAC treated hearts were 28%
heavier in WT TAC mice than TRPV1 KO TAC mice. When normalized to
body weight and tibia length, the heart weight/body weight ratio
and the heart weight/tibia length ratio were also significantly
greater in WT TAC mice than TRPV1 KO TAC mice. (FIGS. 5A and 5B)
Mice lacking functional TR PV1 present preservation of heart
structure and function during pressure overload cardiac
hypertrophy, ( WTTR PV1 KO). FIG. 5A is a graph showing heart
weight/body weight (HW/BW) and heart weight/tibia length (HW/TL).
There is significant difference in HW/BW between WT Sham and TAC
mice (p=0.027), WT TAC and TR PV1 KO TAC mice (p=0.019) and TR PV1
KO Sham and TAC mice (p=0.045). FIG. 5B shows that there is
significant difference in HW/TL between WT Sham and TAC mice
(p=0.034), WT TAC and TR PV1 KO TAC mice (p=0.03), but not between
TR PV1 KO Sham and TR PV1 KO TAC mice (p=0.095).
[0079] Heart Structure and Function are Maintained during Pressure
Overload Cardiac Hypertrophy in Mice Lacking Functional TRPV1
[0080] End-diastolic left ventricular internal diameter (LVIDd) was
analyzed for eight weeks following TAC by transthoracic
echocardiographic analysis. In WT TAC mice, LVIDd began to increase
at two weeks and plateaued at approximately six weeks. The TRPV1 KO
TAC mice showed no change in LVIDd until six weeks. FIG. 5C shows
the analysis of left ventricular internal diameter end-diastolic
(LVIDd) from zero to eight weeks in WT (n=6) and TR PV1 KO mice
(n=8). The TAC WT control mice start increasing their internal
diameter at two weeks, whereas in TAC TR PV1 KO mice there is a
delay until six weeks post TAC treatment. The rate of increase in
LVIDd is significantly greater in WT TAC mice than in TRPV1 KO TAC
mice (FIG. 5D) between weeks two and six post TAC. FIG. 5D shows
the rate of change in LVIDd from zero to eight weeks was
significant (p=0.013) between TAC WT and TAC TR PV1 KO. Heart
function was analyzed by left ventricular ejection fraction (% EF).
Heart function declined in WT mice from approximately two to six
weeks post TAC treatment, but was preserved in TRPV1 KO TAC mice
over the same period of time. FIG. 5E shows a reduction in function
starting at two weeks in TAC WT mice, but TAC TR PV1 KO mice are
protected until six weeks, the percent change in ejection fraction
was significantly different at six weeks (p=0.039). The change in
ejection fraction at six weeks is significantly different between
WT TAC mice and TRPV1 KO TAC mice (FIG. 5F).
[0081] Mice Lacking Functional TRPV1 are Protected from Hypertrophy
and Apoptosis after Modeled Pressure Overload Cardiac
Hypertrophy
[0082] The degree of cellular hypertrophy was examined by staining
of the plasma membranes with fluorescently-labeled wheat germ
agglutinin (WGA). Cell sizes were compared by imaging and computer
aided measurement of the cross-sectional area of cardiomyocytes.
This comparison reflects the degree of cellular hypertrophy between
samples. (Shiojima, 2005) The data show a significant increase in
the cardiomyocyte cross sectional area of WT TAC compared to TRPV1
KO TAC mice (FIG. 6A). Measurement of cardiomyocyte cross sectional
area, was significantly different between TAC WI mice and TAC TR
PV1 KO mice (p=0.025, n=100), 8 weeks post TAC treatment. This
shows that, at the cellular level, TRPV1 KO mice develop less
cardiac hypertrophy than WT mice, in response to modeled pressure
overload cardiac hypertrophy. To further compare the degree of
hypertrophy between TRPV1 and WT mice, additional markers of
hypertrophy, apoptosis and heart failure were assessed. Plasma
concentrations of the circulating hormone atrial natriuretic
peptide (ANP) arid the growth factor TGFbeta increase during heart
failure and are considered late markers of cardiac hypertrophy.
Therefore, expression of ANP and TGFbeta was analyzed by RT-PCR of
mRNA isolated from heart tissue. Significantly greater increases
were shown in ANP and TGFbeta transcript levels in WT TAC mice than
in TRPV1 KO TAC mice. FIG. 6B shows that expression levels of
atrial natriuretic peptide (ANP and TGFbeta transcripts were
significantly greater in TAC WT mice than in TRPV1 KO mice
(p=0.037, p=0.007) relative to control mice. Western blot analysis
confirmed that there was a significant increase (FIG. 6C) in ANP
protein expression in TAC WT mice compared to TAC TRPV1 KO mice.
These data show that protection from the stress and or signaling
systems associated with the hypertrophic transcriptional responses
is observed in the TRPV1 KO mice. The degree of cellular apoptosis
by measurement of cleaved caspase-3 protein in heart tissue lysates
from TAC and sham treated WT and TRPV1 KO mice were assessed.
Analysis of western blot densitometry of heart tissue lysates
showed significantly less caspase-3 cleavage in TRPV1 KO TAC mice
than in WT TAC mice. As expected, WT sham and TRPV KO sham mice
showed no apparent caspase-3 cleavage. (FIG. 6D) These results show
that TAC-induced cardiac apoptosis is reduced in TRPV1 KO mice.
There is protection from the stress and or signaling associated
with cardiac hypertrophy in the TRPV1 KO mice.
[0083] Mice Lacking Functional TRPV1 Show Reduced Fibrosis, Tissue
Remodeling and Inflammatory Markers After Modeled Pressure Overload
Cardiac Hypertrophy
[0084] During the development of ventricular hypertrophy, the
composition of cardiac tissue changes, leading to structural
remodeling of the myocardium. For example, the disruption of the
equilibrium between the synthesis and degradation of collagen
results in an excessive accumulation of collagen type I and III
fibers within the myocardium. As collagen and other extracellular
matrix components accumulate in the interstitial space, myocardial
stiffness increases, and diastolic and systolic dysfunction occurs.
Collagen III levels were analyzed by RT-PCR and total collagen by
histological staining, in heart tissue from Sham and. TAC, WT and
TRPV1 KO mice. It was shown that collagen III transcript levels
(FIG. 7A), and interstitial collagen deposition (FIG. 7B) were
reduced in hearts isolated from TRPV1 KO TAC mice compared to WT
TAC mice. Mice lacking functional TR PV1 present with less
interstitial fibrosis and tissue remodeling enzymes than WT control
mice, eight weeks post TAC treatment. ( WT TR PV1 KO). FIG. 7A
shows an increase in the expression of Collagen III transcript was
significantly greater in TAC WI mice than in TAC TRPV1 KO mice
(p=0.037, 0.007). In FIG. 7B mice lacking functional TR PV1 present
with less interstitial fibrosis and tissue remodeling enzymes than
WT control mice, eight weeks post TAC treatment. ( WT TR PA1 KO).
There was a significant increase in MMP2 transcripts in hearts from
WT TAC mice compared to hearts from TRPV1 TAC mice. FIG. 7C shows
that increases in the expression matrix metalloproteinase-2 (MM
P-2) transcript was significantly less in TAC treated TR PV1 KO
mice than TAC treated WT mice (p=0.049). There was also
significantly less expression of Chymase (CMA1) transcript
(p=0.049). Mast cell chymase, CMA1, is a chymotryptic serine
proteinase that belongs to the peptidase family S1. It functions in
the degradation of the extracellular matrix and in the generation
of vasoactive peptides. In the heart and blood vessels, it is CMA1,
rather than angiotensin converting enzyme (ACE), that is largely
responsible for converting angiotensin I to the vasoactive peptide
angiotensin II. The data in FIGS. 7D and 7E show that CMA1
transcripts and protein are expressed at significantly higher
levels in hearts from WT TAC mice than TRPV1 KO TAC mice. There was
significantly less expression of Chymase (CMA1) transcript
(P=0.049), and Chymase protein (p=0,0218) in isolated heart tissue
from TAC TR PV1 KO mice than TAC LVT mice (CMA1 integrated density
was normalized to GAPDH loading control). The data show that the
functional knockout of TRPV1 in mice allows for the preservation of
heart structure and heart function under modeled pressure overload.
Concomitant with this protection is the down-regulation of multiple
protein and transcriptional markers associated with initiation and
the progression of hypertrophy, apoptosis, fibrosis, and heart
failure. This data show that TRPV1 has a role as either an
initiating stressor, or an upstream signaling transducer of the
hypertrophic transcriptional response in the heart.
Example 5
[0085] Mice Treated with the TRPV1 Antagonist BCTC Present
Preservation of Heart Mass, Structure and Function During Pressure
Overload Cardiac Hypertrophy
[0086] The following tests show that treatment by continuous
administration using osmotic pumps with the TRPV1 antagonist, BCTC,
in WT mice exposed to TAC confirms the findings from tests of the
prior Examples.
[0087] Osmotic pump installation. Long term (up to 42 day) infusion
of drugs can be accomplished by insertion of osmotic pumps without
the need for repeated injection. Mice are placed under a low plane
of anesthesia with an injection of Ketamine/Xylazine anesthetic (50
mg/10 mg.Kg) intraperitoneally (IP) 10 minutes prior to surgery. A
small area between shoulder blades is shaved and sterilized with
Povidine swab. A small incision is made in this area and blunt
dissected below skin to allow placement of an Alzet osmotic pump
(model 2006), previously loaded with the drug of choice under the
skin between the shoulder blades where it is inaccessible to the
mouse. Several stitches are applied to close the incision. The
mouse is placed in regular housing on a warming mat until
completely conscious, after which mice are then returned to regular
housing room.
[0088] The mice were subjected to pressure overload induced cardiac
hypertrophy by TAC while administered 4 mg/kg of BCTC (in 20%
wt/vol 2-Hydroxypropyl)-.beta.-cyclodextrin/PBS) throughout the
entire experiment using osmotic pumps (Alzet, Model 2006) pumping
continuously at a rate of 0.15ul/hr. The test was limited to
.about.42 days (max) by the function of the pumps, as such the
experiment was halted at 36 days post TAG, as pumps arc installed
previous to the TAC to allow recovery before the TAC surgery.
Analysis of heart weights 36 days post TAC revealed that the heart
weight/body weight ratio was significantly greater in vehicle
treated. WT mice than drug treated mice (p=0.035) (FIG. 8A).
Echocardiographic assessment of mice every 9 days for the duration
of the study showed that Vehicle and BCTC treated sham mice
(Vehicle Sham (n=2) and BCTC sham mice (n=2)) show no difference in
their left ventricular internal diameter (LVID,d) (FIG. 8B).
However, BCTC treated TAC mice (n=8) have a significantly smaller
LVIDd than the vehicle treated TAC mice (n=7) (FIG. 8C) from zero
to thirty six days, indicating that the TAG Vehicle control mice
start increasing their internal diameter at 9 days, whereas in TAG
BCTC treated mice, the diameter is maintained for the duration of
the test. The LVIDd is significantly different after 18 days
(p<0.001). This protection from dilation of the left ventricle
translates to a protection in the function of the heart as measured
by ejection fraction (% EF, p<0.05) and fractional shortening (%
FS). Both the % EF and % FS (p<0.01) of vehicle-treated TAG mice
declined steadily over the course of the study and was
significantly diminished at 36 days post TAG compared to
drug-treated mice (FIGS. 8D, 8E).
EXAMPLE 6
[0089] Mice Treated with the TRPV1 Antagonist BCTC Present
Histologically with Less Hypertrophy and Fibrosis than Vehicle
Control Mice, Thirty Six Days Post TAC Treatment
[0090] Histological analysis of the heart 36 days post TAG shows
that BCTC can protect the heart from cellular hypertrophy, and the
deposition of interstitial fibrosis. From stains of plasma
membranes (Wheat germ agglutinin-Alexa488) in heart tissue sections
it is shown that BCTC treated TAG mice, described in Example 5,
have smaller cardiac myocytes and less hypertrophy than vehicle
treated TAG control mice. Measurement of cardiomyocyte cross
sectional area shows significantly smaller myocytes in BCTC treated
TAG mice than vehicle treated TAG control mice (p=<0.01, n=100)
36 days post TAG treatment (FIG. 9A), indicating that BCTC can
protect the heart from hypertrophy at the cellular level. Less
histological staining with Picrosirius red which can indicate areas
of interstitial collagen deposition in isolated heart tissue
sections from BCTC treated TAG mice than vehicle treated control
mice indicates less interstitial collagen deposition. Analysis of
collagen staining by ImageJ (NIH) was used to determine the area of
collagen staining as a percentage of tissue area. The analysis
(FIG. 9B) indicates that there is significantly less interstitial
collagen in BCTC treated TAC mice than vehicle treated. TAC control
mice (p=0.05). This shows that BCTC can protect the heart from
fibrosis during pressure overload cardiac hypertrophy.
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Sequence CWU 1
1
12122DNAArtificialPRIMER FOR ANP 1agaaaccaga gagtgggcag ag
22221DNAArtificialPRIMER FOR ANP 2caagacgagg aagaagccca g
21320DNAArtificialPRIMER FOR TGF BETA 3tggagcaaca tgtggaactc
20418DNAArtificialPRIMER FOR TGF BETA 4cagcagccgg ttaccaag
18520DNAArtificialPRIMER FOR MMP2 5tggtgtggca ccaccgagga
20620DNAArtificialPRIMER FOR MMP2 6gcatcggggg agggcccata
20720DNAArtificialPRIMER FOR MMP9 7cggcacgcct tggtgtagca
20820DNAArtificialPRIMER FOR MMP9 8tcgcgtccac tcgggtaggg
20920DNAArtificialPRIMER FOR COLLAGEN III 9gaccgatgga ttccagttcg
201020DNAArtificialPRIMER FOR COLLAGEN III 10tgtgactcgt gcagccatcc
201122DNAArtificialPRIMER FOR CMA1 11agctcactgt gcgggaaggt ct
221221DNAArtificialPRIMER FOR CMA1 12ctcagggacc aggcagggct t 21
* * * * *