Note: Descriptions are shown in the official language in which they were submitted.
CA 02641871 2008-10-24
PHARMACEUTICAL COMPOSITION FOR TREATING ALCOHOL-INDUCED
LIVER INJURY COMPRISING (4S,5S)-5-FLUOROMETHYL-5-HYDROXY-=1-
( { [(5R)-5-ISOPROPYL-3-(IS OQUINOLIN-I-YL)-4,5-DIHYDRO-5-
ISOXAZOLYL]CARBONYL}AMINO)-DIHYDROFURAN-2-ONE OR
PHARMACEUTICALLY ACCEPTABLE SALT THEREOF
FILED OF THE INVENTION
The present iiivention relates to a pharinaceutical composition for treating
alcohol-induced liver injury comprising (4S,5S)-5-fluoromethyl-5-hydroxy-4-
({[(5R)-5-
isopropyl-3-(isoquinolin-1-yl)-4,5-d ihydro-5-isoxazolyl]carbonyl } amino)-
dihydrofuran-
2-one or pharmaceutically acceptable salt thereof, and a use thereof.
BACKGROUND ART
It is known that the continual alcohol ingestion causes fatty liver and
subsequent alcoliolic steatohepatitis. Alcoliolic liver diseases had been
known to be
caused by nutritional deficiency for the last several decades. But, at
present, alcohol
itself is regarded as a toxic substance to liver. About 15% of alcoholics
suffer from
progression into alcoholic cirrhosis, and most of them die of hepatic failure
or
complications of liver cirrhosis. Alcoholic liver diseases can be divided into
the
followin- 3 classes of diseases according to patholiistoloby: fatty liver,
alcoholic
hepatitis, and alcoholic cirrhosis.
Although more and more evidences for association of chronic inflammation in
alcohol-induced liver injury have been shown, definite inode of action for
alcohol-
induced liver injury is not well-known and yet to be defined. In an individual
of
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alcohol dependence, either imtnunolgical response to antigen originated from
oxidative
stress or lipopolysaccharide (LPS) from large intestine has been reported as
the cause of
liver injury (Nagata K, Suzuki H, Sakaguchi S; Common pathogenic mechanism in
development progression of liver injury caused by non-alcoholic or alcoholic
steatohepatitis; J Toxicol Sci.; 2007 Dec; 32(5):453-468). Differentiated from
viral
hepatitis and autoimmune disease associated hepatitis, causative agents of
immunological response mediated by denatured protein due to oxidative stress
have not
been clearly identified, and yet hepatitis is known to be accelerated by
synergic
interaction witli immunological response mediated by elevation of intestinal
LPS
permeability due to alcohol ingestion. An anti-oxidailt such as silymarin is
used to
alleviate the symptom of such llepatitis, but its treating effect is
uncertain.
Steroids are usually used to treat severe alcoholic hepatitis, but the steroid
treatment inay cause systemic infections resulting in serious conditions.
Moreover,
currently available steroid therapies are still ineffective in about 40% of
patients with
alcoholic hepatitis.
SUMMARY OF THE INVENTION
The purpose of the present invention is to provide a pharinaceutical
composition for effective treatment of alcohol-induced liver injury.
To meet the above purpose, the present invention provides a use of (4S,5S)-5-
fluoromethyl-5-hydroxy-4-({[(5R)-5-isopropyl-3-(isoquinolin-l-yl)-4,5-dihydro-
5-
isoxazolyl]carbonyl}amino)-dihydrofuran-2-one of the following Formula
1(referred to
as CF1 (Compound of Formula 1) hereinafter) or pharmaceutically acceptable
salt
thereof for treating alcohol-induced liver injury.
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[Formula 1]
N-0 H HO F
N
0
iN 0 O
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a graphical presentation of fluorometric assay results of caspase-
3
activity in experiinental groups of control diet (Control), control diet with
CF I (Control
+ CF 1), EtOH diet (EtOH), and EtOH diet with CF 1(EtOH + CF 1).
Figure 2 is a graphical presentation of fluoroinetric assay results of caspase-
8
activity in experimental groups of control diet (Control), control diet with
CF1 (Control
+ CF 1), EtOH diet (EtOH), and EtOH diet with CF 1(EtOH + CF l).
Figure 3 is a graphical presentation showing effect of CFI on serum aspartate
transaminase (AST) and alanine transaminase (ALT) values in experiinental
groups of
control diet (Control), control diet with C F I (Control + CF 1), EtOH diet
(EtOH), and
EtOH diet with CF1 (EtOH + CFl).
Figure 4A is a set of representative photomicrograplis of liver after
heinatoxylin
and eosin (H&E) staining at 100 times magnifcation, and Figure 4B is a set of
representative photomicrographs of liver section processed for Masson's
trichrome at
100 times magnification.
Figure 5 is a set of representative photomicrographs of liver after terminal
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deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nickend
labeling
(TUNEL) staining at 400 times magnification.
Figure 6is a graphical presentation of quantitative results by number of
TUNEL positive cells per field in experimental groups of control diet
(Control), control
diet with CFl (Control + CF1), EtOH diet (EtOH), and EtOH diet with CF1 (EtOH
+
CF 1).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail hereafter.
The present invention relates to a use of (4S,5S)-5-fluoromethyl-5-hydroxy-4-
( { [(5R)-5-isopropyl-3-(isoquinolin-l-yl)-4,5-dihydro=5-isoxazolyl]carbonyl}
amino)-
dihydrofuran-2-one of the following Formula 1 or pharmaceutically acceptable
salt
thereof for treating alcohol-induced liver injury.
[Formula 1]
NI-O H HO F
N O \\
I i
O
According to one embodiment of the present invention, a pharmaceutical
coniposition for treating alcohol-induced liver injury comprising CFI or a
pharmaceutically acceptable salt thereof is provided.
According to another embodiment of the present invention, a method for
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inanufacturing of pharmaceutical composition for treating alcohol-induced
liver injury
comprising admixing CF 1 or a pharmaceutically acceptable salt thereof with
pharmaceutically acceptable carrier is provided.
According to still another embodiment of the present invention, a use of CF1
or
a pharmaceutically acceptable salt thereof for manufacturing a pharmaceutical
co-nposition for treating alcohol-induced liver injury is provided.
According to still another enibodiment of the present invention, a method for
treating alcohol-induced liver injury, comprising administrating an effective
amount of
the pharmaceutical composition comprising CF 1 or a pharmaceutically
acceptable salt
thereof to a patient suffering from alcoliol-induced liver injury is provided.
According to still another embodinient of the present invention, a use of the
pharmaceutical composition comprising CF 1 or a pharmaceutically acceptable
salt
thereof for treating alcohol-induced liver injury is provided.
The manufacturing method of CF1, the manufacturing method of intermediate
of CFl and use of CFl for treating hepatic diseases by hepatitis virus, acute
hepatitis
and hepatic cirrhosis are disclosed in International Publication No. WO
2006/090997.
In the present invention, the alcoholic liver diseases include fatty liver,
alcoholic hepatitis, alcoholic cirrhosis and the like.
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The present inventors have discovered the role of caspase in alcohol-induced
liver injury and that alcohol-iriduced liver injury can be efficiently treated
by inhibitin;
the caspase activity.
CFI of the present invention is usually synthesized from the mixture of the
compound of following Formula 6 and the compound of the following Forinula 7.
[Forinula 6]
N-0 0
H
N_ F
I~ N O
COZH
VI
[Formula 7]
N-O 0
F
H 11
N 0
COZH
VII
In solution phase, CF1 and the compound of Formula 6 exist in equilibrium as
follows:
N-0`HO -F N-0 ~ . 0
H
N_ / II_ ~ N. k F
O = ----- , I
N 0
N 0 O CO2H
VI
The equilibrium state can be verified by solution NMR (nuclear magnetic
resonance), and the structure of CF 1 was determined by X-ray crystallography
and solid
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NMR. CF 1 can be obtained in both amorphous form and crystalline form, and the
crystalline form is more stable. CFI can be obtained in the crystalline form
exclusively by crystallization method.
According to the purpose, CF1 of the present invention nlay be formulated into
various pharmaceutical forms for administration. To prepare the pharmaceutical
coinposition according to the present invention, an effective amount of CFI is
mixed
with a pharmaceutically acceptable carrier that may be selected in various
forms
depending on the formulation to be prepared.
CF 1 of the present invention may be formulated as a parenteral injection, or
percutaneous or oral preparation depending on its application purpose. It is
preferable
to formulate the composition in a unit dosage form in terms of administration
easiness
and dose uniformity.
For the oral preparation, any conventional pharmaceutical carrier may be used.
For example, water, glycols, oils, alcohols and the like may be used for oral
liquid
preparations such as suspensions, syrups, elixirs and solutions. Starches,
sugars,
kaolin, lubricants, binders, disintegration agents and the like may be used
for solid
preparations such as powders, pills, capsules and tablets. Tablets and
capsules are the
most convenient dosage unit forms for their administration easiness. Tablets
and pills
are preferably formulated as enteric-coated preparation.
For the parenteral preparation, sterile water is usually used as the carrier,
and
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other ingredients such as solubility aids may be furtlier used. Injections,
for example,
sterilized aqueous or oily suspension for injection, can be prepared according
to known
procedures usinb suitable dispersing agent, wetting agent, or suspending
agent.
Solvents that can be used for preparing injections include water, Ringer's
fluid, and
isotonic NaCI solution, and sterilized fixing oil may also be used
conveniently as the
solvent or suspending media. Any non-stimulative fixing oil including mono-,
di-
glyceride may be used for this purpose. Fatty acid such as oleic acid may also
be used
for injections.
For the percutaneous preparation, the carrier may include a penetration
enhancing agent and/or a suitable wetting agent, optionally in coinbination
with suitable
additives with no significant irritation to skin. For additives, those
enhancing the
administration through the skin and/or assisting the preparation of a desired
composition
are selected. These percutaneous preparations are adininistered via various
routes, e.g.,
a transdermal patch,.a spot-on, or an ointment.
When CFl is used for clinical purpose, it is preferably administered to the
subject patient in a total ainount ranging from 0.1 to 100 mg per kg of body
weigllt a
day via either single dosabe or divided dosage. Specific adininistration
dosage for a
specific individual patient can vary depending on specific compound to be
used, body
weight, sex, liygienic condition and diet of the subject patient, time and
inethod of
administration, excretion rate, mixing ratio of agent, severity of disease to
be treated, etc.
The present invention is explained in inore detail by the following Examples,
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but is not limited thereto.
Preparatory Experiment: Synthesis of CF1
CF1 of the present invention was synthesized through the following Reaction
Scheme I.
[Reaction Sclieine 1]
Oxalyl chloride Me0
N-O Me0 OMe Et N NO OMe
OH + HzN' V1,,: F ~ ~JrN . ~~ F
C02Et MDC l\\~N O CO Et
N 0
z
II III I v
C
LiOH I l N-O
THF/H20 N,Me0 OMe F HCI N OõN
,
II N O CO H iN O L..C02H
z
V mixture ofVlandVll
Toluene N-O\ \
DIPA HO F
Recrystallization O
O -- ~
O
Preparation Examplc 1
5-fluoro-3-[((R)-5-isopropyl-3-(isoq ui nolin-1-yl)-4,5-dihydro-isoxazole-5-
carbonyl)-amino]-4,4-dimethoxy-pentanoic acid etliyl ester (Compound IV)
15.5 g of the compound of Formula 11 (Compound 11, 54.5 mmol) was
dissolved in 150 mL of inethylene cllforide (MDC), and the temperature was
adjusted to
0 C , and then 7.1 mL (81.7 mmol) of oxalyl chloride and 0.2 mL (2.6 mmol) of
diinethyl formamide (DMF) were added thereto with maintaining the inner
temperature
below 12 C. The reaction mixture was stirred at 20 `C for about 2 hours, and
concentrated under reduced pressure. The concentrated reaction mixture was
diluted
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with 150 mL of methylene chloride (MDC), then the temperature was adjusted to
0 C,
and 16.5 g of triethylamine (Et3N) was added thereto, and a solution of 12.8 g
of the
compound of Formula III (Compound 111, 57.4 mmol) dissolved in 30 mL of
methylene
chloride was slowly added thereto over 20 minutes. The resulting reaction
mixture
was stirred at 25 C for 1.5 hours, and then a mixed solution of 120 mL of 10%
sodium
hydrogen carbonate aqueous solution and 60 mL of 1 N sodiuin hydroxide aqueous
solution was added thereto to terminate the reaction. A fter the separation of
organic
layer, aqueous layer was extracted with methylene chloride (150 mL x 3). The
combined organic layer was concentrated under reduced pressure to give the
title
compound (Compound IV; 30.1 g, quantitative yield). This compound was used in
the
next step without further purification.
'H NMR (500 MHz, CDC13): 9.12 (q, 1 H), 8.53 (m, I H), 7.85-7.25 (rn, 4 H),
4.80 (rn, 1 H), 4.54-4.34 (m, 2 H), 4.14 (q, J= 7.4 Hz, 2 H), 3.99 (2d, J=
18.4 Hz, 1 H),
3.81 (m, 1 H), 3.78 (2d, J=. 8.6 Hz, 1 H), 3.33 (d, 3 H), 3.20 (d, 3 H), 2.75
(in, 3 H),
2.53 (m, 1 H), 2.39 (heptet, J= 6.7 Hz, 1 H), 1.27 (t, J= 7.4 Hz, 1.5 H), 1.07
(m, 6 H),
0.97 (t, J= 7.4 Hz, 1.5 H)
Preparation Fxample 2
5-Fiuoro-3-f ((R)-5-isopropyl-3-(isoquinolin-1-yl)-4,5-dihydro-isoxazole-5-
carbonyl)-Zmino]-4,4-dinietlioxy-pentanoic acid (Compound V)
30.1 g of the coinpound obtained from the above Preparation Example 1
(Compound IV, 61.6 mmol); together with 7.76 g (185 mmol) of lithium hydroxide
inonohydrate, was added to a mixed solvent of 168 mL of tetrahydrofuran (THF)
and 42
mL of water, and stirred at about 40 C for 4 hours. The reaction mixture was
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concentrated under reduced pressure to remove tetrahydrofuran in the solvent.
180 mL
of 1 N sodium hydroxide aqueous solution was added thereto, and the mixture
was
washed with toluene (120 mL x 2). The aqueous layer was acidified with 66 mL
of 6
N hydrochloric acid aqueous solution, and extracted with methylene chloride
(150 mL x
3), and the combined organic layer was concentrated under reduced pressure to
give the
title compound (Compound V, 25.4 g; 89%). This compound was used in the next
step
without further purification.
'H NMR (400 MHz, CDCl3): 9.10-8.92 (in, 1 H), 8.52 (m, 1 H), 7.86-7.13 (m,
4 H), 4.77 (rn, 1 I-I), 4.54-4.34 (m, 2 H), 3.95 (2d, J= 8.0 Hz, I H), 3.75
(2d, J= 18.4
Hz, I H), 3.35-3.16 (2d, 6 H), 2.78 (2dd, J= 16.0, 4.4 Hz 1 H), 2.54 (m, I H),
2.39 (m,
1 H), 2.35 (s, 1 H), 1.06 (rn, 6 H)
Preparation Example 3-1
(-1S,5S)-5-fluoromethyl-5-hydroxy-4-({[(5R)-5-isopropyl-3-(isoquinolin-l-
yl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-dihydrofuran-2-one (Conipound I)
17.0 g (36.9 inmol) of the compound obtained froin the above Preparation
Example 2(Compound V) and 6.6 niL (110 mmol) of acetic acid were dissolved in
123
mL (738 inmol) of 6 N hydrochloric acid aqueous solution, and stirred for
about 4 hours.
The inner temperattire of the reaction mixture was adjusted to 0 C, and 150 mL
of
ethyl acetate was added thereto. 220 mL (660 mmol) of 3 N sodium hydroxide
aqueous solution was slowly added to adjust the pH to about 3. The organic
layer was
separated, and the aqueous layer was extracted with ethyl acetate (150 inL x
2). The
combined organic phase was washed with 100 mL of salt water, and concentrated
under
reduced pressure. The residue was diluted with 50 mL of toluene, and
concentrated
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again under reduced pressure to give a inixture of the compounds of Formula
6(Vl) and
Formula 7 (VII) (15.4 g, quantitative yield, chemical purity: 87.0%).
'H NMR (500 MHz, DMSO-d6): d 8.99 (m, 1 H), 8.65 (m, 1 H), 8.19-7.78 (m,
4 H), 5.15 (m, 1.5 H), 4.77 (m, I H), 4.42 (m, 0.5 H), 3.91 (2d, J= 17.6 Hz, 1
H), 3.74
(m, 1 H), 2.99 (m, 0.2 H), 2.82 (m, I H), 2.63 (in, 0.8 H), 2.33 (m, 1 H),
0.97 (m, 6 H)
146 mL of toluene was added to 14.6 b(35.2 mmol) of the mixture of the
compounds of Formula 6 and Formula 7(cllemical purity: 87.0%), and the mixture
was
heated up to 100 C for complete dissolution. Then, 14 mg of the title compound
(Compound I) was added thereto as a seed. T he temperature was then slowly
lowered
to 20 C, and the reaction mixture was stirred to produce solid. 0.25 mL (1.8
mmol)
of diisopropylamine (DIPA) was added tllereto, and stirred at 20 'C for about
2 weeks.
It was confirnled by HPLC that the ratio between the compound of Formula 6
(Compound VI) and the compound of Formula 7(Compound VII) became 92.8:7.2.
The reaction mixture was concentrated under reduced pressure to remove
toluene. 88
mL of ethyl acetate was added thereto and the mixture was heated up to 65 C
for
coinplete dissolution. Then, 88 mL of normal hexane was added thereto, and the
temperature was slowly lowered and stirred at about 20 C for 2 days. The
resulting
solid was filtered, and washed with a tnixed solution of 15 mL of ethyl
acetate and 15
mL of normal llexane. After drying the solid with nitrogen, the title compound
(Compound I) was obtained as white solid witli 54.7% of yield (8.0 g,
cheinical purity
98.6%) from the compound of Forinula 2(Compound 11).
'H NMR (CDCI3): d 9.02 (bs, 1H), 8.54 (d, J= 5.5 Hz, I H), 7.85 (d, J= 7.95
Hz, 1 H), 7.70 (m, 3 H), 7.60 (bs, 1 H), 4.86 (bs, I H), 4.2-5.2 (bs, 2 H),
4.05 (b, J
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19.0 Hz, 1 H), 3.78 (b, J= 19.0 Hz, 1 H), 2.7-3.1 (bm, 2 H), 2.40 (m, 1 H).
1.08 (dd, J
6.7,4:9Hz,6H)
13C NMR (CDC13): 173.8, 172.4, 160.2, 147.6, 141.7, 136.8, 130.7, 129.0,
127.4, 127.3, 126.8, 122.9, 92.3, 82.7 (d, J= 215 Hz), 48.9 (b), 44.6, 34.4,
33.9, 17.7,
16.3 M ass (ESI): 416.14 (M+1). [u]'S,p= +3.2 (c = 1.0, acetonitrile)
Preparation Example 3-2
(4S,5S)-5-fluoromethyl-5-liydroxy-=4-({ [(SR)-5-isopropyl-3-(isoquinolin-l-
yl)-4,5-d.ihydro-5-isoxazolyl]carbonyl}amino)-dihydrofuran-2-one (Compound I)
12.4 kg (26.86 inol) of the compound obtained from the above Preparation
Example 2(Compound V) and 4.9 kg (90.5 mol) of acetic acid were dissolved in
88.9
kg (52.9 kg of 35% hydrochloric acid + 36.0 kg of water) of hydrochloric acid
aqueous
solution, and stirred for about 2 hours. The inner temperature of the reaction
mixture
was adjusted to 0'C , and 40.0 kg of ethyl acetate was added thereto. 161.0 kg
(19.4
kg of sodium hydroxide + 141.6 kg of water) of sodium hydroxide aqueous
solution was
slowly added to adjust the pH to about 3.5. The organic layer was separated,
and the
aqueous layer was extracted with ethyl acetate (30.0 kg x 2). The combined
organic
phase was washed with 160.0 kg of water, and concentrated under reduced
pressure.
The residue was diluted with 73.0 kg of toluene, and concentrated again under
reduced
pressure. 97.0 kg of toluene and 0.1 kg (1.0 mol) of diisopropylamine were
added to
the resulting concentrated inixture (the nlixture of the compounds of Formula
6 and
Forinula 7), and the inixture was heated up to 100 C for complete dissolution.
Then,
4.0 g of the object coinpound (Compound I) was added thereto as a seed. The
temperature was slowly lowered to 40 'C , and the reaction inixture was
stirred to
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produce solid. The reaction mixture was stirred at about 40 C for about 3
days.
The resultin; solid was filtered, and washed with 10.0 kg of toluene. After
drying the
solid with nitrogen, the title compound (Compound I) was obtained as light
brown solid
with 70.0% of yield. (8.3 kg, chemical purity 94.9%) from the coinpound of
Formula 2
(Compound 11).
Experimental EYainple 1: Induction of Alcoholic Hepatitis
Male Sprabue-Dawley rats (5 weeks of age, 170 g of body weight) were
divided into four experimental groups: control diet group, control diet with
CFI group,
ethanol (EtOH) diet broup and EtOH diet with CF1 aroup. Control diet group,
control
diet with CF1 group, .EtOH diet group, and EtOH diet with CF1 broup were fed,
respectively, with Lieber-Decarli.contrdl diet, Liebet-Decacli control diet
and CF1,
Lieber-Decarli liquid diet containing 6% ethanol, and Lieber-Decarli liquid
diet
containing 6% ethanol and CFI for 12 weeks. CF i was dissolved in PEG
(polyethylene glycol) 400 and 5% ethanol solution (PEG 400:ethanol = 2:1), and
administered daily at 10 mg/kb by oral gavage. About 8 hours prior to rats'
sacrifice,
lipopolysaccharide (LPS) was intraperitoneally injected to increase the liver
injury in
the amount of 1 mg/kb.
Experimental Example 2: Measurement of Change of Body Weiglit and Liver Mass
After the 12 weeks' experiment, all rats were weighed prior to their
sacrifice.
After sacrifice, the livers were extracted and weiglied. The results are shown
in the
following TABLE 1.
TABLE 1
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Group Population (n) Body Weight (g) Liver Mass (g)
Control diet 3 400.0 00.00 10.67 0.32
Control diet with CF 1 3 400.0 20.00 10.37 1.10
EtOH diet 5 366.0 16.73 11.40 1.02
EtOH diet with CF1 5 372.5 17.08 11.36 0.87
As can be seen in the above TABLE 1, in EtOH diet group and EtOH diet with
CF1 group, the body weights decreased slightly, while the liver masses
increased
slightly.
Experimental Example 3: Assay of Activity of Caspase-3 and Caspase-8 in Liver
Tissue
To investigate the role of caspase in alcohol-induced liver injury, the
activities
of caspase-3 and caspase-8 were assayed in cellular extraction of liver
tissue. T he
activities of caspase-3 and caspase-8 were assayed by using EnzChekTM Caspase
Assay
Kit (Molecular Probes, USA) according to the manufacturer's instructions. The
fluorometric assay results of caspase-3 and caspase-8 activities are shown in
Figure 1
and 2, respectively.
As can be seen in Figure 1 and 2, when alcohol was ingested, activities of
both
caspase-3 and caspase-8 were significantly elevated eompared with the control
group.
When CF 1 which is a caspase inhibitor was administered, activities of both
caspase-3
and caspase-8 were decreased to the level of control group.
Eaperimental Example 4: Deterniination of serum liver enzyines in association
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with liver injury
Liver injury was assessed by a biocheinical assay of liver enzyme. After the
12 weeks' experiment, the blood samples were obtained from all rats' heart.
Seruin
aspartate transaminase (AST) and alanine transaminase (ALT) values were
analyzed for
100 ue of plasina froin each blood sample with Hitachi 7050 automatic analyzer
(Hitachi, Japan). The results are shown in Figure 3.
As can be seen in Figure 3, the control diet with CF1 group did not show
significant change of AST and ALT values compared with the control diet ~roup.
The
alcohol diet group showed elevated level of both AST and ALT as compared with
the
control diet group (AST 454 175 lU/L, ALT 336 163 IU/L). The alcohol diet
with CF1 ;roup showed signiticantly decreased level of botli AST and ALT
compared
with the alcohol diet group (AST 194 22 IU/L, ALT 120 10 IU/L).
Experimental Exainple 5: Histological Assessnient
The extracted livers were fixed by 10% neutral formalin in PBS (phosphate
buffered saline). Then, a paraffin section of 4 uni thickness was prepared,
and stained
with H&E (Heinatoxylin & Eosin) and Masson's triclirome. The stained tissue
was
pllotographed at 100 times magnification (Figures 4A and 4B).
From Figure 4A, it can be known that the alcohol diet group showed more fatty
liver than the control diet group, and the administration of CF1 inhibited the
increase of
fatty liver. From Figure 4B, it can be observed that the alcoliol diet group
and the
alcohol diet with CF 1 group sliowed some progression of hepatic fibrosis
compared
with the control diet aroup and the control diet with CF 1 group, but there
was no
statistical significance.
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Experimental Exaniple 6: TUNEL Staining
To investigate apoptosis of liver cell according to alcohol ingestion, TUNEL
staining was processed. TUNEL staining was carried out using in situ Cell
Death
Detection Kit (Roclle Diagnostics, Germany) according to the manufacturer's
instructions.
Stained tissue was photographed at 100 times magnification (Figure 5).
TUNEL positive cells per field were calculated. The results are shown in
Figure 6.
As can be seen in Figures 5 and 6, TUNEL positive cells were rarely observed
in the control diet group and the control diet with CFI group. However, the
alcoliol
diet group showed statistically significant increase of TUNEL positive cells
(alcohol
diet group 25.14 9.1.4, control diet group. 'l 0.3 1.9 (unit: TUNEL
positive
cells/field)). TUNEL positive cells were rarely observed in the alcohol diet
witli CFI
group, like the control diet group (alcollol diet witll CF1 group 6.4 1.1
TUNEL
positive cells/field).
INDUSTRIAL APPLICABILITY
CFI of the present invention efficiently inhibits hepatocyte apoptosis via
inhibition of caspase activity in alcohol-induced liver injury. As a result,
CF1 of the
present invention also lowers levels of AST and ALT which are biochemical
indicators
of liver injury. Also, when CF1 is administered, decreases in fatty liver
lesion can be
observed by histological method, which means that CF1 can inhibit apoptosis
efficiently.
Tlierefore, CF1 of the present invention can be used as a therapeutic agent in
treating
alcohol-induced liver injury such as alcoholic hepatitis.
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