U.S. patent application number 14/612673 was filed with the patent office on 2015-07-30 for method of treating liver disorders.
This patent application is currently assigned to MediciNova, Inc.. The applicant listed for this patent is MediciNova, Inc.. Invention is credited to Kazuko MATSUDA.
Application Number | 20150209314 14/612673 |
Document ID | / |
Family ID | 50826043 |
Filed Date | 2015-07-30 |
United States Patent
Application |
20150209314 |
Kind Code |
A1 |
MATSUDA; Kazuko |
July 30, 2015 |
METHOD OF TREATING LIVER DISORDERS
Abstract
A compound of Formula (I): ##STR00001## or a metabolite thereof,
or an ester of the compound of Formula (I) or the metabolite
thereof, or a pharmaceutically acceptable salt of each thereof,
wherein m, n, X.sup.1 and X.sup.2 are as defined herein, is useful
for inhibiting liver steatosis, lobular inflammation, hepatic
ballooning and hepatic scarring.
Inventors: |
MATSUDA; Kazuko; (Beverly
Hills, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MediciNova, Inc. |
La Jolla |
CA |
US |
|
|
Assignee: |
MediciNova, Inc.
La Jolla
CA
|
Family ID: |
50826043 |
Appl. No.: |
14/612673 |
Filed: |
February 3, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14152924 |
Jan 10, 2014 |
8962687 |
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14612673 |
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13706161 |
Dec 5, 2012 |
8835499 |
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14152924 |
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61568517 |
Dec 8, 2011 |
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Current U.S.
Class: |
514/571 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 31/192 20130101; A61P 1/16 20180101 |
International
Class: |
A61K 31/192 20060101
A61K031/192 |
Claims
1. A method of inhibiting steatosis in a patient suffering from
non-alcoholic steatohepatitis comprising administering to the
patient an effective amount of a compound of Formula (I):
##STR00011## or a metabolite thereof, or an ester of the compound
of Formula (I) or the metabolite thereof, or a pharmaceutically
acceptable salt of each thereof, wherein m is an integer from 2 to
5, and n is an integer from 3 to 8, X.sup.1 and X.sup.2 each
independently represent a sulfur atom, oxygen atom, sulfinyl group
or a sulfonyl group, provided that X.sup.1 and X.sup.2 are not
simultaneously oxygen atom.
2. The method of claim 1, in which the compound of Formula (I) is
of Formula (IA) ##STR00012##
3. The method of claim 1, in which the metabolite of the compound
of Formula (I) is a compound of Formula (IB): ##STR00013##
4. The method of claim 1, in which the compound is administered
orally.
5. The method of claim 4, in which the compound is administered as
a tablet or a capsule.
6. The method of claim 2, in which the compound is present in an
orthorhombic polymorphic form A that is substantially free of other
polymorphic forms.
7. The method of claim 1, in which the compound is administered as
a liquid dosage form.
8. The method of claim 1, in which the compound is administered in
an amount from 100 to 4,000 mg/day, divided into one, two, or three
portions.
9. A method of inhibiting lobular inflammation in a patient
suffering from non-alcoholic steatohepatitis comprising
administering to the patient an effective amount of a compound of
Formula (I): ##STR00014## or a metabolite thereof, or an ester of
the compound of Formula (I) or the metabolite thereof, or a
pharmaceutically acceptable salt of each thereof, wherein m is an
integer from 2 to 5, and n is an integer from 3 to 8, X.sup.1 and
X.sup.2 each independently represent a sulfur atom, oxygen atom,
sulfinyl group or a sulfonyl group, provided that X.sup.1 and
X.sup.2 are not simultaneously oxygen atom.
10. The method of claim 9, in which the compound of Formula (I) is
of Formula (IA) ##STR00015##
11. The method of claim 9, in which the metabolite of the compound
of Formula (I) is a compound of Formula (IB): ##STR00016##
12. The method of claim 9, in which the compound is administered
orally.
13. The method of claim 12, in which the compound is administered
as a tablet or a capsule.
14. The method of claim 10, in which the compound is present in an
orthorhombic polymorphic form A that is substantially free of other
polymorphic forms.
15. The method of claim 9, in which the compound is administered as
a liquid dosage form.
16. The method of claim 9, in which the compound is administered in
an amount from 100 to 4,000 mg/day, divided into one, two, or three
portions.
17. A method of inhibiting hepatic ballooning in a patient
suffering from non-alcoholic steatohepatitis comprising
administering to the patient an effective amount of a compound of
Formula (I): ##STR00017## or a metabolite thereof, or an ester of
the compound of Formula (I) or the metabolite thereof, or a
pharmaceutically acceptable salt of each thereof, wherein m is an
integer from 2 to 5, and n is an integer from 3 to 8, X.sup.1 and
X.sup.2 each independently represent a sulfur atom, oxygen atom,
sulfinyl group or a sulfonyl group, provided that X.sup.1 and
X.sup.2 are not simultaneously oxygen atom.
18. The method of claim 17, in which the compound of Formula (I) is
of Formula (IA) ##STR00018##
19. The method of claim 17, in which the metabolite of the compound
of Formula (I) is a compound of Formula (IB): ##STR00019##
20. The method of claim 17, in which the compound is administered
orally.
21. The method of claim 20, in which the compound is administered
as a tablet or a capsule.
22. The method of claim 18, in which the compound is present in an
orthorhombic polymorphic form A that is substantially free of other
polymorphic forms.
23. The method of claim 17, in which the compound is administered
as a liquid dosage form.
24. The method of claim 17, in which the compound is administered
in an amount from 100 to 4,000 mg/day, divided into one, two, or
three portions.
25. A method of inhibiting hepatic scarring in a patient suffering
from non-alcoholic steatohepatitis comprising administering to the
patient an effective amount of a compound of Formula (I):
##STR00020## or a metabolite thereof, or an ester of the compound
of Formula (I) or the metabolite thereof, or a pharmaceutically
acceptable salt of each thereof, wherein m is an integer from 2 to
5, and n is an integer from 3 to 8, X.sup.1 and X.sup.2 each
independently represent a sulfur atom, oxygen atom, sulfinyl group
or a sulfonyl group, provided that X.sup.1 and X.sup.2 are not
simultaneously oxygen atom.
26. The method of claim 25, in which the compound of Formula (I) is
of Formula (IA) ##STR00021##
27. The method of claim 25, in which the metabolite of the compound
of Formula (I) is a compound of Formula (IB): ##STR00022##
28. The method of claim 25, in which the compound is administered
orally.
29. The method of claim 28, in which the compound is administered
as a tablet or a capsule.
30. The method of claim 26, in which the compound is present in an
orthorhombic polymorphic form A that is substantially free of other
polymorphic forms.
31. The method of claim 25, in which the compound is administered
as a liquid dosage form.
32. The method of claim 25, in which the compound is administered
in an amount from 100 to 4,000 mg/day, divided into one, two, or
three portions.
33. A method of reducing and/or inhibiting elevated liver
hydroxyproline levels in a patient suffering from non-alcoholic
steatohepatitis comprising administering to the patient an
effective amount of a compound of Formula (I): ##STR00023## or a
metabolite thereof, or an ester of the compound of Formula (I) or
the metabolite thereof, or a pharmaceutically acceptable salt of
each thereof, wherein m is an integer from 2 to 5, and n is an
integer from 3 to 8, X.sup.1 and X.sup.2 each independently
represent a sulfur atom, oxygen atom, sulfinyl group or a sulfonyl
group, provided that X.sup.1 and X.sup.2 are not simultaneously
oxygen atom.
34. The method of claim 33, in which the compound of Formula (I) is
of Formula (IA) ##STR00024##
35. The method of claim 33, in which the metabolite of the compound
of Formula (I) is a compound of Formula (IB): ##STR00025##
36. The method of claim 33, in which the compound is administered
orally.
37. The method of claim 36, in which the compound is administered
as a tablet or a capsule.
38. The method of claim 34, in which the compound is present in an
orthorhombic polymorphic form A that is substantially free of other
polymorphic forms.
39. The method of claim 33, in which the compound is administered
as a liquid dosage form.
40. The method of claim 33, in which the compound is administered
in an amount from 100 to 4,000 mg/day, divided into one, two, or
three portions.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/152,924, filed Jan. 10, 2014, (now
pending), which is a continuation in part of U.S. application Ser.
No. 13/706,161, filed Dec. 5, 2012, now U.S. Pat. No. 8,835,499,
the contents of which are incorporated by reference herein in their
entirety.
FIELD OF THE INVENTION
[0002] This invention relates to methods of treating non-alcoholic
fatty liver disease (NAFLD), and/or non-alcoholic steatohepatitis
(NASH), conditions leading to or arising from one or more of them,
and/or negative effects of each thereof by administering
phenoxyalkylcarboxylic acids such as MN-001 and MN-002.
BACKGROUND OF THE INVENTION
[0003] Non-alcoholic fatty liver disease (NAFLD) refers to fat
accumulation in the liver that is not related to alcohol
consumption. Fat may accumulate as a result of obesity, diabetes or
other conditions. In a small number of people, NAFLD progresses to
liver inflammation, scarring and, eventually, liver failure. This
serious form of the disease is sometimes called non-alcoholic
steatohepatitis (NASH). NAFLD and NASH, and conditions leading to
or arising from one or more of them, are a growing problem
worldwide, affecting people of every age. NAFLD and NASH are
currently the fastest-rising indicator for liver transplant.
SUMMARY OF THE INVENTION
[0004] In one aspect, the present invention provides a method of
treating a patient suffering from non-alcoholic fatty liver disease
(NAFLD) or non-alcoholic steatohepatitis (NASH) comprising
administering to a patient in need thereof an effective amount of a
compound of Formula (I):
##STR00002##
or a metabolite thereof, or an ester of the compound of Formula (I)
or the metabolite thereof, or a pharmaceutically acceptable salt of
each thereof, wherein m is an integer from 2 to 5, and n is an
integer from 3 to 8, X.sup.1 and X.sup.2 each independently
represent a sulfur atom, a oxygen atom, a sulfinyl (--S(O)--) group
or a sulfonyl (--S(O).sub.2--) group, provided that X.sup.1 and
X.sup.2 are not simultaneously oxygen atoms.
[0005] In another aspect, the present invention provides a method
of reducing liver inflammation in a patient suffering from NAFLD or
NASH comprising administering to a patient in need thereof an
effective amount of a compound of Formula (I), or an ester thereof,
or a pharmaceutically acceptable salt of each thereof, wherein the
compound of Formula (I) is defined as above.
[0006] In another aspect, the present invention provides a method
of inhibiting one or more of steatosis, lobular inflammation,
hepatic ballooning, and hepatic scarring in a patient suffering
therefrom comprising administering to a patient in need thereof an
effective amount of a compound of Formula (I), or an ester thereof,
or a pharmaceutically acceptable salt of each thereof, wherein the
compound of Formula (I) is defined as above. As used herein,
"steatosis" (also called fatty change, fatty degeneration or
adipose degeneration) is a process describing the abnormal
retention of lipids within a cell, preferably, liver cell. In
another aspect, the present invention provides a method of reducing
and/or inhibiting hydroxyproline formation in a liver of a patient
in need thereof, comprising administering to a patient in need
thereof an effective amount of a compound of Formula (I), or an
ester thereof, or a pharmaceutically acceptable salt of each
thereof, wherein the compound of Formula (I) is defined as above.
In certain preferred embodiments, the steatosis, lobular
inflammation, hepatic ballooning, hepatic scarring, or
liver-hydroxyproline accumulation is not associated with excessive
alcohol intake; in other words, they are substantially
non-alcoholic in nature.
[0007] In a preferred embodiment, the compound of Formula (I) is a
compound of Formula (IA) (or MN-001):
##STR00003##
[0008] In another preferred embodiment, the metabolite of the
compound of Formula (I) and (IA) is a compound of Formula (IB) (or
MN-002):
##STR00004##
[0009] In one embodiment, the patient is suffering from NAFLD. In
another embodiment, the patient is suffering from NASH.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1 graphically illustrates steatosis scores in treated
and untreated mice.
[0011] FIG. 2 graphically illustrates lobular inflammation scores
in treated and untreated mice.
[0012] FIG. 3 graphically illustrates hepatocyte ballooning scores
in treated and untreated mice.
[0013] FIG. 4 graphically illustrates percentages of fibrosis area
in treated and untreated mice.
[0014] FIG. 5 graphically illustrates inflammation area in treated
and untreated mice.
[0015] FIG. 6 graphically illustrates liver hydroxyproline content
in treated and un treated mice.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0016] As used herein, and in the appended claims, the singular
forms "a," "an" and "the" include plural references unless the
context clearly dictates otherwise.
[0017] "Administering" or "Administration of" a drug to a patient
(and grammatical equivalents of this phrase) includes both direct
administration, including self-administration, and indirect
administration, including the act of prescribing a drug. For
example, as used herein, a physician who instructs a patient to
self-administer a drug and/or provides a patient with a
prescription for a drug is administering the drug to the
patient.
[0018] "C.sub.X" when placed before a group refers to the number of
carbon atoms in that group to be X.
[0019] "Alkyl" refers to a monovalent acyclic hydrocarbyl radical
having 1-12 carbon atoms. Non limiting examples of alkyl include
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl,
pentyl, hexyl and the like.
[0020] "Aryl" refers to a monovalent aromatic hydrocarbyl radical
having up to 10 carbon atoms. Non-limiting examples of aryl include
phenyl and naphthyl.
[0021] "Heteroaryl" refers to an aromatic group of from 1 to 10
carbon atoms and 1 to 4 heteroatoms selected from the group
consisting of oxygen, nitrogen, sulfur within the aromatic ring,
wherein the nitrogen and/or sulfur atom(s) of the heteroaryl are
optionally oxidized (e.g., N-oxide, --S(O)-- or --S(O).sub.2--).
Such heteroaryl groups can have a single ring (e.g., pyridyl or
furyl) or multiple condensed rings (e.g., indolizinyl or
benzothienyl) wherein the condensed rings may or may not be
aromatic and/or contain a heteroatom provided that the point of
attachment is through an atom of the aromatic heteroaryl group. Non
limiting examples of heteroaryl include pyridyl, pyrrolyl, indolyl,
thiophenyl, and furyl.
[0022] "Cycloalkyl" refers to a monovalent non-aromatic cyclic
hydrocarbyl radical having 3-12 carbon atoms. Non limiting examples
of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, and the like.
[0023] "Heterocyclyl" refers to a monovalent non-aromatic cyclic
group of 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from
the group consisting of oxygen, nitrogen, sulfur within the cycle,
wherein the nitrogen and/or sulfur atom(s) of the heteroaryl are
optionally oxidized (e.g., N-oxide, --S(O)-- or --S(O).sub.2--).
Such heteroaryl groups can have a single ring (e.g., piperidinyl or
tetrahydrofuranyl) or multiple condensed rings wherein the
condensed rings may or may not be aromatic and/or contain a
heteroatom provided that the point of attachment is through an atom
of the non-aromatic heterocyclyl group. Non limiting examples of
heterocyclyl include pyrrolidinyl, piperidinyl, piperazinyl, and
the like.
[0024] "Amino" refers to --NH.sub.2.
[0025] "Alkylamino" refers to --NHR.sub.B, wherein R.sub.B is
C.sub.1-C.sub.6 alkyl optionally substituted with 1-3 aryl,
heteroaryl, cycloalkyl, or heterocyclyl group.
[0026] "Dialkylamino" refers to --N(R.sub.B).sub.2, wherein R.sub.B
is defined as above.
[0027] "Comprising" shall mean that the methods and compositions
include the recited elements, but not exclude others. "Consisting
essentially of" when used to define methods and compositions, shall
mean excluding other elements of any essential significance to the
combination for the stated purpose. Thus, a composition consisting
essentially of the elements as defined herein would not exclude
trace contaminants from the isolation and purification method and
pharmaceutically acceptable carriers, such as phosphate buffered
saline, preservatives and the like. "Consisting of" shall mean
excluding more than trace elements of other ingredients and
substantial method steps for administering the compositions of this
invention or process steps to produce a composition or achieve an
intended result. Embodiments defined by each of these transitional
terms and phrases are within the scope of this invention.
[0028] "Effective amount" of a compound utilized herein is an
amount that, when administered to a patient with NAFLD or NASH,
will have the intended therapeutic effect, e.g., alleviation,
amelioration, palliation or elimination of one or more
manifestations of the medical condition in the patient. The full
therapeutic effect does not necessarily occur by administration of
one dose (or dosage), and may occur only after administration of a
series of doses. Thus, an effective amount may be administered in
one or more administrations.
[0029] "Non-alcoholic steatohepatitis" or NASH is a common liver
disease, which resembles alcoholic liver disease, but occurs in
people who drink little or no alcohol. The major feature in NASH is
fat in the liver, along with inflammation and damage. NASH can lead
to cirrhosis, in which the liver is permanently damaged and scarred
and is no longer able to work properly. NASH affects 2 to 5 percent
of the U.S. population. Currently, no specific therapies for NASH
exist. An additional 10 to 20 percent of Americans have fat in
their liver, but no substantial inflammation or liver damage, a
condition called "non-alcoholic fatty liver disease" (NAFLD).
Although having fat in the liver is not normal, by itself it
probably causes little harm or permanent damage. If fat is
suspected based on blood test results or scans of the liver, this
problem is referred to as NAFLD. If a liver biopsy is performed in
this case, it will show that some people have NASH while others
have NAFLD.
[0030] NASH is usually first suspected in a person who is found to
have elevations in liver tests that are included in routine blood
test panels, such as alanine aminotransferase (ALT) or aspartate
aminotransferase (AST). When further evaluation shows no apparent
reason for liver disease (such as medications, viral hepatitis, or
excessive use of alcohol) and when x rays or imaging studies of the
liver show fat, NASH is suspected. NASH is diagnosed and separated
from NAFLD by a liver biopsy. For a liver biopsy, a needle is
inserted through the skin to remove a small piece of the liver.
NASH is diagnosed when examination of the tissue with a microscope
shows fat along with inflammation and damage to liver cells. If the
tissue shows fat without inflammation and damage, NAFLD is
diagnosed. An important piece of information learned from the
biopsy is whether scar tissue has developed in the liver.
[0031] NASH can slowly worsen, causing scarring or fibrosis to
appear and accumulate in the liver. As fibrosis worsens, cirrhosis
develops; the liver becomes severely scarred, hardened, and unable
to function normally. Once serious scarring or cirrhosis is
present, few treatments can halt the progression. A person with
cirrhosis experiences fluid retention, muscle wasting, bleeding
from the intestines, and liver failure. Liver transplantation is
the only treatment for advanced cirrhosis with liver failure, and
transplantation is increasingly performed in people with NASH. For
example, NASH ranks as one of the major causes of cirrhosis in the
U.S.A., behind hepatitis C and alcoholic liver disease.
[0032] "Pharmaceutically acceptable" refers to non-toxic and
suitable for administration to a patient, including a human
patient.
[0033] "Pharmaceutically acceptable salts" refer to salts that are
non-toxic and are suitable for administration to patients.
Non-limiting examples include alkali metal, alkaline earth metal,
and various primary, secondary, and tertiary ammonium salts. When
the ester of the compound of Formula (I) includes a cationic
portion, for example, when the ester includes an amino acid ester,
the salts thereof can include various carboxylic acid, sulfonic
acid, and miner acid salts. Certain non limiting examples of salts
include sodium, potassium, and calcium salts.
[0034] "Protecting groups" refer to well known functional groups
which, when bound to a functional group, render the resulting
protected functional group inert to the reaction to be conducted on
other portions of a compound and the corresponding reaction
condition, and which can be reacted to regenerate the original
functionality under deprotection conditions. The protecting group
is selected to be compatible with the remainder of the molecule. A
"carboxylic acid protecting group" protects the carboxylic
functionality of the phenoxyalkylcarboxylic acids during their
synthesis. Non limiting examples of carboxylic acid protecting
groups include, benzyl, p-methoxybenzyl, p-nitrobenzyl, allyl,
benzhydryl, and trityl. Additional examples of carboxylic acid
protecting groups are found in standard reference works such as
Greene and Wuts, Protective Groups in Organic Synthesis., 2d Ed.,
1991, John Wiley & Sons, and McOmie Protective Groups in
Organic Chemistry, 1975, Plenum Press. Methods for protecting and
deprotecting the carboxylic acids disclosed herein can be found in
the art, and specifically in Greene and Wuts, supra, and the
references cited therein.
[0035] "Treating" a medical condition or a patient refers to taking
steps to obtain beneficial or desired results, including clinical
results. For purposes of the various aspects and embodiments of the
present invention, beneficial or desired clinical results include,
but are not limited to, reduction, alleviation, or amelioration of
one or more manifestations of or negative effects of NAFLD and/or
NASH, improvement in one or more clinical outcomes, diminishment of
extent of disease, delay or slowing of disease progression,
amelioration, palliation, or stabilization of the disease state,
and other beneficial results described herein.
Preferred Embodiments
[0036] In one aspect, the present invention provides a method of
treating a patient suffering from non-alcoholic fatty liver disease
(NAFLD) or non-alcoholic steatohepatitis (NASH) comprising
administering to a patient in need thereof an effective amount of a
compound of Formula (I):
##STR00005##
or a metabolite thereof, or an ester of the compound of Formula (I)
or the metabolite thereof, or a pharmaceutically acceptable salt of
each thereof, wherein m is an integer from 2 to 5, and n is an
integer from 3 to 8, X.sup.1 and X.sup.2 each independently
represent a sulfur atom, an oxygen atom, a sulfinyl group or a
sulfonyl group, provided that X.sup.1 and X.sup.2 are not
simultaneously oxygen atom.
[0037] In another aspect, the present invention provides a method
of reducing liver inflammation in a patient suffering from NAFLD or
NASH comprising administering to a patient in need thereof an
effective amount of a compound of Formula (I), or a metabolite
thereof, or an ester of the compound of Formula (I) or the
metabolite thereof, or a pharmaceutically acceptable salt of each
thereof, wherein the compound of Formula (I) is defined as
above.
[0038] As used herein, "a metabolite thereof" refers to a
metabolite that shows substantially similar therapeutic activity as
a compound of Formula (I). Non limiting examples of such
metabolites include compounds where the --COCH.sub.3 group, of a
compound of Formula (I), that is attached to the phenyl containing
the --O--(CH.sub.2)--CO.sub.2H moiety is metabolized to a
1-hydroxyethyl (--CH(OH)Me) group.
[0039] Metabolites containing such a 1-hydroxyethyl group contain
an asymmetric center on the 1-position of the 1-hydroxyethyl group.
The corresponding enantiomers and mixtures thereof, including
racemic mixtures, are included within the metabolites of the
compound of Formula (I) as utilized herein.
[0040] As used herein, "an ester thereof" refers to an ester of the
phenolic hydroxy group and/or an ester of the carboxylic acid shown
in the compound of Formula (I), and an ester of the 1-hydroxyethyl
(an aliphatic hydroxy group) group of a metabolite of the compound
Formula (I). An ester of the phenolic and/or the aliphatic hydroxy
groups can include, without limitation, as the corresponding acid,
a carboxylic acid R.sub.A--CO.sub.2H, wherein R.sub.A is
C.sub.1-C.sub.6 alkyl, aryl, heteroaryl, C.sub.3-C.sub.12
cycloalkyl, or C.sub.2-C.sub.8 heterocyclyl, wherein the alkyl,
aryl, heteroaryl, cycloalkyl, or heterocyclyl are optionally
substituted with 1-4 C.sub.1-C.sub.3 alkyl, aryl, CO.sub.2H, amino,
alkylamino, or dialkylamino groups. Other acids such as mono-, di-,
or tri phosphoric acids are also contemplated. An ester of the
carboxylic acid can include, without limitation, as the
corresponding alcohol, a compound of formula R.sub.A--OH, wherein
R.sub.A is defined as above. In one embodiment, only the carboxylic
acid in Formula (I) is esterified. In another embodiment, only the
phenolic hydroxy group in Formula (I) is esterified. In another
embodiment, R.sub.A is C.sub.1-C.sub.4 alkyl. As will be apparent
to the skilled artisan, such esters act as prodrugs that are
hydrolyzed in vivo to release the compound of Formula (I) or a salt
thereof
[0041] In a preferred embodiment, the compound of Formula (I) is a
compound of Formula (IA):
##STR00006##
In another preferred embodiment, the metabolite of the compound of
Formula (I) and (IA) is a compound of Formula (IB):
##STR00007##
[0042] In one embodiment, the patient is suffering from NAFLD. In
another embodiment, the patient is suffering from NASH. In another
embodiment, the compound is administered orally. In another
embodiment, the compound is administered as a tablet or a capsule.
In another embodiment, the compound of Formula (IA) is present in
polymorphic form A that is substantially free of other polymorphic
forms. In another embodiment, the compound is administered as a
liquid dosage form. In another embodiment, the compound is
administered in an amount from 100 to 4,000 mg/day, divided into
one, two, or three portions.
[0043] Without being bound by theory, the compounds utilized herein
are effective in treating NAFLD and/or NASH due in part to their
anti-inflammatory activity. It is believed that various receptor
sites can be blocked by the compounds utilized in herein. Few, if
any, of the known inhibitors of inflammatory disease embody all of
the following sites of activity in a single molecule: inhibition of
1) leukotriene synthesis, 2) leukotriene D-4 receptors, 3)
leukotriene E-4 receptors, 4) cAMP PDE III, 5) cAMP PDE IV, 6)
synthesis of thromboxaneA-2, 7) eosinophil migration and 8) 20
lymphocyte migration. The above mechanisms are involved and
cooperate in different degrees and with different specificities
among the wide variety of cells interacting in the so-called
"inflammatory cascade," to produce a fission-like result. By
blocking a wide variety of action sites, the compounds utilized
herein are contemplated to be effective for treating NAFLD and/or
NASH.
[0044] The efficacy of a compound utilized herein can be tested by
methods well known to the skilled artisan, e.g., in the STAM mice
model as described herein below, or adapting the procedure
described in "Protection from liver fibrosis by a peroxisome
proliferator-activated receptor .delta. agonist," Keiko Iwaisako et
al., PNAS 2012, 109 (21) E1369-E1376.
Synthesis
[0045] The synthesis and certain biological activity of the
compounds of Formula (I) are described in U.S. Pat. No. 4,985,585
which is incorporated herein in its entirety by reference. For
example, the compound of Formula (IA) is prepared by reacting a
phenol of Formula (II):
##STR00008##
wherein, R is a carboxylic acid protecting group, with a compound
of Formula (III):
##STR00009##
to provide a compound of Formula (IC):
##STR00010##
Non limiting examples of acid protecting groups, or R groups,
include C.sub.1-C.sub.6 alkyl, benzyl, benzhydryl, and trityl,
wherein the benzyl, benzhydryl, or trityl group is optionally
substituted with 1-6 C.sub.1-C.sub.6 alkyl, halo, and/or
C.sub.1-C.sub.6 alkoxy groups. It will be apparent to the skilled
artisan that a leaving group other than the bromo group of Formula
(III) may be used. Non limiting examples of such other leaving
groups include chloro or tosylate.
[0046] Deprotection of the protected carboxylic acid of Formula
(IC) provides the compound of Formula (IA). As is apparent based on
this disclosure, compounds of Formula (IC) are in some embodiments
useful in accordance with this invention. Non-limiting examples of
deprotection methods include, alkaline hydrolysis and
hydrogenolysis under H.sub.2 and a catalyst such as Pd/C or
Pt/C.
[0047] The reactions are carried out in an inert organic solvent,
for example and without limitation, acetone, methylethylketone,
diethylketone, or dimethylformamide. The nucleophilic displacement
reaction may be conducted at a temperature below room temperature
up to the reflux temperature of the solvent, in the presence of an
inorganic base, such as potassium carbonate or sodium carbonate,
and optionally in the presence of potassium iodide. The reactions
are carried out for a period of time sufficient to provide
substantial product as determined by well known methods such as
thin layer chromatography and .sup.1H-NMR. Other compounds utilized
herein are made by following the procedures described herein and
upon appropriate substitution of starting materials, and/or
following methods well known to the skilled artisan. See also, U.S.
Pat. No. 5,290,812 (incorporated herein in its entirety by
reference).
[0048] The compound of Formula (IA) is recrystallized under
controlled conditions to provide an essentially pure orthorhombic
polymorph, referred to as Form A crystals (e.g., 90% or more,
preferably at least 95% Form A). Polymorphic Form A and processes
for producing it are described in U.S. Pat. Nos. 7,060,854 and
7,064,146; which are incorporated herein in their entirety by
reference. All polymorphic forms of the compound of Formula (I) are
active, but polymorphic Form A is preferred. Under certain
conditions, the solubility and the bioavailability of this
polymorph is superior to the other polymorphs and thus Form A may
offer improved solid formulations.
[0049] Form A crystals can be obtained, For example, by dissolving
the compound of Formula (IA) in 5 to 10 parts by weight of ethanol
at 25-40.degree. C. to give a yellow to orange solution. The
ethanol solution is charged with 1-10 parts of water and agitated
at 20-25.degree. C. for about 15-60 minutes and then at
5-10.degree. C. for an additional period of 1-4 hours, preferably
2.0-3.0 hours, resulting in an off-white suspension. To this
suspension is added 5-15 parts of water and the mixture is agitated
at 5-10.degree. C. for an additional 1-4 hours, preferably 1.5-2.0
hours. A solid, white to off-white product is isolated by vacuum
filtration and the filter cake is washed with water and dried in a
vacuum at 25-40.degree. C. for 12-24 hours.
[0050] For compounds utilized herein that exist in enantiomeric
forms, such as certain metabolites of the compound of Formula (I)
(for example, the compound of formula IB), the two enantiomers can
be optically resolved. Such a resolution is performed, for example,
and without limitation, by forming diastereomeric salt of a base
such as (S)-(-)-1-(1-naphthyl)ethylamine with the corresponding
carboxylic acid compound, or by separating the enantiomers using
chiral column chromatography. Intermediates to such compounds,
which intermediates also exist in enantiomeric forms can be
similarly resolved.
Administration and Formulation
[0051] The compounds utilized herein can be administered orally, or
by intravenous, intramuscular, and subcutaneous injection, or
transdermal methods. Effective dosage levels can vary widely, e.g.,
from about 100 to 4000 mg per day. In one embodiment, the daily
dosage range is 250 to 2,000 mg, given in one, two or three
portions. In one embodiment, the daily dosage range is 100 to 500
mg, such as 100, 200, 300, 400, or 500 mg given in one, two or
three portions. In one embodiment, the daily dosage range is 250 to
2,000 mg, such as 250, 500, 750, 1,000, 1,250, 1,500, 1,750, or
2,000 mg given in one, two or three portions. In one embodiment,
the daily dosage range is 1000 to 4,000 mg, such as 1,000, 2,000,
3,000, or 4,000 mg, given in one, two or three portions. In another
embodiment, the dosage is 1000 mg twice a day. In other
embodiments, suitable dosages include 1000 mg qd, 1000 mg bid, and
750 mg tid.
[0052] Actual amounts will depend on the circumstances of the
patient being treated. As those skilled in the art recognize, many
factors that modify the action of the active substance will be
taken into account by the treating physician such as the age, body
weight, sex, diet and condition of the patient, the time of
administration, the rate and route of administration. Optimal
dosages for a given set of conditions can be ascertained by those
skilled in the art using conventional dosage determination
tests.
[0053] The compounds utilized herein can be formulated in any
pharmaceutically acceptable form, including liquids, powders,
creams, emulsions, pills, troches, suppositories, suspensions,
solutions, and the like. Therapeutic compositions containing the
compounds utilized herein will ordinarily be formulated with one or
more pharmaceutically acceptable ingredients in accordance with
known and established practice. In general, tablets are formed
utilizing a carrier such as modified starch, alone or in
combination with 10% by weight of carboxymethyl cellulose (Avicel).
The formulations are compressed at from 1,000 to 3,000 pounds
pressure in the tablet forming process. The tablets preferably
exhibit an average hardness of about 1.5 to 8.0 kp/cm.sup.2,
preferably 5.0 to 7.5 kp/cm.sup.2. Disintegration time varies from
about 30 seconds to about 15 or 20 minutes.
[0054] Formulations for oral use can be provided as hard gelatin
capsules wherein the therapeutically active compounds utilized
herein are mixed with an inert solid diluent such as calcium
carbonate, calcium phosphate or kaolin, or as soft gelatin capsules
in which the compounds are mixed with an oleaginous medium, e.g.,
liquid paraffin or olive oil. Suitable carriers include magnesium
carbonate, magnesium stearate, talc, sugar, lactose, pectin,
dextrin, starch, gelatin, tragacanth, methylcellulose, sodium
carboxymethyl cellulose, a low melting wax, cocoa butter, and the
like.
[0055] The compounds utilized herein can be formulated as aqueous
suspensions in admixture with pharmaceutically acceptable
excipients such as suspending agents, e.g., sodium carboxymethyl
cellulose, methylcellulose, hydroxypropylmethyl cellulose, sodium
alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;
dispersing or wetting agents such as naturally occurring
phosphatide, e.g., lecithin, or condensation products of an
alkaline oxide with fatty acids, e.g., polyoxyethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, e.g, heptadecaethylene-oxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol, e.g., polyoxyethylene sorbitol monoleate or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, e.g., polyoxyethylene
sorbitan monoleate. Such aqueous suspensions can also contain one
or more preservatives, e.g., ethyl- or -n-propyl-p-hydroxy
benzoate, one or more coloring agents, one or more flavoring agents
and one or more sweetening agents, such as glycerol, sorbitol,
sucrose, saccharin or sodium or calcium cyclamate.
[0056] Suitable formulations also include sustained release dosage
forms, such as those described in U.S. Pat. Nos. 4,788,055;
4,816,264; 4,828,836; 4,834,965; 4,834,985; 4,996,047; 5,071,646;
and, 5,133,974, the contents of which are incorporated herein in
their entirety by reference.
[0057] Other forms suitable for oral administration include liquid
form preparations including emulsions, syrups, elixirs, aqueous
solutions, or solid form preparations which are intended to be
converted shortly before use to liquid form preparations. Emulsions
may be prepared in solutions, for example, in aqueous propylene
glycol solutions or may contain emulsifying agents, for example,
such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions
can be prepared by dissolving the active component in water and
adding suitable colorants, flavors, stabilizing, and thickening
agents. Solid form preparations may contain, in addition to the
active component, colorants, flavors, stabilizers, buffers,
artificial and natural sweeteners, dispersants, thickeners,
solubilizing agents, and the like.
[0058] The compounds utilized herein may be formulated for
parenteral administration (e.g., by injection, for example bolus
injection or continuous infusion) and may be presented in unit dose
form in ampoules, pre-filled syringes, small volume infusion or in
multi-dose containers with an added preservative. The compositions
may take such forms as suspensions, solutions, or emulsions in oily
or aqueous vehicles, for example as solutions in aqueous
polyethylene glycol. Examples of oily or nonaqueous carriers,
diluents, solvents or vehicles include propylene glycol,
polyethylene glycol, vegetable oils (e.g., olive oil), and
injectable organic esters (e.g., ethyl oleate), and may contain
formulatory agents such as preserving, wetting, emulsifying or
suspending, stabilizing and/or dispersing agents. Alternatively,
the active ingredient may be in powder form, obtained by aseptic
isolation of sterile solid or by lyophilisation from solution for
constitution before use with a suitable vehicle, e.g., sterile,
pyrogen-free water.
[0059] The compounds utilized herein may be formulated for topical
administration to the epidermis as ointments, creams or lotions, or
as a transdermal patch. Ointments and creams may, for example, be
formulated with an aqueous or oily base with the addition of
suitable thickening and/or gelling agents. Lotions may be
formulated with an aqueous or oily base and will in general also
containing one or more emulsifying agents, stabilizing agents,
dispersing agents, suspending agents, thickening agents, or
coloring agents. Formulations suitable for topical administration
in the mouth include lozenges comprising active agents in a
flavored base, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert base such as gelatin
and glycerin or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0060] The compounds utilized herein may be formulated for
administration as suppositories. In such a formulation, a low
melting wax, such as a mixture of fatty acid glycerides or cocoa
butter is first melted and the active component is dispersed
homogeneously, for example, by stirring. The molten homogeneous
mixture is then poured into convenient sized molds, allowed to
cool, and to solidify.
[0061] The compounds utilized herein may be formulated for vaginal
administration. Pessaries, tampons, creams, gels, pastes, foams or
sprays containing in addition to the active ingredient such
carriers as are known in the art to be appropriate.
[0062] The compounds utilized herein may be formulated for nasal
administration. The solutions or suspensions are applied directly
to the nasal cavity by conventional means, for example, with a
dropper, pipette or spray. The formulations may be provided in a
single or multidose form. The patient can administer an
appropriate, predetermined volume of the solution or suspension via
a dropper or pipette. A spray may be administered for example by
means of a metering atomizing spray pump.
[0063] The compounds utilized herein may be formulated for aerosol
administration, particularly to the respiratory tract and including
intranasal administration. The compound will generally have a small
particle size for example of the order of 5 microns or less. Such a
particle size may be obtained by means known in the art, for
example by micronization. The active ingredient is provided in a
pressurized pack with a suitable propellant such as a
chlorofluorocarbon (CFC), (for example, dichlorodifluoromethane,
trichlorofluoromethane, or dichlorotetrafluoroethane), carbon
dioxide or other suitable gases. The aerosol may conveniently also
contain a surfactant such as lecithin. The dose of drug may be
controlled by a metered valve. Alternatively the active ingredients
may be provided in a form of a dry powder, for example a powder mix
of the compound in a suitable powder base such as lactose, starch,
starch derivatives such as hydroxypropylmethyl cellulose and
polyvinylpyrrolidine. The powder carrier will form a gel in the
nasal cavity. The powder composition may be presented in unit dose
form for example in capsules or cartridges of, for example gelatin
or blister packs from which the powder may be administered by means
of an inhaler.
[0064] When desired, formulations can be prepared with enteric
coatings adapted for sustained or controlled release administration
of the active ingredient. A common type of controlled release
formulation that may be used for the purposes of the present
invention comprises an inert core, such as a sugar sphere, a first
layer, coated with an inner drug-containing second layer, and an
outer membrane or third layer controlling drug release from the
inner layer.
[0065] The cores are preferably of a water-soluble or swellable
material, and may be any such material that is conventionally used
as cores or any other pharmaceutically acceptable water-soluble or
water-swellable material made into beads or pellets. The cores may
be spheres of materials such as sucrose/starch (Sugar Spheres NF),
sucrose crystals, or extruded and dried spheres typically comprised
of excipients such as microcrystalline cellulose and lactose.
[0066] The substantially water-insoluble material in the first
layer is generally a "GI insoluble" or "GI partially insoluble"
film forming polymer (dispersed or dissolved in a solvent). As
examples may be mentioned ethyl cellulose, cellulose acetate,
cellulose acetate butyrate, polymethacrylates such as ethyl
acrylate/methyl methacrylate copolymer (Eudragit NE-30-D) and
ammonio methacrylate copolymer types A and B (Eudragit RL30D and
RS30D), and silicone elastomers. Usually, a plasticizer is used
together with the polymer. Exemplary plasticizers include:
dibutylsebacate, propylene glycol, triethylcitrate,
tributylcitrate, castor oil, acetylated monoglycerides, acetyl
triethylcitrate, acetyl butylcitrate, diethyl phthalate, dibutyl
phthalate, triacetin, fractionated coconut oil (medium-chain
triglycerides).
[0067] The second layer containing the active ingredient may be
comprised of the active ingredient (drug) with or without a polymer
as a binder. The binder, when used, is usually hydrophilic but may
be water-soluble or water-insoluble. Exemplary polymers to be used
in the second layer containing the active drug are hydrophilic
polymers such as polyvinylpyrrolidone, polyalkylene glycol such as
polyethylene glycol, gelatine, polyvinyl alcohol, starch and
derivatives thereof, cellulose derivatives, such as
hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose,
carboxymethyl cellulose, methyl cellulose, ethyl cellulose,
hydroxyethyl cellulose, carboxyethyl cellulose, carboxymethyl
hydroxyethyl cellulose, acrylic acid polymers, polymethacrylates,
or any other pharmaceutically acceptable polymer. The ratio of drug
to hydrophilic polymer in the second layer is usually in the range
of from 1:100 to 100:1 (w/w).
[0068] Suitable polymers for use in the third layer, or membrane,
for controlling the drug release may be selected from water
insoluble polymers or polymers with pH-dependent solubility, such
as, for example, ethyl cellulose, hydroxypropylmethyl cellulose
phthalate, cellulose acetate phthalate, cellulose acetate
trimellitate, polymethacrylates, or mixtures thereof, optionally
combined with plasticizers, such as those mentioned above.
[0069] Optionally, the controlled release layer comprises, in
addition to the polymers above, another substance(s) with different
solubility characteristics, to adjust the permeability, and thereby
the release rate, of the controlled release layer. Exemplary
polymers that may be used as a modifier together with, for example,
ethyl cellulose include: HPMC, hydroxyethyl cellulose,
hydroxypropyl cellulose, methylcellulose, carboxymethylcellulose,
polyethylene glycol, polyvinylpyrrolidone (PVP), polyvinyl alcohol,
polymers with pH-dependent solubility, such as cellulose acetate
phthalate or ammonio methacrylate copolymer and methacrylic acid
copolymer, or mixtures thereof. Additives such as sucrose, lactose
and pharmaceutical grade surfactants may also be included in the
controlled release layer, if desired.
[0070] Also provided herein are unit dosage forms of the
formulations. In such forms, the formulation is subdivided into
unit dosages containing appropriate quantities of the active
component (e.g., and without limitation, a compound of Formula (I)
or an ester thereof, or a salt of each thereof). The unit dosage
form can be a packaged preparation, the package containing discrete
quantities of preparation, such as packeted tablets, capsules, and
powders in vials or ampoules. Also, the unit dosage form can be a
capsule, tablet, cachet, or lozenge itself, or it can be the
appropriate number of any of these in packaged form.
[0071] Other suitable pharmaceutical carriers and their
formulations are described in Remington: The Science and Practice
of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company,
19th edition, Easton, Pa.
EXAMPLES
Example 1
Treatment of Non-Alcoholic Steatohepatitis (NASH)
[0072] 250 adults with nonalcoholic steatohepatitis are randomly
assigned to receive MN-001 or MN-002, each at a daily dose of 500
mg, or placebo, for up to 6 months. The primary outcome is an
improvement in histologic features of nonalcoholic steatohepatitis,
as assessed with the use of a composite of standardized scores for
steatosis, lobular inflammation, hepatocellular ballooning, and/or
fibrosis. The results are analyzed following methods well known to
the skilled artisan.
Example 2
Treatment of Non-Alcoholic Fatty Liver Disease (NAFLD)
[0073] A randomized, double-blind, placebo-controlled study is
performed on 50 patients with NAFLD diagnosed by ultrasound (US)
and confirmed by liver biopsy (40 patients). The patients are
randomized to receive MN-001 or MN-002 (each at a daily dose of 500
mg for up to 6 months) or placebo. All patients participate in an
identical behavioral weight loss program. All patients undergo
monthly evaluation by abdominal US. Liver enzyme levels, lipid
profiles, insulin levels, and anthropometric parameters are also
monitored, and all patients undergo nutritional follow-up
evaluation. Patients also undergo a second liver biopsy examination
at the end of the study. Serum alanine transaminase levels and
steatosis by US are measured as non-limiting endpoints. The results
are analyzed following methods well known to the skilled
artisan.
Example 3
Therapeutically Beneficial Effects of MN-001 in STAM Model of
Non-Alcoholic Steatohepatitis
[0074] STAM.TM. is a model for non-alcoholic steatohepatitis
(NASH), symptoms thereof, and related liver disorders, created by
the combination of chemical and dietary interventions in C57BL/6
mice. Telmisartan has been shown to have anti-NASH, -fibrosis and
-inflammatory effects in STAM mice and therefore was used as the
positive control in the present study. According to this study, and
as described below, treatment with Telmisartan significantly
decreased liver weight, NAS, fibrosis area and inflammation area
compared with the Vehicle group in agreement with reported data,
thereby providing evidence of the usefulness of the STAM mice model
as employed herein for demonstrating the usefulness of a compound
utilized in this invention.
[0075] Treatment with MN-001 significantly reduced fibrosis area
compared with Vehicle in a dose dependent manner, demonstrating
anti-fibrotic effect of MN-001 in the present study. High dose of
MN-001 tended to reduce liver hydroxyproline content, supporting
its anti-fibrotic property. Treatment with high dose of MN-001
significantly decreased the NAFLS activity score (NAS). The
improvement in NAS was attributable, e.g., to the reduction in
lobular inflammation and hepatocyte ballooning. Notably, high dose
of MN-001 significantly reduced ballooning score. Since hepatocyte
ballooning is derived from oxidative stress-induced hepatocellular
damage and is associated with disease progression of NASH (Fujii H
et al. J. Atheroscler. Thromb. 2009; 16:893, Rangwala F et al. J.
Pathol. 2011; 224:401), it is contemplated, without being bound by
theory, that MN-001 can improve NASH pathology by inhibiting
hepatocyte damage and ballooning.
[0076] Treatment with low dose of MN-001 significantly reduced
inflammation area compared with Vehicle, demonstrating
anti-inflammatory effect of MN-001.
[0077] In conclusion, MN-001, administered at various doses, showed
one or more of anti-NASH, anti-fibrotic and anti-inflammatory
effects in the present study. These and other results are discussed
below.
Materials and Methods
Test Substance
[0078] MN-001 was provided by MediciNove Inc. To prepare dosing
solution, MN-001 was weighed and dissolved in 0.2% methylcellulose
(vehicle). Telmisartan (Micardis.RTM.) was purchased from
Boehringer Ingelheim GmbH (Germany) and was dissolved in pure
water.
Induction of NASH
[0079] NASH was induced in 50 male mice by a single subcutaneous
injection of 200 .mu.g streptozotocin (STZ, Sigma-Aldrich, USA)
solution 2 days after birth and feeding with high fat diet (HFD, 57
kcal % fat, cat#: HFD32, CLEA Japan, Japan) after 4 weeks of age.
Ten male littermates, fed with normal diet and without STZ
treatment, were used for the normal group.
Route of Drug Administration
[0080] Vehicle, MN-001, and Telmisartan were administered by oral
route in a volume of 10 mL/kg.
Treatment Doses
[0081] MN-001 was administered at doses of 10, 30, and 100 mg/kg
once daily. Telmisartan was administered at dose of 10 mg/kg once
daily.
Animals
[0082] C57BL/6 mice (15-day-pregnant female) were obtained from
Charles River Laboratories Japan (Kanagawa, Japan). All animals
used in the study were housed and cared for in accordance with the
Japanese Pharmacological Society Guidelines for Animal Use.
Environment
[0083] The animals were maintained in a SPF facility under
controlled conditions of temperature (23.+-.2.degree. C.), humidity
(45.+-.10%), lighting (12-hour artificial light and dark cycles;
light from 8:00 to 20:00) and air exchange. A high pressure
(20.+-.4 Pa) was maintained in the experimental room to prevent
contamination of the facility.
Animal Husbandry
[0084] The animals were housed in polycarbonate cages KN-600
(Natsume Seisakusho, Japan) with a maximum of 4 mice per cage.
Sterilized PULMAS.mu. (Material Research Center, Japan) was used
for bedding and replaced once a week.
Food and Drink
[0085] Sterilized solid HFD was provided ad libitum, being placed
in the metal lid on top of the cage. Pure water was provided ad
libitum from a water bottle equipped with a rubber stopper and a
sipper tube. Water bottles were replaced once a week, cleaned and
sterilized in autoclave and reused.
Animal and Cage Identification
[0086] Mice were identified by numbers engraved on earrings. Each
cage was labeled with a specific identification code.
Measurement of Whole Blood and Plasma Biochemistry
[0087] Non-fasting blood glucose was measured in whole blood using
LIFE CHECK (EIDIA, Japan). For plasma biochemistry, blood was
collected in polypropylene tubes with anticoagulant (Novo-Heparin,
Mochida Pharmaceutical, Japan) and centrifuged at 1,000.times.g for
15 minutes at 4.degree. C. The supernatant was collected and stored
at -80.degree. until use. Plasma ALT and AST levels were measured
by FUJI DRI-CHEM 7000 (Fujifilm, Japan).
Measurement of Liver Biochemistry
Liver Hydroxyproline Content
[0088] To quantify liver hydroxyproline content, frozen liver
samples (32-40 mg) were processed by an alkaline-acid hydrolysis
method as follows. Liver samples were defatted with 100% acetone,
dried in the air, dissolved in 2N NaOH at 65.degree. C., and
autoclaved at 121.degree. C. for 20 minutes. The lysed samples (400
.mu.L) were acid-hydrolyzed with 400 .mu.L of 6N HCl at 121.degree.
C. for 20 minutes, and neutralized with 400 .mu.L of 4N NaOH
containing 10 mg/mL activated carbon. AC buffer (2.2M acetic
acid/0.48M citric acid, 400 .mu.L) was added to the samples,
followed by centrifugation to collect the supernatant. A standard
curve of hydroxyproline was constructed with serial dilutions of
trans-4-hydroxy-L-proline (Sigma-Aldrich) starting at 16 .mu.g/mL.
The prepared samples and standards (each 400 .mu.L) were mixed with
400 .mu.L chloramine T solution (Wako Pure Chemical Industries) and
incubated for 25 minutes at room temperature. The samples were then
mixed with Ehrlich's solution (400 .mu.L) and heated at 65.degree.
C. for 20 minutes to develop the color. After samples were cooled
on ice and centrifuged to remove precipitates, the optical density
of each supernatant was measured at 560 nm. The concentrations of
hydroxyproline were calculated from the hydroxyproline standard
curve. Protein concentrations of liver samples were determined
using a BCA protein assay kit (Thermo Fisher Scientific, USA) and
used to normalize the calculated hydroxyproline values. Liver
hydroxyproline levels were expressed as .mu.g per mg protein.
Histopathological Analyses
[0089] For HE staining, sections were cut from paraffin blocks of
left lateral liver tissue prefixed in Bouin's solution and stained
with Lillie-Mayer's Hematoxylin (Muto Pure Chemicals, Japan) and
eosin solution (Wako Pure Chemical Industries). NAS was calculated
according to the criteria of Kleiner (Kleiner D E. et al.,
Hepatology, 2005; 41:1313). To visualize collagen deposition,
Bouin's fixed left lateral liver sections were stained using
picro-Sirius red solution (Waldeck, Germany).
[0090] For immunohistochemistry, sections were cut from frozen left
lateral liver tissues embedded in Tissue-Tek O.C.T. compound and
fixed in acetone. Endogenous peroxidase activity was blocked using
0.03% H2O2 for 5 minutes, followed by incubation with Block Ace
(Dainippon Sumitomo Pharma, Japan) for 10 minutes. The sections
were incubated with a 200-fold dilution of anti-.alpha.-SMA
(Epitomics, USA) or anti-F4/80 antibody (BMA Biomedicals,
Switzerland) 1 hour at room temperature. After incubation with
secondary antibody (HRP-Goat anti-rat antibody, Invitrogen, USA),
enzyme-substrate reactions were performed using
3,3'-diaminobenzidine/H2O2 solution (Nichirei, Japan).
[0091] For quantitative analysis of fibrosis area, inflammation
area, and semi-quantification of .alpha.-SMA, bright field images
of Sirius red-stained, F4/80 and .alpha.-SMA-immunostained sections
were captured around the central vein using a digital camera
(DFC280; Leica, Germany) at 200-fold magnification, and the
positive areas in 5 fields/section were measured using ImageJ
software (National Institute of Health, USA).
Quantitative RT-PCR
[0092] Total RNA was extracted from liver samples using RNAiso
(Takara Bio, Japan) according to the manufacturer's instructions.
One .mu.g of RNA was reverse-transcribed using a reaction mixture
containing 4.4 mM MgCl.sub.2 (Roche, Switzerland), 40 U RNase
inhibitor (Toyobo, Japan), 0.5 mM dNTP (Promega, USA), 6.28 .mu.M
random hexamer (Promega), 5.times. first strand buffer (Promega),
10 mM dithiothreitol (Invitrogen) and 200 U MMLV-RT (Invitrogen) in
a final volume of 20 .mu.L. The reaction was carried out for 1 hour
at 37.degree. C., followed by 5 minutes at 99.degree. C. Real-time
PCR was performed using real-time PCR DICE and SYBR premix Taq
(Takara Bio). To calculate the relative mRNA expression level, the
expression of each gene was normalized to that of reference gene
36B4 (gene symbol: Rplp0). Information of PCR-primer sets and the
plate layout was described in Table 1.
Statistical Tests
[0093] Statistical analyses were performed using Bonferroni
Multiple Comparison Test on GraphPad Prism 4 (GraphPad Software,
USA). P values <0.05 were considered statistically significant.
A trend or tendency was assumed when a one-tailed t-test returned P
values <0.10. Results were expressed as mean.+-.SD.
Experimental Design and Treatment
Study Groups
Group 1: Normal
[0094] Ten normal mice were fed with a normal diet ad libitum
without any treatment until 9 weeks of age.
Group 2: Vehicle
[0095] Ten NASH mice were orally administered vehicle in a volume
of 10 mL/kg once daily from 6 to 9 weeks of age.
Group 3: MN-001-Low Dose
[0096] Ten NASH mice were orally administered vehicle supplemented
with MN-001 at a dose of 10 mg/kg once daily from 6 to 9 weeks of
age.
Group 4: MN-001-Middle Dose
[0097] Ten NASH mice were orally administered vehicle supplemented
with MN-001 at a dose of 30 mg/kg once daily from 6 to 9 weeks of
age.
Group 5: MN-001-High Dose
[0098] Ten NASH mice were orally administered vehicle supplemented
with MN-001 at a dose of 100 mg/kg once daily from 6 to 9 weeks of
age.
Group 6: Telmisartan
[0099] Six NASH mice were orally administered pure water
supplemented with Telmisartan at a dose of 10 mg/kg once daily from
6 to 9 weeks of age. The table below summarizes the treatment
schedule.
TABLE-US-00001 No. Test Dose Volume Sacrifice Group mice Mice
substance (mg/kg) (mL/kg) Regimens (wks) 1 10 Normal -- -- -- -- 9
2 10 STAM Vehicle -- 10 Oral, once daily, 9 6 wks-9 wks 3 10 STAM
MN-001 10 10 Oral, once daily, 9 6 wks-9 wks 4 10 STAM MN-001 30 10
Oral, once daily, 9 6 wks-9 wks 5 10 STAM MN-001 100 10 Oral, once
daily, 9 6 wks-9 wks 6 10 STAM Telmisartan 10 10 Oral, once daily,
9 6 wks-9 wks
Animal Monitoring and Sacrifice
[0100] The viability, clinical signs and behavior were monitored
daily. Body weight was recorded before the treatment. Mice were
observed for significant clinical signs of toxicity, moribundity
and mortality approximately 60 minutes after each administration.
The animals were sacrificed by exsanguination through direct
cardiac puncture under ether anesthesia (Wako Pure Chemical
Industries).
Results
Histological Analyses
HE Staining and NAFLD Activity Score
[0101] Liver sections from the Vehicle group exhibited severe
micro- and macrovesicular fat deposition, hepatocellular ballooning
and inflammatory cell infiltration. Consistent with these
observations, NAS significantly increased in the Vehicle group
compared with the Normal group. The Telmisartan group showed marked
improvements in hepatocellular ballooning and inflammatory cell
infiltration, with significant reduction in NAS compared with the
Vehicle group. The MN-001-high dose group showed marked
improvements in hepatocellular ballooning and moderate improvements
in inflammatory cell infiltration. NAS significantly decreased in
the MN-001-high group compared with the Vehicle group. The
MN-001-low and -middle groups showed a moderate decrease in
hepatocellular ballooning compared with the Vehicle group. There
was no significant difference in the NAS between the Vehicle group
and any of the other groups (Normal: 0.0.+-.00, Vehicle:
5.3.+-.0.5, MN-001-low: 4.7.+-.0.5, MN-001-middle: 4.7.+-.0.5,
MN-001-high: 3.3.+-.0.8, Telmisartan: 2.6.+-.0.7). See FIGS. 1-3
and the Tables below.
TABLE-US-00002 A Table of NAFLD Activity Score (NAS) Score
Steatosis Lobular inflammation Hepatocyte ballooning NAS Group n 0
1 2 3 0 1 2 3 0 1 2 (mean .+-. SD) Normal 10 10 -- -- -- 10 -- --
-- 10 -- -- 0.0 .+-. 0.0 Vehicle 10 -- 9 1 -- -- -- 8 2 -- -- 10
5.3 .+-. 0.5 MN-001-low 10 -- 10 -- -- -- 1 9 -- -- 2 8 4.7 .+-.
0.5 MN-001-middle 10 -- 10 -- -- -- -- 10 -- -- 3 7 4.7 .+-. 0.5
MN-001-high 10 -- 10 -- -- 1 4 5 -- 2 7 1 3.3 .+-. 0.8 Telmisartan
10 1 9 -- -- -- 10 -- -- 4 6 -- 2.6 .+-. 0.7
TABLE-US-00003 Definition of NAS Components Item score Extent
Steatosis 0 <5% 1 5-33% 2 >33-66% 3 >66% Hepatocyte 0 None
Ballooning 1 Few balloon cells 2 Many cells/prominent ballooning
Lobular 0 No foci Inflammation 1 <2 foci/200x 2 2-4 foci/200x 3
>4 foci/200x
Sirius Red Staining
[0102] Liver sections from the Vehicle group showed increased
collagen deposition in the pericentral region of liver lobule
compared with the Normal group. The percentage of fibrosis area
(Sirius red-positive area) significantly increased in the Vehicle
group compared with the Normal group. The fibrosis area
significantly decreased in both the Telmisartan group and MN-001
treatment groups compared with the Vehicle group (Normal:
0.29.+-.0.08%, Vehicle: 0.97.+-.0.19%, MN-001-low: 0.76.+-.0.19%,
MN-001-middle: 0.76.+-.0.16%, MN-001-high: 0.69.+-.0.18%,
Telmisartan: 0.62.+-.0.09%). See, FIG. 4.
F4/80 Immunostaining
[0103] Liver sections from the Vehicle group showed an increased
number and size of F4/80-positive cells in the liver lobule
compared with the Normal group. The percentage of inflammation area
(F4/80-positive area) significantly increased in the Vehicle group
compared with the Normal group. The inflammation area significantly
decreased in both the Telmisartan group and MN-001-low groups
compared with the Vehicle group. There was no significant
difference in the inflammation area between the Vehicle group and
any of the other groups (Normal: 3.26.+-.0.66%, Vehicle:
6.56.+-.1.19%, MN-001-low: 5.18.+-.0.85%, MN-001-middle:
6.33.+-.0.84%, MN-001-high: 6.31.+-.0.76%, Telmisartan:
4.46.+-.0.88%). See, FIG. 5.
Alpha-SMA Immunostaining
[0104] Liver sections from the Vehicle group showed an increased
.alpha.-SMA-positive cells in the liver lobule compared with the
Normal group. The percentage of .alpha.-SMA-positive area
significantly increased in the Vehicle group compared with the
Normal group. The .alpha.-SMA-positive area tended to decrease in
the MN-001-low and -high groups compared with the Vehicle group.
There were no significant differences in .alpha.-SMA-positive area
between the Vehicle group and any of the other groups (Normal:
0.07.+-.0.03%, Vehicle: 0.15.+-.0.08%, MN-001-low: 0.10.+-.0.05%,
MN-001-middle: 0.11.+-.0.04%, MN-001-high: 0.11.+-.0.04%,
Telmisartan: 0.12.+-.0.05%).
Body Weight Changes and General Condition
[0105] Body weight gradually increased during the treatment period
in all except the Telmisartan group. Mean body weight of Vehicle
group was lower than that of Normal group throughout the treatment
period. Mean body weight of Telmisartan group was significantly
lower than that of Vehicle group from at day 11 to day 22. There
were no significant differences in mean body weight between the
Vehicle group and any of the other groups during the treatment
period. In the present study, none of the animals showed
deterioration in general condition.
Body Weight at the Day of Sacrifice
[0106] Mean body weight at sacrifice was significantly lower in the
Vehicle group compared with the Normal group. The Telmisartan group
showed a significant decrease in mean body weight compared with the
Vehicle group. There were no significant differences in mean body
weight between the Vehicle group and any of the other groups
(Normal: 25.0.+-.0.4 g, Vehicle: 20.5.+-.1.9 g, MN-001-low:
21.1.+-.1.3 g, MN-001-middle: 20.3.+-.1.0 g, MN-001-high:
20.6.+-.1.5 g, Telmisartan: 18.0.+-.1.9 g).
Liver Weight and Liver-to-Body Weight Ratio
[0107] Mean liver weight significantly increased in the Vehicle
group compared with the Normal group. The Telmisartan group showed
a significant decrease in mean liver weight compared with the
Vehicle group. The liver weight tended to decrease in the
MN-001-middle group compared with the Vehicle group. There were no
significant differences in mean liver weight between the Vehicle
group and any of the other groups (Normal: 1083.+-.83 mg, Vehicle:
1555.+-.112 mg, MN-001-low: 1567.+-.165 mg, MN-001-middle:
1439.+-.118 mg, MN-001-high: 1480.+-.145 mg, Telmisartan:
1172.+-.90 mg).
[0108] The liver-to-body weight ratio significantly increased in
the Vehicle group compared with the Normal group. The Telmisartan
group showed a significant decrease in mean liver-to-body weight
ratio compared with the Vehicle group. The liver-to-body weight
ratio tended to decrease in the MN-001-middle and -high groups
compared with the Vehicle group. There were no significant
differences in mean liver-to-body weight ratio between the Vehicle
group and the MN-001-low group (Normal: 4.3.+-.0.3%, Vehicle:
7.6.+-.0.6%, MN-001-low: 7.4.+-.0.8%, MN-001-middle: 7.1.+-.0.5%,
MN-001-high: 7.2.+-.0.6%, Telmisartan: 6.5.+-.0.4%).
Whole Blood and Biochemistry
[0109] Whole blood glucose (FIG. 3.1 and Table 3) Blood glucose
levels significantly increased in the Vehicle group compared with
the Normal group. The Telmisartan group showed a significant
increase in the blood glucose levels compared with the Vehicle
group. There were no significant differences in blood glucose
levels between the Vehicle group and any of the other groups
(Normal: 192.+-.40 mg/dL, Vehicle: 632.+-.95 mg/dL, MN-001-low:
614.+-.98 mg/dL, MN-001-middle: 609.+-.78 mg/dL, MN-001-high:
671.+-.124 mg/dL, Telmisartan: 876.+-.29 mg/dL).
Plasma ALT
[0110] Plasma ALT levels tended to increase in the Vehicle group
compared with the Normal group. Plasma ALT levels tended to
decrease in the Telmisartan group compared with the Vehicle group.
There were no significant differences in plasma ALT levels between
the Vehicle group and any of the other groups (Normal: 31.+-.10
U/L, Vehicle: 51.+-.22 U/L, MN-001-low: 71.+-.60 U/L,
MN-001-middle: 48.+-.23 U/L, MN-001-high: 54.+-.11 U/L,
Telmisartan: 37.+-.6 U/L).
Plasma AST
[0111] Plasma AST levels tended to decrease in the Vehicle group
compared with the Normal group. Plasma AST levels tended to
increase in the MN-001-middle and -high groups compared with the
Vehicle group. There were no significant differences in plasma AST
levels between the Vehicle group and the MN-001-low group (Normal:
300.+-.77 U/L, Vehicle: 193.+-.95 U/L, MN-001-low: 214.+-.210 U/L,
MN-001-middle: 270.+-.114 U/L, MN-001-high: 385.+-.183 U/L,
Telmisartan: 190.+-.28 U/L).
Liver Hydroxyproline Content
[0112] There were no significant differences in liver
hydroxyproline content between the Normal group and the Vehicle
group. The liver hydroxyproline content tended to increase in the
Telmisartan group compared with the Vehicle group. The liver
hydroxyproline content tended to decrease in the MN-001-high group
compared with the Vehicle group. There were no significant
differences in liver hydroxyproline content between the Vehicle
group and any of the other groups (Normal: 0.61.+-.0.12 .mu.g/mg
protein, Vehicle: 0.67.+-.0.16 .mu.g/mg protein, MN-001-low:
0.78.+-.0.34 .mu.g/mg protein, MN-001-middle: 0.63.+-.0.12 .mu.g/mg
protein, MN-001-high: 0.55.+-.0.14 .mu.g/mg protein, Telmisartan:
0.87.+-.0.23 .mu.g/mg protein). See, FIG. 6.
Gene Expression Analysis
Alpha-SMA
[0113] Alpha-SMA mRNA expression levels tended to be up-regulated
in the Vehicle group compared with the Normal group. Alpha-SMA mRNA
expression levels tended to be up-regulated in the Telmisartan
group compared with the Vehicle group. There were no significant
differences in .alpha.-SMA mRNA expression levels between the
Vehicle group and any of the other groups (Normal: 1.00.+-.0.44,
Vehicle: 4.08.+-.2.56, MN-001-low: 36.8.+-.111, MN-001-middle:
3.13.+-.2.52, MN-001-high: 5.78.+-.3.45, Telmisartan:
5.21.+-.1.43).
TNF-.alpha.
[0114] TNF-.alpha. mRNA expression levels tended to be up-regulated
in the Vehicle group compared with the Normal group. There were no
significant differences in TNF-.alpha. mRNA expression levels
between the Vehicle group and any of the other groups (Normal:
1.00.+-.0.48, Vehicle: 9.88.+-.19.3, MN-001-low: 3.42.+-.2.53,
MN-001-middle: 7.97.+-.9.30, MN-001-high: 9.74.+-.3.34,
Telmisartan: 8.35.+-.2.84).
CCR2
[0115] CCR2 mRNA expression levels were significantly up-regulated
in the Vehicle group compared with the Normal group. CCR2 mRNA
expression levels were significantly down-regulated in the
MN-001-low and -middle groups compared with the Vehicle group.
There were no significant differences in CCR2 mRNA expression
levels between the Vehicle group and any of the other groups
(Normal: 1.00.+-.0.27, Vehicle: 6.83.+-.9.89, MN-001-low:
0.13.+-.0.09, MN-001-middle: 0.22.+-.0.35, MN-001-high:
3.86.+-.1.43, Telmisartan: 3.21.+-.0.85).
MCP-1
[0116] MCP-1 mRNA expression levels were significantly up-regulated
in the Vehicle group compared with the Normal group. MCP-1 mRNA
expression levels were significantly down-regulated in the
MN-001-low group compared with the Vehicle group. MCP-1 mRNA
expression levels tended to be down-regulated in the MN-001-high
and Telmisartan groups compared with the Vehicle group.
[0117] There were no significant differences in MCP-1 mRNA
expression levels between the Vehicle group and any of the other
groups (Normal: 1.00.+-.0.35, Vehicle: 2.17.+-.42.2, MN-001-low:
1.97.+-.2.06, MN-001-middle: 4.00.+-.7.78, MN-001-high:
3.64.+-.1.52, Telmisartan: 2.69.+-.0.95).
Collagen Type 1
[0118] Collagen Type 1 mRNA expression levels tended to be
up-regulated in the Vehicle group compared with the Normal group.
Collagen Type 1 mRNA expression levels were significantly
TIMP-1
[0119] TIMP-1 mRNA expression levels were significantly
up-regulated in the Vehicle group compared with the Normal group.
TIMP-1 mRNA expression levels were significantly down-regulated in
the MN-001-low and -middle groups compared with the Vehicle group.
There were no significant differences in TIMP-1 mRNA expression
levels between the Vehicle group and any of the other groups
(Normal: 1.00.+-.0.37, Vehicle: 9.78.+-.7.28, MN-001-low:
2.20.+-.1.52, MN-001-middle: 3.64.+-.1.66, MN-001-high:
10.6.+-.5.83, Telmisartan: 7.82.+-.2.62).
[0120] In conclusion, MN-001, administered at various doses, showed
one or more of anti-NASH, anti-fibrotic and anti-inflammatory
effects in the present study.
* * * * *