U.S. patent application number 15/633719 was filed with the patent office on 2017-10-26 for method for the treatment of fatty liver disease.
The applicant listed for this patent is Golden Biotechnology Corporation. Invention is credited to Chih-Ming Chen, Sheng-Yung Liu, Wu-Che Wen.
Application Number | 20170304224 15/633719 |
Document ID | / |
Family ID | 51529924 |
Filed Date | 2017-10-26 |
United States Patent
Application |
20170304224 |
Kind Code |
A1 |
Liu; Sheng-Yung ; et
al. |
October 26, 2017 |
METHOD FOR THE TREATMENT OF FATTY LIVER DISEASE
Abstract
The invention provides a method of treating, inhibiting and/or
preventing fatty liver disease in a patient in need thereof,
comprising administering an effective amount of a cyclohexenone
compound of the following formula (I) to said patient,
##STR00001##
Inventors: |
Liu; Sheng-Yung; (New Taipei
City, TW) ; Wen; Wu-Che; (New Taipei City, TW)
; Chen; Chih-Ming; (New Taipei City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Golden Biotechnology Corporation |
JERSEY CITY |
NJ |
US |
|
|
Family ID: |
51529924 |
Appl. No.: |
15/633719 |
Filed: |
June 26, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13801697 |
Mar 13, 2013 |
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15633719 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 36/07 20130101;
A61K 9/0056 20130101; A61K 9/4875 20130101; A61K 31/575 20130101;
A61K 9/0078 20130101; A61K 31/575 20130101; A61P 1/16 20180101;
A61K 2300/00 20130101; A61P 1/00 20180101; A61K 2300/00 20130101;
A61P 43/00 20180101; A61K 31/122 20130101; A61K 31/122
20130101 |
International
Class: |
A61K 31/122 20060101
A61K031/122; A61K 36/07 20060101 A61K036/07 |
Claims
1. A method of treating or inhibiting collagen deposition in a
fatty liver condition of a patient, comprising administering an
effective amount of a cyclohexenone compound of the following
formula (I) to said patient in need thereof, ##STR00014## wherein
each of X and Y independently is oxygen, NR.sub.5 or sulfur; R is a
hydrogen or C(.dbd.O)C.sub.1-C.sub.8 alkyl; each of R.sub.1,
R.sub.2 and R.sub.3 independently is a hydrogen, methyl or
(CH.sub.2).sub.m--CH.sub.3; R.sub.4 is NR.sub.5R.sub.6, OR.sub.5,
OC(.dbd.O)R.sub.7, C(.dbd.O)OR.sub.5, C(.dbd.O)R.sub.5,
C(.dbd.O)NR.sub.5R.sub.6, halogen, 5 or 6-membered lactone,
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, aryl, glucosyl, wherein the 5 or 6-membered
lactone, C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, aryl, and glucosyl are optionally
substituted with one or more substituents selected from
NR.sub.5R.sub.6, OR.sub.5, OC(.dbd.O)R.sub.7, C(.dbd.O)OR.sub.5,
C(.dbd.O)R.sub.5, C(.dbd.O)NR.sub.5R.sub.6, C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.3-C.sub.8
cycloalkyl, and C.sub.1-C.sub.8 haloalkyl; each of R.sub.5 and
R.sub.6 is independently a hydrogen or C.sub.1-C.sub.8alkyl;
R.sub.7 is a C.sub.1-C.sub.8alkyl, OR.sub.5 or NR.sub.5R.sub.6;
m=1-12; and n=1-12; or a pharmaceutically acceptable salt,
metabolite, solvate or prodrug thereof.
2. The method of claim 1, wherein said compound decreases blood
glucose and/or triglyceride level.
3. The method of claim 1, wherein said compound decreases fibrosis
of steatohepatitis liver cells.
4. The method of claim 1, wherein said fatty liver condition is
caused by non-chemical injury.
5. The method of claim 3, wherein said fatty liver condition is a
nonalcoholic fatty liver disease.
6. The method of claim 1, wherein the cyclohexenone compound is
isolated from the organic solvent extracts of Antrodia camphorata,
or prepared synthetically or semi-synthetically.
7. The method of claim 1, wherein R is a hydrogen,
C(.dbd.O)C.sub.3H.sub.8, C(.dbd.O)C.sub.2H.sub.5, or
C(.dbd.O)CH.sub.3.
8. The method of claim 1, wherein R.sub.1 is a hydrogen or
methyl.
9. The method of claim 1, wherein R.sub.2 is a hydrogen, methyl,
ethyl, propyl, butyl, pentyl or hexyl.
10. The method of claim 1, wherein R.sub.4 is halogen, NH.sub.2,
NHCH.sub.3, N(CH.sub.3).sub.2, OCH.sub.3, OC.sub.2H.sub.5,
C(.dbd.O)CH.sub.3, C(.dbd.O)C.sub.2H.sub.5, C(.dbd.O)OCH.sub.3,
C(.dbd.O)OC.sub.2H.sub.5, C(.dbd.O)NHCH.sub.3,
C(.dbd.O)NHC.sub.2H.sub.5, C(.dbd.O)NH.sub.2, OC(.dbd.O)CH.sub.3,
OC(.dbd.O)C.sub.2H.sub.5, OC(.dbd.O)OCH.sub.3,
OC(.dbd.O)OC.sub.2H.sub.5, OC(.dbd.O)NHCH.sub.3,
OC(.dbd.O)NHC.sub.2H.sub.5, or OC(.dbd.O)NH.sub.2.
11. The method of claim 1, wherein R.sub.4 is
C.sub.2H.sub.5C(CH.sub.3).sub.2OH,
C.sub.2H.sub.5C(CH.sub.3).sub.2OCH.sub.3, CH.sub.2COOH,
C.sub.2H.sub.5COOH, CH.sub.2OH, C.sub.2H.sub.5OH, CH.sub.2Ph,
C.sub.2H.sub.5Ph, CH.sub.2CH.dbd.C(CH.sub.3)(CHO),
CH.sub.2CH.dbd.C(CH.sub.3)(C(.dbd.O)CH.sub.3), 5 or 6-membered
lactone, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, aryl, and glucosyl, wherein 5 or 6-membered
lactone, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, aryl, and glucosyl are optionally
substituted with one or more substituents selected from
NR.sub.5R.sub.6, OR.sub.5, OC(.dbd.O)R.sub.7, C(.dbd.O)OR.sub.5,
C(.dbd.O)R.sub.5, C(.dbd.O)NR.sub.5R.sub.6, C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.3-C.sub.8
cycloalkyl, and C.sub.1-C.sub.8 haloalkyl.
12. The method of claim 1, wherein R.sub.4 is
CH.sub.2CH.dbd.C(CH.sub.3).sub.2.
13. The method of claim 1, wherein the compound is ##STR00015##
14. The method of claim 1, wherein the fatty liver condition is a
primary fatty liver disease or a secondary fatty liver disease.
15. The method of claim 14, wherein the primary fatty liver disease
is NASH.
16. The method of claim 1, wherein the fatty liver condition is
cirrhosis or fibrosis.
17. The method of claim 1, wherein the cyclohexenone compound, or a
pharmaceutically acceptable salt, metabolite, solvate or prodrug
thereof, is administered orally, parenterally or intravenously.
18. The method of claim 1, wherein the cyclohexenone compound, or a
pharmaceutically acceptable salt, metabolite, solvate or prodrug
thereof, is administered orally.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for the treatment
of fatty liver disease, and more particularly, to a method of
administering a cyclohexenone compound.
BACKGROUND OF THE INVENTION
[0002] Fatty liver refers to a pathogenic condition where fat
comprises more than 5% of the total weight of the liver. Fatty
liver and steatohepatitis are frequently found in people who intake
excessive alcohols and who have obesity, diabetes, hyperlipidemia,
etc. Nonalcoholic fatty liver disease (NAFLD) refers to a wide
spectrum of liver disease ranging from simple fatty liver
(steatosis), to nonalcoholic steatohepatitis (NASH), to cirrhosis
(irreversible, advanced scarring of the liver). All of the stages
of NAFLD have in common the accumulation of fat (fatty
infiltration) in the liver cells (hepatocytes). In NASH, the fat
accumulation is associated with varying degrees of inflammation
(hepatitis) and scarring (fibrosis) of the liver. The NAFLD
spectrum is thought to begin with and progress from its simplest
stage, called simple fatty liver (steatosis). That is, fatty liver
is the initial abnormality in the spectrum of NAFLD. Simple fatty
liver involves just the accumulation of fat in the liver cells with
no inflammation or scarring. The fat is actually composed of a
particular type of fat (triglyceride) that accumulates within the
liver cells. Fatty liver is a harmless (benign) condition. The next
stage and degree of severity in the NAFLD spectrum is NASH. As
mentioned, NASH involves the accumulation of fat in the liver cells
as well as inflammation of the liver. The inflammatory cells can
destroy the liver cells (hepatocellular necrosis). In the terms
"steatohepatitis" and "steatonecrosis", steato refers to fatty
infiltration, hepatitis refers to inflammation in the liver, and
necrosis refers to destroyed liver cells. Strong evidence suggests
that NASH, in contrast to simple fatty liver, is not a harmless
condition. This means that NASH can ultimately lead to scarring of
the liver (fibrosis) and then irreversible, advanced scarring
(cirrhosis). Cirrhosis that is caused by NASH is the last and most
severe stage in the NAFLD spectrum.
[0003] There are few therapeutically effective drugs for treating
fatty liver. Exercise and controlled diet are recommended, but
these are not so effective in treating fatty liver.
[0004] Accordingly, development of a fatty liver treatment having
superior effect and safety with no adverse reactions is in
need.
SUMMARY OF THE INVENTION
[0005] The invention provides a method of treating, inhibiting
and/or preventing fatty liver disease in a patient in need thereof,
comprising administering an effective amount of a cyclohexenone
compound of the formula (I) as described herein to said patient. In
an exemplary embodiment, the cyclohexenone compound is administered
with a second ingredient.
[0006] In one aspect provides methods of treating, inhibiting
and/or preventing fatty liver disease in a patient in need thereof,
comprising administering an effective amount of a cyclohexenone
compound of the following formula (I) to said patient,
##STR00002## [0007] wherein each of X and Y independently is
oxygen, NR.sub.5 or sulfur; [0008] R is a hydrogen or
C(.dbd.O)C.sub.1-C.sub.8alkyl; [0009] each of R.sub.1, R.sub.2 and
R.sub.3 independently is a hydrogen, methyl or
(CH.sub.2).sub.m--CH.sub.3; [0010] R.sub.4 is NR.sub.5R.sub.6,
OR.sub.5, OC(.dbd.O)R.sub.7, C(.dbd.O)OR.sub.5, C(.dbd.O)R.sub.5,
C(.dbd.O)NR.sub.5R.sub.6, halogen, 5 or 6-membered lactone,
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, aryl, glucosyl, wherein the 5 or 6-membered
lactone, C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, aryl, and glucosyl are optionally
substituted with one or more substituents selected from
NR.sub.5R.sub.6, OR.sub.5, OC(.dbd.O)R.sub.7, C(.dbd.O)OR.sub.5,
C(.dbd.O)R.sub.5, C(.dbd.O)NR.sub.5R.sub.6, C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.3-C.sub.8
cycloalkyl, and C.sub.1-C.sub.8 haloalkyl; [0011] each of R.sub.5
and R.sub.6 is independently a hydrogen or C.sub.1-C.sub.8alkyl;
[0012] R.sub.7 is a C.sub.1-C.sub.8alkyl, OR.sub.5 or
NR.sub.5R.sub.6; [0013] m=1-12; and [0014] n=1-12; or a
pharmaceutically acceptable salt, metabolite, solvate or prodrug
thereof.
INCORPORATION BY REFERENCE
[0015] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWING
[0016] FIG. 1(A-D) shows representative photomicrographs of
HE-stained sections of livers of Group A (vehicle control) and
Group B (test compound, Compound 1) in a Fatty Liver Disease model.
FIGS. 1(A) and 1(B) show HE-stained sections of livers of Group A
at an enlargement ratio of 50.times. enlargement ratio of
200.times., respectively. FIGS. 1(C) and 1(D) show HE-stained
sections of livers of Group B at an enlargement ratio of 50.times.
enlargement ratio of 200.times..
[0017] FIG. 2(A-D) shows representative photomicrographs of
Sirius-red stained sections of livers of Group A and Group B Fatty
Liver Disease model. FIGS. 2(A) and 2(B) show Sirius-red stained
sections of livers of Group A at an enlargement ratio of 50.times.
enlargement ratio of 200.times., respectively. FIGS. 2(C) and 2(D)
show Sirius-red stained sections of livers of Group B at an
enlargement ratio of 50.times. enlargement ratio of 200.times..
[0018] FIG. 3(A-D) shows representative photomicrographs of
collagen Type 3-immunostained sections of livers of Group A and
Group B Fatty Liver Disease model. FIGS. 3(A) and 3(B) show
collagen Type 3-immunostained sections of livers of Group A at an
enlargement ratio of 50.times. enlargement ratio of 400.times.,
respectively. FIGS. 3(C) and 3(D) show Sirius-red stained sections
of livers of Group B at an enlargement ratio of 50.times.
enlargement ratio of 400.times..
[0019] FIG. 4 shows diagrams of the whole blood glucose
concentration (mg/dL) of Group A and Group B.
[0020] FIG. 5 shows diagrams of the plasma triglyceride
concentration (mg/dL) of Group A and Group B.
[0021] FIG. 6 and FIG. 7 respectively show diagrams of plasma AST
and plasma ALT concentration (U/dL) of Group A and Group B.
[0022] FIG. 8(A-D) shows representative photomicrographs of
HE-stained sections of livers of Group A and Group B regarding
fibrosis of steatohepatitis liver cells. FIGS. 8(A) and 8(B) show
HE-stained sections of livers of Group A at an enlargement ratio of
50.times. enlargement ratio of 200.times., respectively. FIGS. 8(C)
and 8(D) show HE-stained sections of livers of Group B at an
enlargement ratio of 50.times. enlargement ratio of 200.times..
[0023] FIG. 9(A-D) shows representative photomicrographs of
Sirius-red stained sections of livers of Group A and Group B
regarding fibrosis of steatohepatitis liver cells. FIGS. 9(A) and
9(B) show Sirius-red stained sections of livers of Group A at an
enlargement ratio of 50.times. enlargement ratio of 200.times.,
respectively. FIGS. 9(C) and 9(D) show Sirius-red stained sections
of livers of Group B at an enlargement ratio of 50.times.
enlargement ratio of 200.times..
[0024] FIG. 10(A-D) shows representative photomicrographs of
collagen Type 3-immunostained sections of livers of Group A and
Group B regarding fibrosis of steatohepatitis liver cells. FIGS.
10(A) and 10(B) show collagen Type 3-immunostained sections of
livers of Group A at an enlargement ratio of 50.times. enlargement
ratio of 400.times., respectively. FIGS. 10(C) and 10(D) show
Sirius-red stained sections of livers of Group B at an enlargement
ratio of 50.times. enlargement ratio of 400.times..
[0025] FIG. 11 shows diagrams of the whole blood glucose
concentration (mg/dL) of Group A and Group B.
[0026] FIG. 12 shows diagrams of the plasma triglyceride
concentration (mg/dL) of Group A and Group B.
[0027] FIG. 13 and FIG. 14 respectively show diagrams of plasma AST
and plasma ALT concentration (U/dL) of Group A and Group B.
[0028] FIG. 15(A-F) shows representative photomicrographs of
HE-stained sections of livers in a fatty liver condition assay in
Group A (vehicle control), Group B (test compound & ergosterol)
and Group C (test compond alone). FIGS. 15(A) and 15(B) show
HE-stained sections of livers of Group A at an enlargement ratio of
50.times. enlargement ratio of 200.times., respectively. FIGS.
15(C) and 15(D) show HE-stained sections of livers of Group B at an
enlargement ratio of 50.times. enlargement ratio of 200.times..
FIGS. 15(E) and 15(F) show HE-stained sections of livers of Group C
at an enlargement ratio of 50 .times. enlargement ratio of
200.times..
[0029] FIG. 16(A-F) shows representative photomicrographs of
Sirius-red stained of livers from Group A to Group C. FIGS. 16(A)
and 16(B) show Sirius-red stained sections of livers of Group A at
an enlargement ratio of 50.times. enlargement ratio of 200.times.,
respectively. FIGS. 16(C) and 16(D) show Sirius-red stained
sections of livers of Group B at an enlargement ratio of 50.times.
enlargement ratio of 200.times.. FIGS. 16(E) and 16(F) show
Sirius-red stained sections of livers of Group C at an enlargement
ratio of 50.times. enlargement ratio of 200.times..
[0030] FIG. 17(A-F) shows representative photomicrographs of
collagen Type 3-immunostained sections of livers from Group A to
Group C. FIGS. 17(A) and 17(B) showcollagen Type 3-immunostained
sections of livers of Group A at an enlargement ratio of 50.times.
enlargement ratio of 400.times., respectively. FIGS. 17(C) and
17(D) show Sirius-red stained sections of livers of Group B at an
enlargement ratio of 50.times. enlargement ratio of 400.times..
FIGS. 17(E) and 17(E) show Sirius-red stained sections of livers of
Group C at an enlargement ratio of 50.times. enlargement ratio of
400.times..
DETAILED DESCRIPTION OF THE INVENTION
[0031] In some embodiments, the invention surprisingly found that
cyclohexenone compounds described herein effectively treat, inhibit
and/or prevent fatty liver disease.
[0032] The terms "a" and "an" refer to one or to more than one
(i.e., to at least one) of the grammatical object of the
article.
[0033] The term "treat," "treatment" or "treating" means reducing
the frequency, extent, severity and/or duration with which symptoms
of fatty liver disease are experienced by a patient.
[0034] The term "prevent," "prevention" or "preventing" means
inhibition, risk reduction, reducing the onset of or the averting
of symptoms associated with fatty liver disease.
[0035] The term "pharmaceutically acceptable salt" refers to those
salts which retain the biological effectiveness and properties of
the free bases and which are obtained by reaction with inorganic or
organic acids such as hydrochloric acid, hydrobromic acid, sulfuric
acid, nitric acid, phosphoric acid, methanesulfonic acid,
ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic
acid, maleic acid, succinic acid, tartaric acid, citric acid, and
the like.
[0036] The term "effective amount" means an amount of the compound
described herein effective to treat, inhibit and/or prevent fatty
liver diseases. For example, the effective amount of the compound
described herein reduces the number of fat cells; reduces the liver
size; inhibits (i.e., slow to some extent and preferably stop)
fatty cell infiltration; inhibits (i.e., slows at least to some
extent and preferably stops) inflammation (hepatitis), scarring
(cirrhosis) or necrosis; and/or relieves to some extent one or more
of the symptoms associated with the disease.
[0037] Plants and mushrooms are a valuable resource for the
discovery and development of novel, naturally derived agents to
treat cancer. Antrodia camphorata is also called Chang-Zhi, Niu
Chang-Gu, red camphor mushroom and the like, which is a perennial
mushroom belonging to the order Aphyllophorales, the family
Polyporaceae. It is an endemic species in Taiwan growing on the
inner rotten heart wood wall of Cinnamomum kanehirae Hay. C.
kanehirai Hay is rarely distributed and being overcut unlawfully,
which makes A. camphorata growing inside the tree in the wild
became even rare. The price of A. camphorata is very expensive due
to the extremely slow growth rate of natural A. camphorata that
only grows between Junes to October. Traditionally, A. camphorata
is used as a Chinese remedy for food, alcohol, and drug
intoxication, diarrhea, abdominal pain, hypertension, skin itches,
and liver cancer. Triterpenoids are the most studied component
among the numerous compositions of A. camphorata.
[0038] U.S. Pat. No. 7,385,088 is directed to a novel compound and
use thereof, in particular to Antroquinonol B and Antroquinonol C
isolated from A. camphorata extracts which effectively inhibit the
growth of certain cancer cells. U.S. Pat. No. 7,342,137 provides
cyclohexenone compounds and their uses in tumor growth inhibition,
which is an extract isolated and purified from A. camphorate, in
particular to
4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-dodeca-2,6,10-trienyl-
)-cyclohex-2-enone. Furthermore, several uses of the compound,
4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-dodeca-2,6,10-trienyl-
)-cyclohex-2-enone were developed. U.S. Pat. No. 7,411,003
discloses the use of
4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-dodeca-2,6,10--
trienyl)-cyclohex-2-enone in inhibiting HBV. U.S. Pat. No.
7,456,225 discloses the use of
4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-dodeca-2,6,10-trienyl-
)-cyclohex-2-enone in liver protection such as alleviating liver
injury and fibrosis induced by chemicals and reduces the serum
levels of alanine aminotransferase (ALT) and aspartate
aminotransferase (AST). U.S. Pat. No. 7,468,392 discloses the use
of
4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-dodeca-2,6-10-trienyl-
)-cyclohex-2-enone in delaying fatigue. U.S. Pat. No. 7,501,454
relates to the use of
4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-dodeca-2,6,10-trienyl-
-)-cyclohex-2-enone in treating autoimmune diseases. U.S. Pat. No.
8,236,860 provides the use of
4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyl-dodeca-2,6,10-trieny-
l)-cyclohex-2-enone in inhibiting the survival of pancreatic cancer
cells. US 20110060055, US 20110060056, US 20110060057, US
20110060058 and US 20110060059 disclose the use of
4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-dodeca-2,6,10-trienyl-
-)-cyclohex-2-enone in inhibiting the survival of lymphoma, gastric
cancer, skin cancer, ovarian cancer and bladder cancer cells,
respectively.
[0039] However, none of prior references teaches and suggests that
the above-mentioned cyclohexenone compound can be used in the
treatment and/or prevention of fatty liver diseases, in particular
NAFLD.
[0040] Accordingly, the invention provides a method of treating,
inhibiting and/or preventing fatty liver diseases in a patient in
need thereof, comprising administering an effective amount of a
cyclohexenone compound of the following formula (I) to said
patient,
##STR00003## [0041] wherein each of X and Y independently is
oxygen, NR.sub.5 or sulfur; [0042] R is a hydrogen or
C(.dbd.O)C.sub.1-C.sub.8alkyl; [0043] each of R.sub.1, R.sub.2 and
R.sub.3 independently is a hydrogen, methyl or
(CH.sub.2).sub.m--CH.sub.3; [0044] R.sub.4 is NR.sub.5R.sub.6,
OR.sub.5, OC(.dbd.O)R.sub.7, C(.dbd.O)OR.sub.5, C(.dbd.O)R.sub.5,
C(.dbd.O)NR.sub.5R.sub.6, halogen, 5 or 6-membered lactone,
C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, aryl, glucosyl, wherein the 5 or 6-membered
lactone, C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, aryl, and glucosyl are optionally
substituted with one or more substituents selected from
NR.sub.5R.sub.6, OR.sub.5, OC(.dbd.O)R.sub.7, C(.dbd.O)OR.sub.5,
C(.dbd.O)R.sub.5, C(.dbd.O)NR.sub.5R.sub.6, C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.3-C.sub.8
cycloalkyl, and C.sub.1-C.sub.8 haloalkyl; each of R.sub.5 and
R.sub.6 is independently a hydrogen or C.sub.1-C.sub.8alkyl; [0045]
R.sub.7 is a C.sub.1-C.sub.8alkyl, OR.sub.5 or NR.sub.5R.sub.6;
[0046] m=1-12; and [0047] n=1-12; or a pharmaceutically acceptable
salt, metabolite, solvate or prodrug thereof.
[0048] In some embodiments, the fatty liver diseases are the
primary fatty liver diseases or the secondary fatty liver diseases.
In some embodiments, the secondary fatty liver disease is alcohol
liver disease, fatty liver associated with chronic hepatitis
infection, total parental nutrition (TPN), Reye's Syndrome,
gastrointestinal disorders, or gastroparesis and irritable bowel
(IBS) disorders. In certain embodiments, the fatty liver disease is
cirrhosis or fibrosis.
[0049] In some embodiments, the cyclohexenone compound having the
following structure
##STR00004##
is prepared synthetically or semi-synthetically from any suitable
starting material. In other embodiments, the cyclohexenone compound
is prepared by fermentation, or the like. For example, Compound 1
(also known as Antroquinonol.TM. or "Antroq") or Compound 3, in
some instances, is prepared from
4-hydroxy-2,3-dimethoxy-6-methylcyclohexa-2,5-dienone. The
non-limited exemplary compounds are illustrated below.
##STR00005## ##STR00006## ##STR00007##
[0050] In other embodiments, the cyclohexenone compound having the
structure
##STR00008##
is isolated from the organic solvent extracts of A. camphorata. In
some embodiments, the organic solvent is selected from alcohols
(e.g., methanol, ethanol, propanol, or the like), esters (e.g.,
methyl acetate, ethyl acetate, or the like), alkanes (e.g.,
pentane, hexane, heptane, or the like), halogenated alkanes (e.g.,
chloromethane, chloroethane, chloroform, methylene chloride, or the
like), and the like. For example, exemplary Compounds 1-7 are
isolated from organic solvent extracts. In certain embodiments, the
organic solvent is alcohol. In certain embodiments, the alcohol is
ethanol. In some embodiments, the cyclohexenone compound is
isolated from the aqueous extracts of A. camphorata.
[0051] In some embodiments, R is a hydrogen,
C(.dbd.O)C.sub.3H.sub.8, C(.dbd.O)C.sub.2H.sub.5, or
C(.dbd.O)CH.sub.3. In some embodiments, R.sub.1 is a hydrogen or
methyl. In certain embodiments, R.sub.2 is a hydrogen, methyl,
ethyl, propyl, butyl, pentyl or hexyl. In some embodiments, R.sub.3
is a hydrogen, methyl, ethyl, propyl, butyl, pentyl or hexyl. In
some embodiments, R.sub.4 is halogen, NH.sub.2, NHCH.sub.3,
N(CH.sub.3).sub.2, OCH.sub.3, OC.sub.2H.sub.5, C(.dbd.O)CH.sub.3,
C(.dbd.O)C.sub.2H.sub.5, C(.dbd.O)OCH.sub.3,
C(.dbd.O)OC.sub.2H.sub.5, C(.dbd.O)NHCH.sub.3,
C(.dbd.O)NHC.sub.2H.sub.5, C(.dbd.O)NH.sub.2, OC(.dbd.O)CH.sub.3,
OC(.dbd.O)C.sub.2H.sub.5, OC(.dbd.O)OCH.sub.3,
OC(.dbd.O)OC.sub.2H.sub.5, OC(.dbd.O)NHCH.sub.3,
OC(.dbd.O)NHC.sub.2H.sub.5, or OC(.dbd.O)NH.sub.2. In some
embodiments, R.sub.4 is C.sub.2H.sub.5C(CH.sub.3).sub.2OH,
C.sub.2H.sub.5C(CH.sub.3).sub.2OCH.sub.3, CH.sub.2COOH,
C.sub.2H.sub.5COOH, CH.sub.2OH, C.sub.2H.sub.5OH, CH.sub.2Ph,
C.sub.2H.sub.5Ph, CH.sub.2CH.dbd.C(CH.sub.3)(CHO),
CH.sub.2CH.dbd.C(CH.sub.3)(C(.dbd.O)CH.sub.3), 5 or 6-membered
lactone, C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, aryl, and glucosyl, wherein the 5 or
6-membered lactone, C.sub.1-C.sub.8alkyl, C.sub.2-C.sub.8alkenyl,
C.sub.2-C.sub.8alkynyl, aryl, and glucosyl are optionally
substituted with one or more substituents selected from
NR.sub.5R.sub.6, OR.sub.5, OC(.dbd.O)R.sub.7, C(.dbd.O)OR.sub.5,
C(.dbd.O)R.sub.5, C(.dbd.O)NR.sub.5R.sub.6, C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.3-C.sub.8
cycloalkyl, and C.sub.1-C.sub.8 haloalkyl. In certain embodiments,
R.sub.4 is CH.sub.2CH.dbd.C(CH.sub.3).sub.2. In certain
embodiments, the compound is
##STR00009##
[0052] According to the invention, in some embodiments, the
compounds provided herein are used in the treatment, inhibition
and/or prevention of fatty liver disease. Examples of fatty liver
diseases or liver disorders include the primary fatty liver
diseases NAFLD and NASH and the secondary fatty liver diseases
(e.g., alcoholic liver disease (ALD), fatty liver associated with
chronic hepatitis infection, total parental nutrition (TPN), Reye's
Syndrome, and gastrointestinal disorders such as Intestinal
Bacterial Overgrowth (IBO), gastroparesis, irritable bowel (IBS)
disorders, and the like). These examples are listed as examples
only and the list is not intended to limit the treatment to these
diseases.
Combination Treatments
[0053] In some embodiments, therapeutically-effective dosages vary
when the drugs are used in treatment combinations. Combination
treatment further includes periodic treatments that start and stop
at various times to assist with the clinical management of the
patient. For combination therapies described herein, dosages of the
co-administered compounds vary depending on the type of co-drug
employed, on the specific drug employed, on the disease, disorder,
or condition being treated and so forth.
[0054] It is understood that in some embodiments, the dosage
regimen to treat, prevent, or ameliorate the condition(s) for which
relief is sought, is modified in accordance with a variety of
factors. These factors include the disorder from which the subject
suffers, as well as the age, weight, sex, diet, and medical
condition of the subject. Thus, in other embodiments, the dosage
regimen actually employed varies widely and therefore deviates from
the dosage regimens set forth herein.
[0055] Combinations of compounds (i.e., the cyclohexenone compound
described herein) with other fatty liver disease therapeutic agents
are intended to be covered. In some embodiments, examples of fatty
liver disease therapeutic agents include, but are not limited to,
the following: ergosterol, vitamin E, selenium, betaine, insulin
sensitizers (e.g., Metformin, Pioglitazone, Rosiglitazone,
Thiazolidinediones, or the like), and statins, or the like.
[0056] The combinations of the cyclohexenone compounds and other
fatty liver disease therapeutic agents described herein encompass
additional therapies and treatment regimens with other agents in
some embodiments. Such additional therapies and treatment regimens
include another fatty liver disease therapy in some embodiments.
Alternatively, in other embodiments, additional therapies and
treatment regimens include other agents used to treat adjunct
conditions associated with fatty liver disease or a side effect
from such agent in the combination therapy. In further embodiments,
adjuvants or enhancers are administered with a combination therapy
described herein.
[0057] In some embodiment, the cyclohexenone compound of the
invention is administered with a second ingredient. According to
the invention, the second ingredient is ergosterol. The amounts of
the cyclohexenone compound in combination with ergosterol range
from 50% (w/w) to 90% (w/w) and 50% (w/w) to 10% (w/w),
respectively. In general, the compositions described herein and, in
embodiments where combinational therapy is employed based on the
mode of action described herein, other agents do not have to be
administered in the same pharmaceutical composition, and in some
embodiments, because of different physical and chemical
characteristics, are administered by different routes. In some
embodiments, the initial administration is made according to
established protocols, and then, based upon the observed effects,
the dosage, modes of administration and times of administration is
modified by the skilled clinician.
[0058] In some embodiments, the compounds provided herein are
administered by any convenient route, including oral, parenteral,
subcutaneous, intravenous, intramuscular, intra peritoneal, or
transdermal. The dosage administered depends upon the age, health,
and weight of the recipient, kind of concurrent treatment, if any,
and the nature of the effect desired.
[0059] In some embodiments, for administration, the compounds
provided herein are mixed with one or more physiologically
acceptable carriers comprising excipients and auxiliaries, which
facilitate processing of the active compounds into preparations
which are used pharmaceutically. Proper formulation is dependent
upon the route of administration chosen.
[0060] In some embodiments, for oral administration, the compounds
provided herein are formulated as discrete units such as capsules,
cachets or tablets each containing a predetermined amount of the
compound of the invention; as a powder or granules; as solution or
a suspension in an aqueous liquid or a non-aqueous liquid; or as an
oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
[0061] In some embodiments, for injection, the compounds provided
herein are formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological saline buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants, for example
polyethylene glycol, are generally known in the art. In some
embodiments, pharmaceutical compositions which are used orally,
include push-fit capsules.
[0062] For administration by inhalation, the molecules for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from a pressurized pack
or a nebulizer with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit is determined by providing a
valve to deliver a metered amount in some embodiments. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflator,
is formulated containing a powder mix of the polypeptide and a
suitable powder base such as lactose or starch in some
embodiments.
[0063] The dose will be determined by the activity of the compound
produced and the condition of the subject, as well as the body
weight or surface area of the subject to be treated. The size of
the dose and the dosing regiment also will be determined by the
existence, nature, and extent of any adverse side effects that
accompany the administration of a particular compound in a
particular subject. In determining the effective amount of the
compound to be administered, the physician needs to evaluate
circulating plasma levels, toxicity, and progression of the
disease.
EXAMPLE
Example 1
Isolation of Exemplary Cyclohexenone Compounds
[0064] 100 g of mycelia, fruiting bodies or mixture of both from A.
camphorata were placed into a flask. A proper amount of water and
alcohol (70-100% alcohol solution) was added into the flask and
were stirred at 20-25 degrees Celsius (.degree. C.) for at least 1
hour. The solution was filtered through a filter and a 0.45 .mu.m
(micrometer) membrane and the filtrate was collected as the
extract. The filtrate of A. camphorata was subjected to a High
Performance Liquid chromatography (HPLC). The HPLC was performed
using a RP18 column, methanol (A) and 0.1.about.0.5% acetic acid
(B) as the mobile phase, with gradient of 0.about.10 min in
95%.about.20% B, 10-20 min in 20%.about.10% B, 20.about.35 min in
10%.about.10% B, 35-40 min in 10%.about.95% B, at a flow rate of 1
ml/min. The effluent was monitored with a UV-visible detector.
[0065] The fractions at 25.about.30 min were collected and
concentrated to yield Compound 1,
4-hydroxy-2,3-dimethoxy-6-methyl-5
(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone, a
product with the appearance of light yellow oil. The molecular
formula, molecular weight and melting point of
4-hydroxy-2,3-dimethoxy-6-methyl-5
(3,7,11-trimethyl-dodeca-2,6,10-trienyl)-cyclohex-2-enone are
C.sub.24H.sub.38O.sub.4, 390, and 48.degree. C. to 52.degree. C.,
respectively. NMR spectra of the compound showed that .sup.1H-NMR
(CDCl.sub.3).delta.(ppm)=1.51, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07,
2.22, 2.25, 3.68, 4.05, 5.07, and 5.14; .sup.13C-NMR
(CDCl.sub.3).delta.(ppm)=12.31, 16.1, 16.12, 17.67, 25.67, 26.44,
26.74, 27.00, 39.71, 39.81, 4.027, 43.34, 59.22, 60.59, 120.97,
123.84, 124.30, 131.32, 135.35, 135.92, 138.05, 160.45, and
197.12.
[0066] The fractions collected at 21.2 to 21.4 min were collected
and concentrated to yield compound 5, a product of pale yellow
liquid. Compound 5 was analyzed to be
4-hydroxy-5-(11-hydroxy-3,7,11-trimethyldodeca-2,6-dienyl)-2,3-dimethoxy--
6-methylcyclohex-2-enone with molecular weight of 408 (Molecular
formula: C.sub.24H.sub.40O.sub.5). .sup.1H-NMR (CDCl.sub.3) .delta.
(ppm)=1.21, 1.36, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25,
3.68, 4.05, 5.71 and 5.56. .sup.13C-NMR(CDCl.sub.3).delta.(ppm):
12.31, 16.1, 16.12, 17.67, 25.67, 26.44, 26.74, 27.00, 30.10,
40.27, 43.34, 59.22, 60.59, 71.8, 120.97, 123.84, 124.30, 131.32,
134.61, 135.92, 138.05, 160.45, and 197.11.
##STR00010##
Compound 5:
4-hydroxy-5-(11-hydroxy-3,7,11-trimethyldodeca-2,6-dienyl)-2,3-dimethoxy--
6-methylcyclohex-2-enone
[0067] The fractions collected at 23.7 to 24.0 min were collected
and concentrated to yield compound 7, a product of pale yellow
liquid. Compound 7 was analyzed to be
4-hydroxy-2,3-dimethoxy-5-(11-methoxy-3,7,11-trimethyldodeca-2,6-dienyl)--
6-methylcyclohex-2-enone with molecular weight of 422
(C.sub.25H.sub.42O.sub.5). .sup.1H-NMR (CDCl.sub.3) .delta.
(ppm)=1.21, 1.36, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25, 3.24,
3.68, 4.05, 5.12, 5.50, and 5.61.
.sup.13C-NMR(CDCl.sub.3).delta.(ppm): 12.31, 16.1, 16.12, 17.67,
24.44, 26.44, 26.74, 27.00, 37.81, 39.81, 40.27, 43.34, 49.00,
59.22, 60.59, 120.97, 123.84, 124.30, 135.92, 138.05, 160.45 and
197.12.
##STR00011##
Compound 7:
4-hydroxy-2,3-dimethoxy-5-(11-methoxy-3,7,11-trimethyldodeca-2,6-dienyl)--
6-methylcyclohex-2-enone
[0068] Compound 6, a metabolite of Compound 1, was obtained from
urine samples of rats fed with Compound 1 in the animal study.
Compound 6 was determined to be
4-hydroxy-2,3-dimethoxy-6-methyl-5-(3-methyl-2-hexenoic
acid)cyclohex-2-enone with molecular weight of 312
(C.sub.16H.sub.24O.sub.6). Compound 4 which was determined as
3,4-dihydroxy-2-methoxy-6-methyl-5-(3,7,11-trimethyldodeca-2,6,10-trienyl-
)cyclohex-2-enone (molecular weight of 376,
C.sub.23H.sub.36O.sub.4), was obtained when Compound 1 was under
the condition of above 40.degree. C. for 6 hours.
##STR00012##
[0069] Alternatively, the exemplary compounds may be prepared from
4-hydroxy-2,3-dimethoxy-6-methylcyclohexa-2,5-dienone, or the like.
Similarly, other cyclohexenone compounds having the structure
##STR00013##
are isolated from Antrodia camphorata or prepared synthetically or
semi-synthetically from the suitable starting materials. An
ordinary person skilled in the art would readily utilize
appropriate conditions for such synthesis.
Example 2
Reduction of Fatty Liver Condition by Compound 1
(4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-dodeca-2,6-10-trieny-
l)-cyclohex-2-enone)
[0070] In order to simulate an unhealthy diet tendency in humans,
such as excess consumption of high caloric food, the example
constructs an animal model using rats fed a high fat diet to
evaluate the effects of chronic liver injury. Thereafter, the
effects can be revealed through biochemical assays to prove the
fatty liver reduction of exemplary compounds provided herein such
as Compound 1,
4-hydroxy-2,3-dimethoxy-6-methyl-5(3,7,11-trimethyl-dodeca-2,6-10-trienyl-
)-cyclohex-2-enone (the test compound).
[0071] The assay simulates liver disease caused by a high fat diet
through a "Metabolic Syndrome" model. That is, the model is
different from conventional chemical induced models caused by toxic
components such as CCl.sub.4. This model is distinguishable from
the models caused by virus or alcohol.
[0072] The establishment of a long term high-fat diet is described
as follows: First, the C57BL/6 mice were obtained from Charles
River Laboratories Japan (Kanagawa, Japan). Animals were housed
under specific-pathogen-free (SPF) conditions. The liver injury was
developed in 18 male mice by subcutaneous injection of
Streptozotocin (STZ) 2 days after birth (day 2). After 4 weeks, a
high-fat commercial rodent diet ad libitum (CLEA JAPAN) were
supplied. These mice were randomized into 3 groups before the
treatment. The test compound was administered in a volume of 10
ml/kg twice a day for three weeks. In the control group (group A),
six rats were fed with vehicle (corn oil from Sigma Chemical Co.)
via intubation. Six mice in group B were orally administered
vehicle and the test compound at a dose of 48 mg/kg twice a day (96
mg/kg per day). Table 1 summarizes the study timetable.
TABLE-US-00001 TABLE 1 The timetable of the study Time Process Day
0 Birth Day 2 STZ treatment Day 28 Feeding high fat diet Day 62
Randomization Day 63-Day 76 Test compound administration Day 77
Sacrifice
[0073] Table 2 summarizes the treatment schedule.
TABLE-US-00002 TABLE 2 Experimental groups with the substances and
dosages fed No. Dosage Volume Group mice Test substance (mg/kg)
(ml/kg) Regiments A 6 Vehicle control -- 10 Oral, twice a day, B 6
The test 48 9-11 weeks compound
Histology Analysis
[0074] (1) HE Staining. The HE staining method is performed as
follows: Liver slices are cut from the livers' left side, embedded
in Tissue-Tek.RTM. OCT.TM. Compounds (Sakura Finetek, Japan), snap
frozen in liquid nitrogen, and stored at -80.degree. C. 5 .mu.m
sections are cut, air-dried, fixed in acetone, air dried again and
finally washed with phosphate buffered saline (PBS). Then, in the
hematoxylin and eosin staining method, liver sections will be
prefixed in a Bouin solution (formalin-acetic acid) for one week
and then stained with Lillie-Mayer's Hematoxylin (Muto Pure
Chemicals, Japan) and eosin solution (Wako, Japan) to visualize the
lipid deposition, inflammations, cell necrosis and fibrosis, or
stained with Masson's trichromic solution to visualize extra
cellular matrix and collagen fibers of liver fibrosis
development.
[0075] (2) Sirius Red Staining. The Sirius Red staining method is
performed as follows: Liver slices are cut from the livers' left
side, embedded in Tissue-Tek.RTM. OCT.TM. Compounds (Sakura
Finetek, Japan), snap frozen in liquid nitrogen, and stored at
-80.degree. C. 5 .mu.m sections are cut, air-dried, fixed in
acetone, air dried again and finally washed with phosphate buffered
saline (PBS). Then, liver sections are stained with a Picro-Sirius
Red Solution (Waldeck GmbH & KG, Germany) to observe collagen
deposition.
[0076] (3) Immunohistochemistry of Collagen Type 3. The
immunohistochemistry assay for collagen Type 3 is performed as
follows: Liver slices are cut from the livers' left side, embedded
in Tissue-Tek.RTM. OCT.TM. Compounds (Sakura Finetek, Japan), snap
frozen in liquid nitrogen, and stored at -80.degree. C. 5 .mu.m
sections are cut, air-dried, fixed in acetone, air dried again and
finally washed with phosphate buffered saline (PBS). For
immunohistochemistry, endogenous peroxidase activity will be
blocked by using 0.03% H.sub.2O.sub.2 for 5 minutes, followed by
incubation with Block Ace (Dainippon Sumitomo Pharm, Japan) for 10
minutes. The sections will be incubated with the optimal dilution
of anti-Type 3 collagen antibody overnight at 4.degree. C. After
incubation with an appropriate secondary antibody, the substrate
reactions are performed using DAB/H.sub.20.sub.2 solution
(Nichirei, Japan).
Whole Blood and Plasma Biochemistry
[0077] (1) Whole Blood Glucose. Blood samples were collected in
heparinized syringes (Novo-Heparin 5,000 units/5 ml, Mochida
Pharmaceutical, Japan) by cardiac puncture, kept on ice and
centrifuged at 1,000.times.g at 4.degree. C. for 15 minutes. The
supernatant was collected and stored at -80.degree. C. until use.
Blood glucose was measured in whole blood samples using G Checker
(Sanko Junyaku Co. Ltd., Japan).
[0078] (2) Plasma Aspartate aminotransferase (AST) and Alanine
aminotransferase (ALT). The detection of plasma AST and ALT are
performed as follows: Blood samples were collected in heparinized
syringes (Novo-Heparin 5,000 units/5 ml, Mochida Pharmaceutical,
Japan) by cardiac puncture and kept on ice then centrifuged at
1,000.times.g at 4.degree. C. for 15 minutes. The supernatant was
collected and stored at -80.degree. C. until use. AST, ALT levels
were measured by FUJI DRY CHEM 7000 (Fuji Film, Japan).
Fatty Liver Disease Caused by Non-Chemical Injury
[0079] FIG. 1 to FIG. 7 have proved that the exemplary compound
extracted from A. camphorata (Compound 1) effectively decreases the
extent of fatty liver disease caused by non-chemical injury. The
decrease is assessed by evaluating the extent of liver injuries of
fat deposition by histological analyses such as HE staining, Sirius
red staining and collagen immunostaining, and further by plasma
biochemical marker such as blood glucose, plasma triglyceride,
plasma ALT and plasma AST.
[0080] HE staining reveals the morphology of hepatic cells under
inflammatory cell infiltration, and macro- and micro-vesicular fat
deposition. FIG. 1(A-D) shows the representative photomicrographs
of HE-stained sections of livers of Group A and Group B, wherein
FIGS. 1(A) and 1(C) show an enlargement ratio of 50.times. and
FIGS. 1(B) and 1(D) show an enlargement ratio of 200.times.. As
shown in FIGS. 1(A) and 1(B), the Group A (vehicle) diagrams
revealed infiltration of inflammatory cells, macro- and micro
vesicular fat deposition, proliferated bile ducts and
hepato-cellular ballooning in the liver sections. As shown in FIGS.
1(C) and 1(D), group B (the test compound, Compound 1) treatment
tended to decrease the infiltration of inflammatory cells and also
tended to decrease the macro-vesicular fat deposition compared to
Group A.
[0081] Sirius Red Staining is utilized to detect collagen
deposition. FIG. 2 shows the representative photomicrographs of
Sirius-red staining of livers of Group A and Group B, wherein FIGS.
2(A) and 2(C) show an enlargement ratio of 50.times. and FIGS. 2(B)
and 2(D) show an enlargement ratio of 200.times.. As shown in FIGS.
2(A) and 2(B), in group A, Sirius red staining demonstrates
collagen deposition around central veins, bile ducts and
degenerative hepatocytes. As shown in FIGS. 2(C) and 2(D), in Group
B (the test compound, Compound 1) treatment, the collagen
deposition around the central veins and the bile ducts was
reduced.
[0082] Collagen Type 3 staining is used to detect the distribution
of collagen fibers. FIG. 3 shows the representative
photomicrographs of collagen Type 3-immunostained sections of
livers of Group A and Group B, wherein FIGS. 3(A) and 3(C) show an
enlargement ratio of 50.times. and FIGS. 3(B) and 3(D) show an
enlargement ratio of 400.times.. As shown in FIGS. 3(A) and 3(B),
Collagen Type 3 staining shows accumulation of collagen fibers in
the sinusoidal area and around bile ducts and central veins in the
Vehicle control group A. As shown in FIGS. 3(C) and 3(D), the Group
B (the test compound, Compound 1) treatment tended to reduce the
thickness and/or the length of the collagen fibers in the
sinusoidal area.
[0083] FIG. 4 shows the diagram of the whole blood glucose
concentration (mg/dL) of Group A and Group B. As shown in FIG. 4,
Group B treatment (the test compound, Compound 1) showed a decrease
in the whole blood glucose level compared to the results of Group A
treatment (Group A: 636.+-.137 mg/dL, Group B: 580.+-.122
mg/dL).
[0084] FIG. 5 shows the diagram of the plasma triglyceride (TG)
concentration (mg/dL) of Group A and Group B. As shown in FIG. 5,
Group B treatment (the test compound, Compound 1) showed a
significant decrease in the plasma TG compared to the results of
Group A treatment (Group A: 643.+-.402 mg/dL, Group B: 229.+-.144
mg/dL).
[0085] The aspartate aminotransferase (AST) and the alanine
aminotransferase (ALT) are important enzymes in the amino acid
synthesis for the human organs such as liver, heart, muscles, etc.
AST and ALT are commonly measured clinically as a part of a
diagnostic evaluation of hepatocellular injury, to determine liver
health. These enzymes' content in serum is low under normal
conditions. Significantly elevated levels of AST and ALT often
suggest the existence of liver injury. FIG. 6 and FIG. 7 show
diagrams of plasma AST and plasma ALT concentration (U/dL) for
Group A and Group B, respectively. As shown in FIG. 6, Group B (the
test compound, Compound 1) treatment seems to slightly reduce the
plasma AST level compared to Group A (vehicle) treatment (Group A:
202.+-.177 U/L, Group B: 141.+-.35 U/L). As shown in FIG. 7, Group
B (the test compound, Compound 1) treatment seems to slightly
reduce the plasma ALT level compared to Group A (vehicle) treatment
(Group A: 64.+-.64 U/L, Group B: 34.+-.10 U/L).
[0086] In summary, the test compound treatment decreased the plasma
TG levels, tending to inhibit blood glucose levels, and slightly
decreased the plasma AST and ALT. The test compound treatment also
decreased the infiltration of inflammatory cells and fat deposition
in the liver lobule. Moreover, the test compound tended to inhibit
collagen deposition, shown in both Sirius red staining and collagen
Type 3 staining, suggesting that the test compound has an
anti-fibrotic effect on liver fibrosis steatohepatitis.
Fibrosis of Steatohepatitis Caused by Non-Chemical Injury
[0087] FIG. 8 to FIG. 14 show that the test compound, Compound 1
effectively decrease fibrosis of steatohepatitis liver cells
induced by non-chemical injury. The decrease is assessed by
evaluating the extent of liver injuries in liver fibrosis by
histological analyses such as HE staining, Sirius red staining and
collagen immunostaining, and further by plasma biochemical marker
such as blood glucose, plasma alanine aminotransferase and plasma
aspartate aminotransferase.
[0088] FIG. 8 shows the representative photomicrographs of
HE-stained sections of livers of Group A and Group B, wherein FIGS.
8(A) and 8(C) show an enlargement ratio of 50.times., and FIGS.
8(B) and 8(D) show an enlargement ratio of 200.times.. As shown in
FIGS. 8(A) and 8(B), the Group A (vehicle) diagrams reveal
infiltration of inflammatory cells, macro- and micro vesicular fat
deposition, proliferated bile ducts, and hepatocellular ballooning
in the liver sections. As shown in FIGS. 8(C) and 8(D), group B
(the test compound, Compound 1) treatment tended to decrease the
macro-vesicular fat deposition compared to Group A.
[0089] FIG. 9 shows the representative photomicrographs of
Sirius-red staining of livers of Group A and Group B, wherein FIGS.
9(A) and 9(C) show an enlargement ratio of 50.times., and FIGS.
9(B) and 9(D) show an enlargement ratio of 200.times.. As shown in
FIGS. 9(A) and 9(B), in Group A, Sirius red staining demonstrates
collagen deposition around central veins, bile ducts and
degenerative hepatocytes. In Group A treatment, central-to-central
bridging fibrosis is observed, showing serious fibrosis in liver
cells. As shown in FIGS. 9(C) and 9(D), in Group B (the test
compound, Compound 1) treatment, the collagen deposition around the
central veins and the bile ducts was reduced.
[0090] FIG. 10 shows the representative photomicrographs of
collagen Type 3-immunostained sections of livers of Group A and
Group B, wherein FIGS. 10(A) and 10(C) show an enlargement ratio of
50.times., and FIGS. 10(B) and 10(D) show an enlargement ratio of
400.times.. As shown in FIGS. 10(A) and 10(B), Collagen Type 3
staining shows accumulation of collagen fibers in the sinusoidal
area, and around bile ducts and central veins in the Vehicle
control Group A. As shown in FIGS. 10(C) and 10(D), the Group B
(the test compound, Compound 1) treatment tended to reduce the
thickness and/or the length of the collagen fibers in the
sinusoidal area.
[0091] FIG. 11 shows the diagram of the whole blood glucose
concentration (mg/dL) of Group A and Group B. As shown in FIG. 11,
Group B treatment (the test compound, Compound 1) showed a
significant decrease in the whole blood glucose level compared to
the results of Group A treatment (Group A: 728.+-.109 mg/dL, Group
B: 566.+-.65 mg/dL).
[0092] FIG. 12 shows the diagram of the plasma triglyceride (TG)
concentration (mg/dL) of Group A and Group B. As shown in FIG. 12,
Group B treatment (the test compound, Compound 1) showed a slight
decrease in the plasma TG compared to the results of Group A
treatment (Group A: 758.+-.877 mg/dL, Group B: 704.+-.450
mg/dL).
[0093] FIG. 13 and FIG. 14, respectively show diagrams of plasma
AST and plasma ALT concentration (U/dL) for Group A and Group B. In
FIG. 13, Group B (the test compound, Compound 1) treatment slightly
increase the plasma AST level compared to Group A (vehicle)
treatment, but the change is not significant (Group A: 143.+-.42
U/L, Group B: 167.+-.87 U/L). As shown in FIG. 14, Group B (the
test compound, Compound 1) treatment reduces the plasma ALT level
compared to Group A (vehicle) treatment (Group A: 47.+-.22 U/L,
Group B: 38.+-.11 U/L).
[0094] In summary, the treatment with the test compound (e.g.,
Compound 1) decreased the blood glucose levels, the plasma TG
levels, and tends to decrease the fat deposition in the liver
lobule and the ALT levels, suggesting that the cyclohexenone
compounds provided herein have an ameliorating effect on lipid and
carbohydrate metabolism and a protective effect on liver injury
caused by a high-fat diet. Also, the exemplary cyclohexenone
compound (e.g., Compound 1) tended to inhibit collagen deposition,
shown in both Sirius red staining and collagen Type 3 staining,
showing that the cyclohexenone compounds provided herein have an
anti-fibrotic effect on liver steatohepatitis.
[0095] Taken together, the present invention successfully
demonstrated that the test compound treatment effectively decrease
the extents of fatty liver disease and liver fibrosis induced by
metabolic syndrome, especially from high-fat diet treatment.
Example 3
Reduction of Fatty Liver Condition by the Composition Comprising
Compound 1 and Ergosterol
[0096] Except for the testing groups and treatment schedule, the
materials and processes of Metabolic Syndrome" model, Histology
analysis, Whole blood and plasma biochemistry and Fatty Liver
Disease Caused by Non-Chemical Injury are similar to those stated
in Example 2.
[0097] In control group (group A), six rats were fed with vehicle
(corn oil from Sigma chemical co.) by means of stomach tubes. Six
rats in group B were orally administered vehicle and the test
compound (Compound 1) at a dose of 48 mg/kg twice a day (96 mg/kg
per day) and ergosterol at a dose of 12 mg/kg twice a day (24 mg/kg
per day). Six mice in group C were orally administered vehicle and
the test compound (Compound 1) at a dose of 48 mg/kg twice a day
(96 mg/kg per day).
[0098] Table 1 below shows the study timetable summary.
TABLE-US-00003 TABLE 1 The timetable of the study Time Process Day
0 Birth Day 2 STZ treatment Day 28 Feeding high fat diet Day 62
Randomization Day 63-Day 76 Test substance administration Day 77
Sacrifice
[0099] Table 2 shows the treatment schedule.
TABLE-US-00004 TABLE 2 Experimental groups with the substances they
were fed with and their dosages No. Dosage Volume Group mice Test
substance (mg/kg) (ml/kg) Regiments A 6 Vehicle control -- 10 Oral,
twice a day, B 6 The test 48 9-11 weeks compound Ergosterol 12 C 6
The test 48 compound
HE Staining
[0100] FIG. 15 shows the representative photomicrographs of
HE-stained sections of livers from Group A to Group C, wherein
FIGS. 15(A), 15(C), 15(E) show an enlargement ratio of 50.times.
and FIGS. 15(B), 15(D), 15(F) show an enlargement ratio of
200.times.. As shown in FIGS. 15(A) and 15(B), the Group A figures
revealed infiltration of inflammatory cells, macro- and micro
vesicular fat deposition, proliferated bile ducts and
hepatocellular ballooning in the liver sections. As shown in FIGS.
15(C) and 15(D), treatment with combination of the test compound
(Compound 1) and ergosterol decreased the macro vesicular fat
deposition and the infiltration of inflammatory cells. The number
of mitotic figures in hepatocytes is higher in group B compared to
the group A. As shown in FIGS. 15(E) and 15(F), the treatment with
the test compound alone tended to decrease the macro vesicular fat
deposition but did not affect the infiltration of inflammatory
cells.
[0101] From FIG. 15, it is recognized that the treatment with the
test compound (Compound 1) and ergosterol (Group B) have the
ability of reducing the macro vesicular fat deposition. Compared to
the treatment with the test compound alone (Group C), the treatment
with the test compound and ergosterol (Group B) further inhibit the
inflammatory cells infiltration, thereby showing better
anti-inflammatory effects with respect to Group C.
Sirius Red Staining
[0102] FIG. 16 shows the representative photomicrographs of
Sirius-red stained of livers from Group A to Group C, wherein FIGS.
16(A), 16(C), 16(E) show an enlargement ratio of 50.times. and
FIGS. 16(B), 16(D), 16(F) show an enlargement ratio of 200.times..
As shown in FIGS. 16(A) and 16(B), in group A, Sirius red staining
show collagen deposition around central veins, bile ducts and
degenerative hepatocytes, showing central-to-central bridging
fibrosis and indicating serious fibrosis steatohepatitis. As shown
in FIGS. 16(C) and 16(D), the treatment with the combination of the
test compound (Compound 1) and ergosterol tended to decrease the
collagen deposition. As shown in FIGS. 16(E) and 16(F), the
cyclohexenone alone treatment tended only slightly to decrease the
collagen deposition compared to that in the control group.
[0103] In summary, both the treatments with the cyclohexeone
compound provided herein (e.g., Compound 1) and ergosterol
treatment (Group B) and the treatment with the test compound alone
(Group C) tend to inhibit collagen deposition, and thus prevent
liver fibrosis. More specifically, the treatment with the
combination of the cyclohexenone compound provided herein (e.g.,
Compound 1) and ergosterol provides a better anti-liver-fibrosis
ability compared to the treatment with the test compound alone.
Immunohistochemistry of Collagen Type 3
[0104] FIG. 17 shows the representative photomicrographs of
collagen Type 3-immunostained sections of livers from Group A to
Group C, wherein FIGS. 17(A), 17(C), 17(E) show an enlargement
ratio of 100.times. and FIGS. 17(B), 17(D), 17(F) show an
enlargement ratio of 400.times.. As shown in FIGS. 17(A) and 17(B),
Collagen Type 3 staining shows accumulation of collagen fibers in
the sinusoidal area and around bile ducts and central veins in the
Vehicle control group. As shown in FIGS. 17(C), 17(D), 17(E) and
17(F), the combination of cyclohexenone (e.g., Compound 1) and
ergosterol treatment and the cyclohexenone alone treatment tended
to reduce the collagen deposition proved by the decrease of the
thickness of the collagen fibers in the sinusoidal area, indicating
their anti-fibrosis ability.
Example 4
Study of Compound 1 in Patients With Diabetes and Presumed
NAFLD
[0105] The primary objectives of this study are to assess, in
patients with Type 2 diabetes mellitus (DM) and presumed
nonalcoholic fatty liver disease (NAFLD), the following: [0106] The
safety and tolerability of multiple doses of Compound 1; [0107] The
effects of 2 dose levels (50 mg and 100 mg) of Compound 1 on
insulin resistance and glucose homeostasis; [0108] Effects of
Compound 1 on hepatocellular function as measured by assessment of
liver enzymes and biochemical markers of hepatic and metabolic
function and inflammation.
[0109] Study Type: Interventional [0110] Study Design: Allocation:
Randomized [0111] Endpoint Classification: Safety/Efficacy Study
[0112] Intervention Model: Parallel Assignment [0113] Masking:
Double Blind (Subject, Investigator) [0114] Primary Purpose:
Treatment
Primary Outcome Measures:
[0115] Insulin Resistance and Glucose Homeostasis [ Time Frame:
baseline and 6 weeks] [ Designated as safety issue: No ]
[0116] The primary objective of assessing changes in insulin
resistance and glucose homeostasis will be attained by performing a
euglycemic clamp procedure at baseline (Day 0) and at the end of 6
weeks of treatment (Day 43).
Secondary Outcome Measures:
[0117] Hepatocellular Function [ Time Frame: baseline and 6 weeks]
[ Designated as safety issue: Yes ]
[0118] Hepatocellular function as measured by assessment of liver
enzymes and biochemical markers of hepatic and metabolic
function
TABLE-US-00005 Arms Assigned Interventions Active Comparator: Drug:
Compound 1 50 mg Compound 1 50 mg by mouth once daily, 100 mg by
mouth once daily Active Comparator: Drug: Compound 1 100 mg
Compound 1 100 mg by mouth once daily, 200 mg by mouth once daily
Placebo Comparator: Drug: Placebo Placebo Placebo
Detailed Description:
[0119] This is a multi-center, double-blind, randomized,
placebo-controlled, multiple-dose, parallel-group study. Three (3)
cohorts of 12 patients each will receive either placebo, 50 mg
Compound 1, or 100 mg Compound 1 by mouth daily for 6 weeks.
[0120] The primary objective of assessing changes in insulin
resistance and glucose homeostasis will be attained by performing a
euglycemic clamp procedure at baseline (Day 0) and at the end of 6
weeks of treatment (Day 43). Other endpoints will be evaluated by
monitoring adverse experiences; vital signs; clinical laboratory
values; plasma drug and metabolite concentrations; and general
health and well-being.
Eligibility
[0121] Ages Eligible for Study: 18 Years to 75 Years [0122] Genders
Eligible for Study: Both [0123] Accepts Healthy Volunteers: No
Inclusion Criteria:
[0124] Type 2 diabetes, defined by the American Diabetes
Association (ADA), as one of the following criteria:
[0125] Symptoms of diabetes plus casual plasma glucose
concentration>200 mg/dL (11.1 mmol/L) or Fasting plasma
glucose>126 mg/dL (7.0 mmol/L) or 2-hour post-load
glucose>200 mg/dL (11.1 mmol/L) during a 75 g oral glucose
tolerance test (GTT).
[0126] Presumed NAFLD, defined by one of the following criteria:
[0127] Alanine aminotransferase (ALT).gtoreq.47 U/L for females and
.gtoreq.56 U/L for males [0128] Aspartate aminotransferase
(AST).gtoreq.47 U/L for females and .gtoreq.60 U/L for males [0129]
Enlarged liver (demonstrated by ultrasound or other imaging
technique) [0130] Diagnostic histological findings shown on prior
biopsy (in the last 5 years).
Exclusion Criteria:
[0131] Bilirubin>2.times.ULN [0132] ALT>155 U/L for females
and >185 U/L for males. [0133] AST>155 U/L for females and
>200 U/L for males. [0134] Patients taking any antidiabetic
medications, with the exception of metformin and sulfonylureas. If
the HbA1c is <11%, patients may be enrolled who have been
withdrawn from all other diabetic medications as specified in the
protocol, at the discretion of the Principal Investigator.
Example 5
Oral Formulation
[0135] To prepare a pharmaceutical composition for oral delivery,
100 mg of an exemplary Compound 1 is mixed with 100 mg of corn oil.
The mixture is incorporated into an oral dosage unit in a capsule,
which is suitable for oral administration.
[0136] In some instances, 100 mg of a compound described herein is
mixed with 750 mg of starch. The mixture is incorporated into an
oral dosage unit for, such as a hard gelatin capsule, which is
suitable for oral administration.
Example 6
Sublingual (Hard Lozenge) Formulation
[0137] To prepare a pharmaceutical composition for buccal delivery,
such as a hard lozenge, mix 100 mg of a compound described herein,
with 420 mg of powdered sugar mixed, with 1.6 mL of light corn
syrup, 2.4 mL distilled water, and 0.42 mL mint extract. The
mixture is gently blended and poured into a mold to form a lozenge
suitable for buccal administration.
Example 7
Inhalation Composition
[0138] To prepare a pharmaceutical composition for inhalation
delivery, 20 mg of a compound described herein is mixed with 50 mg
of anhydrous citric acid and 100 mL of 0.9% sodium chloride
solution. The mixture is incorporated into an inhalation delivery
unit, such as a nebulizer, which is suitable for inhalation
administration.
[0139] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *