Note: Descriptions are shown in the official language in which they were submitted.
DESCRIPTION
Medicines for Use in the Therapy and Prevention of
Kidney and Liver Diseases
TECHNICAL FIE:LD
The present invention relates to medicines for use in the
therapy and prevention of kidney and liver diseases, and more
particularly to medicines of this )cind which can exhibit
excellent therapeutic and preventive effects with respect to
kidney or liver diseases individually developed or to
complications resulting from diseases of these organs.
BACKGROUND ART
As is well known, the kidney in a vital organism performs
at its renal tubule and with its filtration and secretion
mechanism the function of excreting into the urine waste
substances from blood and noxious substances taken up by the
vital organism, such as drugs and toxic substances.
On the other hand, the liver in a vital organism performs
three major functions, as listed below. Namely, the liver first
acts as a digestive gland and secretes 500 to 1000 cc of bile per
day to assist in the digestion and absorption of fats by the
small intestines. Its second function is to process chemical
changes, storage, and use of various nutrients within the body;
- 1 - $~k
~2~
in brief, it is an intermediate metabolism. Thirdly, the liyer
performs detoxication by subjecting noxious substances, such as
toxin, from outside the body and toxic substances produced within
the body, to detoxication treatments such as oxidization,
reduction and inclusion, and by mixing these substan-ces with bile
to dispose of them or by sending them to the kidney for excretion
into the urine.
In this way, although the kidney and the liver perform
their individual functions, the above-mentioned waste substances
and drugs tend to accumulate locally, and therefore *hese
substances often cause kidney diseases and liver diseases to
develop, not only individually but also as complications
resulting from diseases of these organs. Specific substances
which can cause such individual or complicated kidney and liver
diseases are of various types. ~or instance, drugs which can act
as such substances are listed below and are classified according
to the organs to be affected. In the following list, the drugs
marked with the symbol ~ are those which can cause injury to both
the kidney and the liver.
~Drugs possessing the tendency to cause kidney injury)
Analgestics, antipyretics, anti-inflammatory drugs, and
antirheumatic drugs:
phenacetin, aspirin~ indomethacin, mefenamic acid,
fenprofen, gold compounds, D-penicillamine, etc.
Antibiotics:
aminoglycosides, polypeptides, polyenes, cephalosporins,
penicillines, etc.
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~z~s~
Chemotherapeutic drugs:
sulfamides, etc.
Anti-cancer drugs:
mitomycin C, daunomycin, cis-platinum, nitrosoureas, etc.
. Immunosuppressants
cyclophosphamide, etc.
Anesthetics:
methoxyflurane, etc.
Diuretics:
thiazides, etc.
. Contrast media:
(Drugs possessing the tendency to cause liver injury)
Analgestics, antipyretics, anti-inflammatory drugs, and
antirheumatic drugs:
acetaminophen, aspirin, phenylbutazone, sulindac,
ibufenac, gold compounds, etc.
Antibiotics:
aminoglycosides, polypeptides, cephalosporins,
penicillines, tetracyclines, etc.
Chemotherapeutic agents:
sulfa drugs, isoniazides, etc.
Anti-cancer drugs:
mitomycin C, cis-platinum, 6-MP, nitrosoureas, etc.
Q Anesthetics:
halothane, methoxyflurane, etc.
. Psychotropic drugs:
chlorpromazines, diazepams, barbitals, etc.
Diuretics:
thiazides, etc.
The above-listed examples of drugs which can cause kidney
and liver diseases are arranged in order based on the following
publications:
RINSHOH SEIJINBYOH (Clinical Studies of Adult Diseases),
No.8, vol, 16, (1986), pages 45 to 62 and 85 to 103; _AISHIN
IGAXU BUNKO, 42 "Mansei Kan'en no Shinryoh (Diagnosis and
Treatment of Chronic Hepatitis)" by Mikio Nishioka, published by
Shinkoh Igaku Shuppan-sha K.K. on January ~6, 1987, pages 29 to
31; RINSHOH TO KENKYU8 (Clinical Studies), No. 4, vol. 63
(published in April 1986), pages 38 to 39; KANZOH NO BYOHKI
(Liver Diseases), by Toshitsugu Orita et a]., published by Chuhgai
Igaku-sha on Qctober 20, 1980, pages 323 to 333; and IYAKUHIN
YOHRAN tHandbook of Medical Articles), edited by Osaka-Fu Byohin
Yakuzaishi-Kai, published by Yakugyo Jihoh Sha on Ncvember 10,
1983.
Hereinabove, kidney and liver diseases have been discussed
from the viewpoint of the metabolic functions of these organs.
However, some kidney diseases are considered to be caused by the
intermediary of the immunological mechanism.
Kidney diseases specified herein are the following:
(a) a functional disorder of the kidney which is caused by
abnormality in the above-mentioned meta~olic functions, including
acute hepatitis caused by drugs, etc., and chronic nephritis
developed from such acute nephritis; (b) acute nephritis caused
by the intermediary of the immunological mechanism and chronic
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'
~Z95~ ~7
nephritis developed from such acute nephritis; and (c) acute
nephritis caused by bacteria and virus infection and chronic
nephritis developed from such acute nephritis, including a wide
range of functional disorders of kidney sites such as the
glomerulus, renal tubule and the lupus.
Similarly, liver diseases specified herein are the
following: (a) a functional disorder of the liver which is
caused by abnormality in the above-mentioned metabolic functions
and in the biosynthetic functions, including acute hepatitis
caused by drugs, etc., and chronic hepatitis developed from such
acute hepatitis; (b) fatty liver caused by drinking, etc. and
chronic hepatitis developed from such fatty liver; and (c) viral
hepatitis caused by virus infection and chronic hepatitis
developed from such acute hepatitis, including liver cirrhosis as
a result of diseases involving a wide range of such functional
disorders of the liver.
Known therapeutic methods of treating kidney diseases are
adopted in accordance with the seriousness of the disease with a
view to maintaining the functions of the liver, and they include
rest therapy, dietetic therapy, drug therapy, hemodialysis, and
kidney transplantation. Hemodialysis is considered to be the
final treatment available for patients suffering from kidney
disease. However, this treatment can only remove from the blood
waste substances stored within the body due to the functional
disorders of the kidney, and cannot improve the kidney function
per se. Thus, such patients have to receive hemodialysis
throughout their life and, in the end, most of them will suffer
~959 ~7
from a complication accompanied by cardiac insufficiency, an
infectious disease, etc., and may die from such a complication.
Some forms of the drug therapy practiced at present use, for
example, diuretics, immunosuppressants, and corticoid, while
others use mannitol and lactulose. ~owever, these forms are all
symptomatic treatments, and their therapeutic effects are not
sufficient. Thus, the present situation is that the patients are
forced to rely on dietetic therapy and rest therapy. For
example, diuretics are used in treating kidney diseases but their
pharmacolo~ical effects are only to compensate for the lowered
functions of the kidney but not to prevent nor cure kidney
diseases. In addition, among diuretics, most of the thiazides
and furosemide which is a non-thiazide diuretic have side effects
which induce functional disorders of the liver and, in this
respect, they are not desirable for therapy.
Therefore, with the present situation of drugs available
for use in the therapy of kidney diseases, it is apparent that no
fully satisfactory drug exists, and the development of a more
effective drug is desired.
Similarly, known therapeutic methods of treating liver
diseases are methods which are adopted with a view to maintaining
the liver functions, and they include rest therapy, dietetic
therapy, and drug therapy. Among these types of therapy,
specific examples of drug therapy include those using, for
instance, amino acid such as aspartic acid, thioctic acid,
methionine, thioproline and glycine, and peptides such as
glutathione, as wlell as extract from the liver, hydrolysate rom
the liver, hydrolysate from the placenta, glucuronic acid
derivatives, and glycyrrhizin extracted from Glycyrrhiza and
refined. Among these agents, glycyrrhizin is known to he
effective or improving the liver functions which have been
disordered by chronic hepatitis. ~owever, this agent involves
problems in its administration that it is ineffective when orally
administered.
Since, the development mechanisms of various liver
diseases have not ~een clarified except those of vixal hepatitis
and of toxic hepatitis, the methods of treating liver diseases
have mainly been rest therapy and dietetic therapy. Recently,
however, malotilate has been developed, which suppresses the
changing of liver tissues into fibrous tissues by activating the
protein synthesis and RNA synthesis. However, the only
application of malotilate that is permitted is for the therapy of
liver cirrhosis.
Further, cianidanol has been developed as an immunity
activating agent although the mechanism of this action is not
clarified, and this agent is considered to be effective in the
treatment of B-type viral hepatitis. ~owever, because accidents
involving the death of patients that were treated with cianidanol
have occurred in Italy and Portugal, the sale of cianidanol is
prohibited at present in Japan.
Therefore, in the present situation, development of a drug
which is truly effective in the therapy and prevention of a wide
range of liver diseases, and which is excellent in terms of
safety is desired.
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~29~
Furthermore, as described above, the kidney and the liver
play important roles in the metabolism of a vital organism, and
are interrelated to each other. This fact means that, when the
functions of one of these organs are disordered, the other will
have to bear an increased burden i;n terms of the metabolic
functions, often resulting in both organs becoming functionally
disordered. Particularly in the case of a patient suffering from
a functional disorder of the liver, as the functional disorder
proceeds, it will often be accompanied by a functional disorder
of the kidney. Therefore, a drug for the therapy which can
improve the functions of both organs simultaneously is desired.
DISCLOSURE OF INVENTION
The present inventors have made various studies to solve
the problems of the prior art. After examining various types of
substan-ces, the present inventors have come to take up
isoliquiritigenin and achieved the following findings.
It has been known for long that isoliquiritigenin exists
in nature as a componel~t of the plant Glycyrrhiza. However,
because the content of the isoliquiritigenin in Glycyrrhiza is
small, and moreover the greater part of this component exists in
the form of glycosides with a lot of analogous homologues
coexisting, the separation and refining of the substance has been
difficult, and the question of whether isoliquiritigenin has any
pharmacological effects has not hitherto been solved~ For
instance, Japanese Patent Publication No. 8485/1973 reports that
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i9 ~
mixtures of liquiritin, isoliquiritin, liquiritigenin, and
isoliquiritigenin extracted from Glycyrrhiza have an
anti-ulcerous activity. However, the Publication gives no clear
report to the effect that isoliquiritigeninl which coexists with
other components and is contained Ln a very small amount, is the
active component responsible to the anti-ulcerous activity.
A report (YAKUGAKU ZASSHI (Pharmaceutics Journal) vol, 80,
pages 620 to 624, 1960) by Shibata et al states that
isoliquiritigenin has a relatively strong antispasmodic activity,
though the purity of the isoliquiritigenin used is not known.
In recent years, chalcones have attracted attention
because of their pharmacological activities. A report (ENSHO~
(INFLAMMATION), vol 4, pages S54 to 556, 1984) has been given by
Nakadate, et al., concerning the activity of suppressing the rise
in permeability of the skin by a carcinogenesis promotor TPA.
Japanese Patent Application No.178815/1985 reports concerning
their activity of inducing the differentiation of tumor cells of
animals, as well as their usefulness as a carcinostatic
substance.
Based on these findings, the present inventors have made
their own studies, and previously made a proposal (Japanese
Patent Application No. 49530/1986) concerning the use of
isoliquiritigenin as an anti-allergic drug. As a result of
further studies made on the basis of this finding, the present
inventors have newly found that isoliquiritigenin is effective in
the therapy and prevention of kidney and liver diseases.
~.Z9~9)47
The present invention has been accomplished on the basis
of the above-mentioned findinqs. That is, the present invention
provides a medicine for use in the therapy and prevention of
kidney and liver diseases, which contains as the active
ingredient isoliquiritigenin or pharmaceutically acceptable salts
thereof.
As described above, kidney diseases specified herein are
the following: (a) a functional disorder of the kidney which is
caused by abnormality in the metabolic functions, including acute
nephritis caused by drugs, etc., and chronic n.ephritis developed
from such acute nephritis; (b) acute nephritis caused by the
intermediary of the immunological mechanism and chronic nephritis
developed from such acute nephritis; and (c) acute nephritis
caused by bacteria and virus infection and chronic nephritis
developed from such acute nephritis, including a wide range of
functional disorders at kidney sites such as the glomerulus,
renal tubule and the lupus.
Similarly, liver diseases specified herein are the
following: ~a) a functional disorder of the liver which is
caused by abnormality in the metabolic functions and in the
biosynthetic functions, including acute hepatitis caused by
drugs, etc., and chronic hepatitis developed from such acute
hepatitis; (b) fatty liver caused by drinking or the like and
chronic hepatitis developed rom such acute hepatitis; and (c)
viral hepatitis caused by virus infection and chronic hepatitis
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4~
developed from such acute hepatitis, including liver cirrhosis as
a result of diseases involving a wide range of such functional
disorders of the liver.
Salts of isoliquiritigenin which are pharmaceutically
acceptable inclu~e non-toxic salts such as salts of alkali metals
and alkaline earth metals, for example, sodium salt, potassium
salt, magnesium salt and calcium salt, and non-toxic amine salts
such as ammonium salt.
The medicine for use in the therapy and prevention of
kidney and liver diseases in accordance with the present
invention can be administered either orally or parenterally (such
as venous injection, subcutaneous injection or rectal
administration, etc.) r and can be prepared in a formulation which
is suitable for the administration method chosen.
The medicine in accordance with the present invention can
be prepared in a formulation such as a tablet, a capsule, a
granule, a powder, a fine granule, a pillr a troche, a buccal, a
suppository, an ointment, an injection, an emulsion, a
suspension, or a syrup, depending on the use.
The preparation can be performed by known methods by using
a non-toxic additive or additives that are usually employed in
the preparation of a medicine of this kind, such as an excipient,
a binder, a disintegrator, a lubricant, a preservative, an
antioxidantr an isotonizing agent, a bufferr a coating agentr a
corrigent, a solution adjuvant, a base, a dispersing agent, a
stabilizer, or a colorant.
Specific examples of non-toxic additives that are usable
are listed below.
Examples of suitable excipients are starches and their
derivatives (dextrin, carboxymethyl starch, etc.), celluloses and
their derivatives (methyl cellulose, hydroxypropylmethyl
cellulose, etc.), sugars (lactose, saccharose, glucose, etc.),
silicic acid and siLicates (natural aluminum silicate, rnaynesium
silicate, magnesium metasilicate aluminate, etc.), carbonates
(calcium carbonate, magnesium carbonate, sodium bicarbonate,
etc.), aluminum hydroxide-magnesium, synthetic hydrotalcite,
polyoxyethylene deviratives, glycerin monostearate, and sorbitan
monooleate, etc.
Examples o~ suitable binders are starches and their
derivatives ( -starch, dextrin, etc.), celluloses and their
derivatives (ethyl cellulose, sodium carboxymethyl cellulose,
hydroxypropylmethyl cellulose, etc.), gum arabic, tragacanth,
gelatin, sugars (glucose, saccharose), ethanol, polyvinyl
alcohol, and polyvinyl pyrrolidone, etc.
Examples of suitable disintegrators are starches and their
derivatives (carboxymethyl starch, hydroxypropyl starch, etc.),
celluloses and their derivatives (carboxymethyl cellulose, sodium
carboxymethyl cellulose, crystalline cellulose,
hydroxypropylmethyl cellulose, etc.), carbonates (calcium
carbonate, sodium bicarbonate, etc.), tragacanth, gelatin, and
agar, etc.
~2~5~7
Examples of suitable lubricants are stearie acid, caleium
stearate, magnesium stearate, tale, silieie acid and its salts
(light anhydrous silieie acid, natural aluminum silieate, ete.),
titanium oxide, caleium hydrogenphosphate, dry aluminum hydroxide
gel, and Maerogol, etc.
Examples of suitable preservatives are
para-hydroxybenzoate esters, sulfites (sodium sulfite, sodium
pyrosulfite, etc.), phosphates (sodium phosphate, ealeium
polyphosphate, sodium polyphosphate, sodium metaphosphate, etc.),
aleohols (-ehlorobutanol, benzyl aleohol, ete.), benzalkonium
ehloride, benzethonium chloride, phenol, eresol, ehloroeresol,
dehydroaeetic acid, sodium dehydroaeetate, glycerin sorbate, and
sugars, ete.
Examples of suitable antioxidants are sulfites (sodium
sulfite, sodium bisulfite, ete.), rongalit, erithorbie acid,
L-ascorbie aeid, cysteine, acetylcysteine, thioglyeerol, butyl
hydroxyanisole, dibutyl hydroxytoluene, propyl gallate, aseorbyl
palmitate, d~ tocopherol, and nordihydroguaiaretic aeid, etc.
Examples of suitable isotonizing agents are sodium
ehloride, potassium nitrate, sodium nitrate, dextran, glyeerin,
and glueose, etc.
Suitable examples of buffers are sodium earbonate,
hydrochloride, boric acid, and phosphates (sodium phosphates,
etc.) ete.
Examples oE suitable eoating agents are derivatives of
eelluloses (hydroxypropyl cellulose, eellulose acetate phthalate,
hydroxypropylmethyl eellulose phthalate, ete.), shellae,
~LZ~
polyvinyl pyrrolidone, polyvinyl pyridines (poly-2-vinylpyridine,
poly-2-vinyl-5-ethylpyridine, etc.), polyvinyl ace~aldiethylamino
acetate, polyvinyl alcohol phthalate, and methacrylate~
methacrylic acid copolymer, etc.
Examples of suitable corrigents are sugars (glucose,
saccharose, lactose, etc.), sodium saccharin, and sugar alcohols,
etc.
Examples of suitable solution adjuvants are
ethylenediamine, nicotinamide, sodium saccharin, citric acid,
citrates, sodium benzoate, soaps, polyvinyl pyrrolidone,
polysorbates, sorbitan fatty acid esters, glycerin, propylene
glycol, benzyl alcohol, glycerol 1,3-diethyl ether, and sugar
esters, etc.
Examples of suitable bases a~e fats (lard, etc.),
vegetable oils (olive oil, sesame oil, etc.), animal oils,
lanolin, vaseline, paraffin, wax, resin, bentonite, glycerin,
glycols, higher alcohols (stearyl alcohol, cetanol, etc.), and
cellulose derivatives, etc.
Examples of suitable dispersing angents are gum arabic,
tragacanth, cellulose derivatives (methyl cellulose, etc.),
stearic acid polyoxyls, sorbitan sesquioleate, aluminum
monostearate, sodium alginate, polysorbates, and sorbitan fatty
acid esters, etc.
Finally, examples of suitable stabili~ers are sulfites
(sodium bisulfite, etc.~, nitrogen, and carbon dioxide, etc.
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~2~ 7
The content of isoliquilitigenin in the medicine varies
depending on the formulation of the medicine. In general, it
should preferably be contained at a concentration of 0.1 to 100%
by weight.
The dosage of the medicine in accordance with the present
invention can be varied within a wide range in accordance with
the kind of warm-blooded animal, including human beings, to which
it is to be administered, the seriousness of the disease, and the
diagnosis of the doctor, but the dosage in terms of the active
ingredient ranges, when the medicine is orally administered, in
general, from 0.01 to 300 mg, preferably from 0.01 to S0 mg, per
kilogram body weight per day, and when the medicine is
parenterally administered it ranges from 0.01 to 150 mg,
preferably 0.01 to 20 mg, per kilogram body weight per day.
However, these dosage ranges can be varied in accordance with the
seriousness of the disease and the diagnosis of the doctor. Each
of the above-mentioned dosages can be divided into portions so
that the dosage is administered one to several times a day.
Certain examples of the present invention will now be
described, though the present invention is not limited to these
examples.
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~5~
BEST MODE FOR CARRYING OUT TEIE INVENTION
Example 1
(Toxicity Tests)
In this example, tests were conducted to confirm the
safety of isoliquiritigenin, which is the active ingredient of
the medicine in accordance with the present invention.
Isoliquiritigenin was administered by oral administration
or intraperitoneal injection to five 5-week-old male ddy mice.
As a result, the minimum lethal dose was found to be not less
than 3000 mg/kg (oral administration) or not less than 1000 mg/kg
(intraperitoneal injection).
Example 2
(Activity against acute kidney injury induced by gentamycin)
In this example, gentamycin, which is an antibiotics of
the aminoglycoside type and is known to have a very strong effect
of inducing functional disorders of the kidney, was selected as a
model of inducer for drug-induced acute nephritis, which is one
of functional disorders of the kidney caused by abnormality in
metabolic functions, and the activity of isoliquiritigenin
against the acute nephritis induced by gentamycin was examined,
to confirm the efficacy of the medicine of the present invention.
The tests were conducted in accordance with the method
reported in NIHON YAKURIGAKUKAI SHI, vol.84, pages 463 to 469,
1984, by Suæuki et al. That is, Wistar strain male rats
(produced by Nihon Charles River K.K., 5 rats in each group),
each weighing 160 to 180 g, were subcutaneously injected with 80
~z63 ~ ~
mg per kilogram body weight per day of gentamycin once a day
through the back for 15 days, thereby inducing injury to the
kidneys, and then orally administered with 5 ml per kilo~ram body
weight of a mixture in which 50, 150 or 300 mg per kilogram body
weight per day of isoliquiritigenin was suspended in aqueous
solutions of l~-carboxymethyl cellulose (hereinafter abbreviated
as 1%-CMC), for 15 continuous days. Meanwhile, rats in a
solution control group were a~ministered with gentamycin in the
same manner as the isoliquiritigenin administration groups, and
orally administered with 5 ml per kilogram body weight of aqueous
solutions of l~-CMC, for 15 continuous days. Samples of the
urine were collected using metabolism cages for a period of 24
hours from the day before the initial administration to the next
day, and for 24 hours from the day of the last administration to
the next day. After the amounts of urine were measured, the
urine samples were centrifuged at 3000 rpm for 15 minutes, and
the supernatant liquids were examined by measuring the activity
of lactate dehydrogenase tLDE~) within the urine by using an
automatic analyzer tAU-550, a product of Olympus Kohgaku
Kohgyoh). The activity of N-acetyl-~-D-glucosaminldase (NAG)
within the urine was also measured, by using an NAG Test Shionogi
(a product of Shionogi Seiyaku). After the completion of the
collection of the urine samples but before the initial
administration, blood samples were collected from the tail veins.
At the day after the last administration, blood samples were
co~lected from the aorta ab~ominalis while the rats were
anesthetized with ether, and the rats were victimized by loss of
~z~
blood. They were subjected to abdominal section and the ~idneys
were removed. After the blood samples were centrifuged at 3000
rpm for 15 minutes, the serum was examined by measuring the urea
nitrogen within the blood (BUN) by using an automatic analyzer.
The removed kidneys were each divided into two, and, after being
fixed in 10~-buffer formalin, samp:Les dyed with hematoxylin-
eosin stain were prepared and were observed under a microscope.
The results are shown in Tables 1 and 2.
-18-
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--19--
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--20--
~ ~9~ 7
(Results)
(1) It was found that the control group which received
only gentamycin showed remarkable increase in NAG and LDH values
after 15 days of administration of gentamycin, as well as
increase in BUN value. Thus, it ~as clearly recognized that
kidney injury was caused by gentamycin.
(2) Isoliquiritigenin acted to remarkably suppress the
increase in NAG, LD~, and BUN values almost dose-dependently as
compared to the control group in which gentamycin induced kidney
injury.
(3) Also, in the pathological views of the kidney
tissues, the group which received 300 mg per kilogram body weight
of isoliquiritigenin showed remarkable improvement with respect
to the necrosis, degeneration, regeneration, division, and
infiltration of the epithelium cells of the renal tubule as
compared to the control group in which gentamycin induced Xidney
injury. In addition, the groups which received 50 and lSO mg per
kilogram body weight, respectively, of isoliquiritigenin also
showed improvement in the necrosis, degeneration, regeneration,
and division of the epithelium cells of the renal tubule.
It is clear from the above-stated results of the tests
that isoliquiritigenin remarkably improves acute nephritis
induced by a drug, and it can be considered that
isoliquiritigenin is very useful in treating acute nephritis
caused by drugs.
-21-
Example 3
(Activity of isoliquiritigenin against acute kidney injury
induced by cis-platinum)
In this Example, tests were conducted by choosing
cis-platinum which is an anti-cancer drug and is known to have a
very strong effect of inducing functional disorders of the kidney
as the inducer of a model drug-induced acute nephritis, a
functional disorder of the kidney caused by abnormality in the
metabolic functions, and by examining the action of
isoliquiritigenin against the acute nephritis induced by
cis-platinum, to confirm the efficacy (effects in the prevention
and therapy) of the medicine in accordance with the present
invention.
(1) Prevention Tests
A group of ten ddy male mice each weighing 20 to 25 g were
orally administered with 200 mg per kilogram body weight per day
of isoliquiritigenin suspended in l~-CMC aqueous solutions, ~or S
continuous days, subcutaneously injected with 16 mg per kilogram
body weight of cis-platinum dissolved in physiological salines at
a ratio of 2 mg per 10 ml, once on the fifth day, and were
treated moreover for 5 continuous days orally administered with
isoliquiritigenin in the same manner as above. Thereafter, blood
samples were collected, and, after they were centrifuged at 3-00
rpm for lS minutes, BUN in the serum was measured.
(2) Therapy Tests
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~.Z~ 7
A group of ten ddy male mice each weighing 20 to 25 g were
subcutaneously injected with 16 mg per kilogram body weight of
cis-platinum dissolved in physiological saline solution at a
ratio of 2 mg / 10 ml once, and with oral adrninistration of 200
mg per kilogram body weight per day of isoliquiritigenin
suspended in 1%-CMC aqueous solution, for 5 continuous days.
Thereafter, blood samples were col]ected, and after they were
centrifuged at 3000 rpm for 15 minutes, BUN in the serum was
measured.
In each of the tests (1) and (2), an isoliquiritigenin
only administration control group and a cis-platinum induced
nephritis control group were employed and were compared with the
cis-platinum and isoliquiritigenin administration group. The
isoliquiritigenin only administration control group consisted of
ten ddy male mice, each weighing 20 to 25 g, which were orally
administered with 200 mg per kilogram body weight per day of
isoliquiritigenin, for continuous days. The cis-platinum induced
nephritis control group consisted of ten ddy male mice, each
weighing 20 to 25 g, which were treated in exactly the same
manner as that of the cis-platinum and isoliquiritigenin
administration group except that the mice in this control group
were subcutaneously injected with 16 mg per kilogram body weight
of cis-platinum once, and oralIy administered with 1%-CMC aqueous
sol-utions, in place of isoliquilirigenin, for continuous days.
The results of the tests are shown in Ta~le 3.
-23-
Table 3
Activity of Isoliquiritigenin Against
Cis-platinum-Induced Acute Nephritis
B U N (mg/dl)
(1) Prevention Fest (2) Therapy Test
mean + SD mean ~ SD
Non-Treated Control Group 18.6 ~ 0.9 19.2 + 0.9
Isoliquiritigenin only 8
Administration Control Group 21- + 0-6 19.8 -~ 0.9
Kidney Injury-Induced 142.8 + 17.2 128.4 ~ 19.0
Control Group - -
Isoliquiritigenin
Administered Kidney48.9 + 13.463.0 + 12.4
Injury-Induced Group
(Results)
(1) The nephritis-induced control groups which received
cis-platinum showed remarkable increases in BUN values when
-compared with non-treated control groups. Thus, it was clearly
recognized that acute kidney injury was caused by cis-platinum.
-24-
~2~ 7
(2) Isoliquiritigenin was found to remarkably supress
increases in BUN value in both the prevention tests and the
therapy tests when compared with the nephritis-induced control
groups.
(3) The isoliquiritigenin only administration control
groups wherein no kidney injury was caused showed results which
are not much different from those shown by non-treated contro~
groups. Thus, it was clearly found that isoliquiritigenin
specifically supresses increase in BUN value caused by kidney
injury.
It is clear from the above-stated results of the tests
that isoliquiritiyenin remarkably improves acute nephritis
induced by drugs. Thus, it can be considered that
isoliquiritigenin is very useful in the prevention and therapy of
acute kidney injury caused by drugs.
Example 4
(Activity of isoliquiritigenin against rat anti-GBM (glomerular
basement membrane) antibody nephritis~.
It is considered that 70% of chronic kidney diseases of
human beings is caused by the intermediary of the immunological
mechanism. Observations suggesting the involvement of the
immunological mechanism in lot of cases of glomerular nephritis
have been made, particularly concerning glomerular nephritis. On
the basis of these facts, various studies have been made to
prepare animal test models as models of chronic nephritis of
human beings. Among these models, the Masugi nephritis model,
which has been developed in Japan, is a nephritis model in which
-25-
~2~59~7
nephritis is caused by the antibody against the glomerular tissue
antigen tGBM), and this mode] is used widely in the world.
Therefore, in this example, the present inventors chose the
Masugi nephritis model as a model of chronic nephritis of human
beings, and conducted tests according to the method report~d in
NIHON JINZOH GAKUKAI SHI, vol. 23, pages 323 to ~31, 1981, and in
NIHON YAKURI GAKUKAI SHI, volO 77, pages 407 to 417, 1981, so as
to confirm th~ efficacy of the medicine of the present invention.
SD strain male rats each weighing 180 to 200 g (produced
by Nihon Charles River K.K., 7 rats for a nephritis-induced group
and 6 rats each for the other groups) were intravenously injected
with 0.5 ml per rat of rabbit serum causative of the antibody
against 5BM within the rats, and immediately thereafter orally
administered with 5 ml per kilogram body weight of suspensions in
which 30, 100, or 300 mg per kilogram body weight per day of
isoliquiritigenin was suspended in l~-C~C aqueous solutions,
everyday for 3 continuous weeks. The rats in the
nephritis-induced control group were intravenously injected with
0.5 ml per rat of rabbit serum causative of the antibody against
GBM within the rats, and orally administered with 5 ml per
kilogram body weight of 1%-CMC aqueous solutions, everyday for 3
continuous weeks. Samples of the urine of the rats were
collected using metabolism cages for a period of 24 hours from
the final administration day which is 3 weeks after the injection
of the rabbit serum to the next day. After the amounts of the
urine were measured, the urine samples were centrifuged at 3000
rpm for 15 minutes, and the supernatant liquids were examined by
~29~7
measuring the activities of lactate dehydrogenase (~DH), the
amount of alkaline phosphatase (ALP), and the amount of protein
by means of an automatic analyzer (AU-550, a product of Olympus
Kohgaku Kohgyoh). After the completion of the collection of the
urine samples, blood samples were collected from the aorta
abdominalis while the rats were anesthetized with ether. The
serum of the blood samples was centrifuged at 3000 rpm for 15
minutes and was examined by measuring BUN, creatinine, ALP, total
cholesterol, total protein, and albumin. The results of the
tests are shown in Table 4.
c ~ ~
r
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c~ ~ rl o rl rl r
~ C~ 0 0 ~
~ ^ O ,~ O O
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~ro~ ¢ 5~
. ~
W '1 ~ O
C , ~, + I , N cn ~ ~r~
~i: E~ ~ Q~
~u~ c ~o
r r r1 L~. O
Q~ - o r ~ 0
Q , E Q~ [~ cn N cn ,~, T
C C l E~ _~ a c (D 0 o o
Q~ C ~ ~ ~ ~ N r a ~ ~ ~ O 01
E~0 ' ~ ~ a c r~ I o I + 1 01 ID I 1. ~ '~1 ~
~,, u u '~ C W ,1 ~ cn ~ u~
E ~ C N N H O o r h v~ I ,11 H 1 0 0 0
~, h C ~ O O o O c; c) O à c) ~ N~ ~) N O
V~ .~ ~ C 111 ~ N N (a E~ ~
E ~ . ~ E ~ . ~t
S-. Q h _ _ o o o O O ~ C ,
u~ cn o o N r~ ~D ~ 0 r~
r~ cn ,~ 0~ I c) ~ N (~ 10 rl
o z C ~1 ~1 +I +I I E ,~ a +I ~1 0 o t~ cn
s a E ~ N N ~ N N m E a) ~ (N) NO 1
m o ~ o
h C ~ h ~ O
O O
L. ~ C L. ~ C
C '' C
~ 0 ~ ~0 t,o +,
E~ no ~ E Eh ~ cr E E E
O Q, O O O O ~ U) rl (~')
Z Z t~l Z Z H
--28--
(Results)
(1) The nephritis-induced control group showed, after 3
weeks, remarkable increases in total protein, total ALP, and
total LDH in the urine, when compared with a non-treated control
group. Although no remarkable changes in BUN and creatinine in
blood were recognized, decrease in ALP and increase in total
cholesterol were observed. In addition, although no change in
total protein in the serum was recognized, decrease in the
albumin content was observed, and decrease in the A/G ratio
ac-companying this was recognized. It is clearly recognized from
these results that kidney injury was caused by the intermediary
of the immune system.
(2) The groups administered with isoliquiritigenin in the
respective dosages showed remarkable suppression of increase in
total protein, total ALP, and total LDH in the urine, when
compared with the nephritis-induced control group. In additlon,
remarkable improvements were found in the amounts of ALP, total
cholesterol, and albumin in the blood.
It can be considered from the above-mentioned results
that isoliquiritigenin is very useful in the prevention and
therapy of kidney injury ca~sed by the intermediary of the
immunological mechanism.
Example 5
(Activity of isoliquiritigenin against liver injury induced by
d-galactosamine)
-29-
~2gs~'~7
d-Galactosamine is a compound capable of inducing injury
which is similar to the lesion of viral hepatitis of human
beings, and is used to induce a model of hepatitis. Therefore,
in this example, the present inventors conducted tests in
accordance with the method reported by Sakamoto et al in
SHINYAKU-KAIHATSU NO TAMENO YAKUKOH SCREENING HOHOH, VOL 1
(Methods of Screening Drug Effe-cts for DevRloping Novel
Medicines), pages 69 to 82, 1984, by Kohji Sakamoto, published by
Seish7 Shoin, so as to confirm the efficacy of the medicine in
accordance with the present invention.
Wistar strain male rats each weighing 180 to 200 g
(produced by Nihon Charles River K.K., 6 rats in each group1 were
orally administered with 5 ml per kilogram body weight of a
mixture in which 10 or 30 mg per kilogram body weight per day of
isoliquiritigenin was suspended in l~-CMC a~ueous solutions~ for
5 days, and intraperitoneally injected with 250 mg per kilogram
body weight of d-galactosamine dissolved in 5 ml per kilogram
body weigh~ of physiological saline solutions, one hour after the
administration of isoliquiritigenin on the 4th day. 48 hours
after the administration of d-galactosamine, blood samples were
collected from aorta abdominalis while the rats were anesthetized
with ether. The serum of the blood samples centrifuged at 3000
rpm for 15 minutes were examined by measuring
glutamic-oxaloacetic transaminase (~OT), glutamic-pyruvic
transaminase (GPT), and ALP by an automatic analyser ~AU~550).
-30-
5~7
A liver injury-induced control group was treated in
exactly the same manner as that of the isoliquiritigenin
administration groups except that rats in this control group were
orally administered with 1%-CMC aqueous solutions in place of the
suspensions of isoliquiritigenin in l~-CMC aqueous solutions.
Positive control groups were treated in exactly the same
manner as that of the isoliquiritigenin administration groups
except that rat-s in two positive control groups were orally
administered with 30 or 100 mg per kilogram body weight per day
of malotilate, in place of isoliquiritigenin, suspended in 1%-CMC
aqueous solutions, while rats in the other two positive control
groups were orally administered with 30 or 100 mg per kilogram
body weight per day of cianidanol, in place of isoliquiritigenin,
suspended in similar aqueous solutions. The results of the tests
are shown in Table 5.
-31-
~2~
o a~ r~ o ~ ~ ,~
o o~
~ ~ C~ +l +l +l +l +l +l +l +l
o o) o
=~ C ) ~ ~ I~ o ~ ~ r~
h ~1 H ~ u-) ~ ~D 1~ 00 1~ ) ~D
_ ~IJ
~: ~ C
h 6 a
oC~ ~ ~ +I +I ~ I +I +I +I +I
:~ ~ ~ ~ ~ o ~ o ~ O
(~ H Q) ~ ~ ~) ~ u-l
-- 6
g V
a
m E~ ~ u~ +l +l +l +l +l +l +l +
~,~ + I ~ O O ~D ~1 0 ~D O
In 4~ oo I ~ I~ ~ O ,~
~ Q~ o ~1 ` ~ ~ ~ ~ ~
l H e
~: Q h -1
a) ,
X ~ X ~ ~C X
. h 6~ e e 6 e e
: 6 ~a ~ ~ o
~1 ~ ~I a
~ ~ O ~
t~ h ~ ~ :
-,~ ~ ~ ,~ ,
J c ~ ~ c c:
: S ~ I
~: ~ O al ~ h -1 -~1 a~ a) ~ .
: m J~ ~ h ~1 ~ ~
S~ O ~ ~ ~1 ~1 ~ ~ :
: I ~J ~ ~1 ~1 0 0 ~:
o ~ o U~
Z ~ ~_) H H
:
~ ~ : : : :
::
.
,'
.
.
~z~
~Results)
(1) The liver injury-induced control group showed obvious
increase in GOT, GPT, and ALP when compared with a non-treated
control group.
(2) The isoliquiritigenin administration group of rats
which received 10 mg per kilogram body weight of
isoliquiritigenin showed suppression of increase in GOT, GPT, and
ALP, wh~n compared with the liver-injury induced control group,
and the isoliquiritigenin administration group of rats which
received 30 mg per kilogram body weight of isoliquiritigenin
showed remarkable improvement in GOT and GPT values.
(3) The positive control groups which received 100 mg per
kilogram body weight of malotilate or cianidanol showed
substantially the same levelc of suppression of increase in GOT
and GPT values as those showed by the isoliquiritigenin
administration group which received 10 mg per kilogram body
weight of isoliquiritigenin.
It is clearly recognized from the above-mentioned results
that isoliquiritigenin suppresses d-galactosamine-induced injury
to the liver in dosages smaller than those of malotilate and
cianidanol. Thus, it can be considered that isoliquiritigenin is
useful in the prevention and therapy of liver injury such as
viral hepatitis.
Examples 6 and 7
It is considered that carbon tetrachloride generates free
radicals with a very high reactivity by the action of drug
metabolizing enzyme systems in liver cells, and these free
~59~
radicals may strongly depress the cell activity by ~ombining with
protein of the liver cell membranes or may cause peroxidation of
membrane lipids of the organelles, thus leading to necrosis of
liver cells and accumulation of liver fats. Accordingly, carbon
tetrachloride is widely used to induce test models of acute
drug-induced hepatitis of human beings, fatty liver, chronic
hepatitis, and liver cirrhosis.
Therefore, in these examples 6 and 7, the present
inventors conducted tests in aeeordanee with a method reported in
JAPAN JOU~NAL OF PHARMACOLOGY, vol.31, pages 15 to 21, 1981, by
Imaizumi et al, and a method reported in NIHON YAKURI~AKUKAI SHI,
vol.80, pages 83 to 91, 1982, by Minoru Katoh et al, to eonfirm
the efficacy of the medicine in accordance with the present
invention.
(Example 6)
(~ctivity of isoliquiritigenin against acute liver injury
induced by carbon tetrachloride (CC14))
Wistar strain male rats each weighing 180 to 200 g
~produced by Nihon Charles river R.K., 6 rats in each group)
orally administered with 10 or 30 mg per kilogram body weight per
day of isoliquiritigenin suspended in l~-CMC aqueous solutions,
for 4 continuous days. Rats in a solution control group were
orally administered with 5 ml per kilogram body weight per day of
1%-CMC aqueous solutions, for 4 continuous days. Two positive
control groups were orally administered with 30 or 100 mg per
kilogram body weight per day of malotilate suspended in 1%-CMC
aqueous solutions, for 4 continuous days, and other two positive
-34-
~L~9~ 7
control groups were orally administered with 30 or 100 mg per
kilogram body weight per ~ay of cianidanol suspended in l~-CMC
aqueous solutions, for 4 continuous days.
The rats were subcutaneously injected with 0~2 ml per
kilogram body weight per day of carbon tetrachloride, which was a
reagent for precision analysis, everyday for three consecutive
days from the s~cond day of the isoliquiritigenin administration.
24 hours after the last administration of carbon
tetrachloride, blood samples were collected from aorta
abdominalis while the rats were anesthetized with ether. The
blood samples were centrifuged at 3000 rpm for 15 minutes and the
serum was examined by measuring GOT and GPT values using an
automatic analyser (.~U-550, a product of Olympus Kohgaku
~ohgyoh). The results of the tests are shown in Table 6.
~s
-35-
~295~3~7
Q
E~ -~ + l ~ a~
h O P. ~ Il] + ¦ + ¦ + ¦ + ¦ .1- l + I + l +
H a) r-l ~r O ~D ~ ~ Ul 0
~ C ~ ~ E~
0 ,C
h v
O
E~ ~ "~
O O ~1 + I + I + I + I + I + I + I + I + I
r~ ~ ~ ~ ~ ~ ~ ~ ~ O ~ U~
O ~ ~ ~ o ~ o u~
m c~ H ~
~D ~ ~
O l
R Sl O ~ ~ ~ X
E~ Ll O o O oE~ ~ o o
h u ~ 7 o r~ o
,~ ¦ O v
v O H ~L)
s ~ ~ h o 3 ~ ~ ~
O ~ ~r- ~ h h ~ ~ O
m h ~0 ~ C5' -1 -
O ~ ~1 ~ O C:
O O
O ~ O Ul U~
Z ~ ) H H ~ ) U
--36--
~Z~ 7
(Results)
(1) It was found that the liver injury-induced control
group showed remarkable increases in GOT and GPT values when
compared with a non-treated control group. Thus, it was clearly
recognized that liver injury was c:aused by carbon tetrachloride.
(2) Each of the isoliquiritigenin administration groups
with the respective amounts used showed remarkable improvement in
GOT and GPT values when compared to the liver injury-induced
control group.
(3) Each of the positive control groups which received 30
or 100 mg 2er kilogram body weight of malotilate or cianidanol
showed suppression of increase in GOT and GPT values to
substantially the s3me extent as that showed by the
isoliquiritigenin administration group which received 10 mg per
kilogram body weight of isoliquiritigenin.
It can be considered from the above-mentioned results that
isoliquiritigenin suppresses liver injury induced by carbon
tetrachloride in smaller amounts than those of malotilate and
cinanidanol, and is thus useful in the prevention and therapy of
acute liver injury.
(Example 7)
~ Activity of isoliquiritigenin against chronic liver
injury induced by carbon tetrachloride)
Wistar strain male rats each weighing 180 to 200 g
(produced by Nihon Charles river K.K., 6 rats in each group) were
orally administered with 30 mg per kilogram body weight per day
of isoliquiritigenin suspended in 1%-CMC aqueous solutions,
-37-
~2~5~
everyday for 3 continuous weeks. Rats in a solution control
group were orally administered with S ml per kilogram body weight
per day of l~-CMC aqueous solutions, every day for 3 continuous
weeks. Rats in a positive control group were orally administered
with 30 mg per kilogram body weight per day of malotilate
suspended in l~-CMC aqueous solutions everyday for 3 continuous
weeks, and rats in another positive control groups were orally
administered with 30 mg per kilogram body weight per day of
cianidanol suspended in 1%-CMC aqueous solutions, everyday for 3
continuous weeks.
The rats were intraperitoneally injected with 0.5 ml per
kilogram body weight per day of carbon tetrachloride, for three
consecutive weeks from the third day of the isoliquiritigenin
administration twice a week.
On the 4th day after the last administration of carbon
tetrachloride, blood samples were collected from aorta
abdominalis while the rats were anesthetized ~ith ether. The
blood samples were centrifuged at 3000 rpm for 15 minutes and the
serum was examined by measuring GOT and GPT values using an
automatic analyser (AU-550, a product of Olympus Kohgaku
Kohgyoh). The results of the tests are shown in Table 7.
-38-
s~
~ l
o ^ a ,~
IE~ ~ u~ ~ ~ ~ ~ o
I~ ~ +~1 +1+i ~1 +1 +1
;~ ~
IC~ H ~ l 0 ~ 01)
l ~
h
a
O
I
a ~D "
u~ I E~ u~ OD O ~ I` ~
x ~ o ~ +l +l +l +i +l +l
Q) ~ ~ ~ ~ ~ ~r ~ ~
3 O H ~J ~` O O 0 ~1
_
r
O C
O H
h
~` ~
4~ ~ O
O _l h
.-1
Q u~ ~ ~ t
115 h L~ ~1
E~ ~ . I O
c: . ~. h ~ t7
a) o
h 0 1:: 0 0 0
1~ I h O ('~
h
J ~1
I o a)
U
~ _ ~ ~ C
O O
.,1 . I O ~ ~:
E~ I ~ H
~) I I
.
o I a
o ~ ~ ~ ~ a
rl ~ ~ \ O
c
H
E~ ~ -1
C ~ ~1 0
O ~ O
Z
~_:1 H ~ O
--39--
~2~ 7
(Results)
(1~ The liver injury-induced control group showed
remarkable increase in GOT and GPT when compared with the
non-treated control group. Thus, it was clearly recognized that
serious injury to the liver was caused by carbon tetrachloride~
(2) The isoliquiritigenin administration group showed
substantially the same level of suppression o~ increase in GOT
and GPT as that showe~ by the positive control group which
received cianidanol. The positive control group which received
malotilate showed remarkable suppression in this series of tests.
It can be considered from the above-mentioned results of
the tests that isoliquiritigenin is useful in the prevention and
therapy of chronic liver injury.
Example 8
(Activity of isoliquiritigenin against acute liver injury induced
by cis-platinum)
It is known that cis-platinum, a carcinostatic drug,
causes injury to the liver as well as to the kidney (CANCER
TREATMENT REPORTS, vol. 62, No. 12, pages 2125 to 2126, 1978, by
F. Cawalli et al). The present inventors have found that the
carcinostatic drug cis-platinum causes injury to the liver
besides it causes injury to the kidney and that it causes liver
injury in a lower dosage than that in which it causes kidney
injury. In this example, the activity of isoliquiritigenin
against acute liver injury induced by cis-platinum was examined,
to confirm the efficacy of the medicine of the present invention.
-40-
~LZ9~;~9'4`~7
A group consisting of six Wistar strain male rats
(produced by Nihon Charles River K.K.), each weighing 150 to 180
g, were subcutaneously injected with 8.5 mg per kilogram body
weight of cis-platinum dissolved in physiological saline solution
at a ratio of 2mg per 10 ml once, and orally administered with
200 mg per kilogram body weight per day of isoliquiritigenin
suspended in 1%-CMC aqueous solutions, for 10 continuous days.
The rats were then dissected, and ]iver samples dyed with
hematoxylin- eosin were prepared and observed under a microscope.
A liver injury-induced control group consisted of six
Wistar strain male rats (produced by Nihon Charles River K.K.),
each weighing 150 to 180 g, were treated in exactly the same
manner as that of the isoliquiritigenin administration group
except that the rats in this control group were orally
administered with l~-CMC aqueous solutions alone, in place of
isoliquiritigenin, for 10 continuous days. The results of the
tests are shown in Table 8.
41
9~7
Table 8
Pathological Views of Isoliquiritigenin against
Acute Liver Injury Induced by Cis-platlnum
Pathological Views
of Liver Injury
Degeneration Grade
Group (n=6) _ f ++ -
Liver Injury-Induced Control Group 0 3 3
Isoliquiritigenin 200mg/Kg 3 3 0
Non-Treated Control Group 6 0 0
12~ 7
(Results)
(1) It was clearly recognized that the liver injury-
induced control group which received cis-platinum caused injury
to the livers when compared with the non-~reated control group.
(2) Isoliquiritigenin remarkably suppressed injury to the
livers induced by cis-platinum.
It is clear from the above-mentioned results that
isoliquiritigenin remarkably improves cis-platinum-indu~ed acute
liver injury. Thus, it can be considered that isoliquiritigenin
is very useful in therapy of acute liver injury induced by drugs.
Example 9
(Activity of isoli~uiritigenin against acute kidney and liver
injuries induced by cis-platinum)
As described above, it has become clear that
isoliquiritigenin is useful in the prevention and therapy of both
kidney in~ury and liver lnjury~ Therefore, in this example, the
present lnventors conducted the following prevention tests and
therapy tests using the same test models as used in examples 3
and 8 wherein kidney or liver injury was caused by cis-platinum,
a carcinostatic drug, to confirm the efficacy of the medicine in
accordance with the present invention agalnst a complication
resulting from kidney and liver injuries.
(1) Prevention Tests
Fihser strain male rat (produced by Nihon Charles River
K.K., 3 rats in each groups), each weighing 215 to 240 g, were
either orally administered or intraperitoneally injected with 20
mg per kilogram body weight per day of isoliquiritigenin
-43-
~z~s~
suspended in l~-CMC aqueous solutions once a day, for 12
continuous days. From the 6th day after the start of the
isoliquiritigenin administration, the rats in these groups
subcutaneously injected with 2 mg ~per kilogram body weight per
day of cis-platinum dissolved in physiological saline solution at
a ratio of 2mg per 10 ml, once a day for ~ continuous days.
Similar isoliquiritigenin administration was effected during the
cis-platinum injection days immediately after the injection of
cis-platinum, and during 3 days following the completion of the
cis-platinum administration.
Rats in a kidney and liver injury-induced control group
were injected with cis-platinum in the same manner as that of the
isoliquiritigenin administration groups, and were treated in the
same manner as that of these groups except that rats in this
control group were orally administered with 1%-CMC aqueous
solutions, in place of isoliquiritigenin suspended in l~-CMC
aqueous solutio~s.
24 hours after the completion of the administration of
isoliquiritigenin, blood samples were collected from the aorta
abdominalis while the rats were anesthetized with ether, and
autopsy was performed. After the livers were removedf liver
samples were fixed in 10%-buffer formalin, dyed with
hematoxylin-eosin and with PAS, and observed under a microscope.
The collected blood was centrifuged at 3000 rpm for 15 minutes,
and the serum was examined by measuring BUN using an automatic
analyser (AU-550, a product of Olympus Kohgaku Kohgyoh).
(2) Therapy Tests
-44-
5~
Fisher strain male rats (produced by Nihon Charles River
K.K., 3 rats in each group), each weighing 2l5 to 240 g, were
first divided into two groups. Rats in these groups were
subcutaneously injected with 2mg per kilogram body weight per day
of cis-platinum dissolved in physiological saline solution at a
ratio of 2mg per 10 ml, once a day for 4 days, thereby inducing
kidney and liver injuries. Everyday, immediately after the
injection of cis-platinum, the rats in the two ~roups were either
orally administered or intraperitoneally injected with 20 mg per
kilogram body weight per day of isoliquiritigenin suspend~d in
1%-CMC aqueous solutions, once a day for 4 continuous days, and
the rats were also administered with isoliquiritigenin once a day
for 3 continuous days after the last injection of cis-platinum.
The rats in a kidney and liver injury-indu~ed control group were
injected with cis-platinum in the same manner as those of the
isoliquiritigenin administration groups, and were treated in the
same manner as the rats of these groups except that rats in this
control group were orally administered with l~-CMC aqueous
solutions, in place of isoliquiritigenin suspended in 1%-CMC
aqueous solutions.
24 hours af~er the completion of the administration of
isoliquiritigenin, blood samples were collected from the aorta
abdominalis wile the rats were anesthetized with ether, and
autopsy was performed. After the livers were removed, liver
samples were fixed in 10%-buffer formalin, dyed wi~h
hematoxylin-eosin and with PAS, and observed through a
microscope. The collected blood was centrifuged at 3000 rpm for
-45-
~Z~S~'7
15 minutes, and the serum was examined by measuring BUN using an
automatic analyser (AU-550, a product of Olympus Kohgaku
Kohgyoh).
A non-treated control group consisted of Fisher strain
male rats (produced by Nihon Charles River K.K., 3 rats in each
group), each weighing 215 to 240 g, which were orally
administered with 1%-CMC aqueous solutions, in place of
isoliquiritigenin suspended in 1%-CMC aqueous solution, once a
day for 12 days. 24 hours after the completion of the
administration, blood samples were collected, and autopsy was
performed. Also, measurement of BUN and observation of liver
samples were performed in similar manners as those described
above. The results of the t-ests are shown in Tables 9 and 10.
-46-
~2~S9 ~
Table 9
Urea Nitrogen in Blood Samples after 4 Days
of Cis-platinum Administration
__ B U N (mg/dl)
(l)Prevention (2)Therapy
Test _ _ Test
mean+SD mean+SD
Non-Treated Control Group23+ 3 23+ 3
Kidney and Liver Injury-Induced 84+27 66+13
Control Group
Isoliquiritigenin
(Oral Administration) 20mg/Kg 51+18 47+16
Isoliquiritigenin
(Intraperitoneal Injection) 41+ 1 38+ 2
-47-
~2~S~7
Table 10
Pathological Views of Liver Injury after
4 days of Cis-platinum Administration_
Pathological Views of
Liver-Injury ___
Group (n=3) Degeneration Grade
+ ..
(1) Prevention Test
Kidney and Liver Injury- 1 2 0
Induced Control Group
Isoliquiritigenin 2 1 0
(Oral Administration) 20mg/Kg
Isoliquiritigenin 2 1 0
(Intraperitoneal Injection)
20mg/Kg
(2) Therapy Test
Kidney and Liver Injury- 1 2 0
Induced Control Group
Isoliquiritigenin 2 1 0
(Oral Administration) 20mg/Kg
Isoliquiritigenin 3 0 0 :
(Intraperitoneal Injection)
: 2Omg~Kg
Non-Treated control GFoup 3
-48 -
:
~-z9~
(Results)
(1) It was clearly recognized from the fact that the
kidney and liver injury-induced groups showed increase in BUN and
from the pathological views of liver tissues and cells, i.e.,
from the views of oxyphilic degeneration and vacuolar
degeneration of the liver cells, that injury to the kidney and
the liver was caused by cis-platinum when compared with the
non-treated groups.
(2) In both the prevention tests and the therapy tests,
the isoliquiritigenin administration groups, which received
isoliquiritigenin either orally or by intraperitoneal injection,
showed remarkable suppression of increase in BUN, and suppression
of development of liver injury was also recognized when compared
with the kidney and liver injury-induced groups.
It can be considered from the above-mentioned results and
from the results of examples 3 and 8 that isoliquiritigenin is
very useful in the prevention and therapy for complications
resulting from kidney and liver injuries.
The foregoing examples show the efficacy of the medicine
in accordance with the present invention in treating individual
kidney and liver diseases and complications thereof.
The following examples give examples of prescription in
which the formulation and the components of the medicine are
specified.
It is to be understood that the present invention are not
limited by the following examples.
-49-
~s~
Example 10
Examples of prescription of tablets each containing 5 or
25 mg of the active ingredient are as given below:
Prescription Example 1 (5mg tablet)
isoliquiritigenin 5
lactose 137
starch 45
calcium carboxylmethyl cellulose 10
talc 2
magnesium stearate
200 mg per tablet
Prescription Example 2 (25 mg tablet)
isoliquiritigenin 25
lactose 120
starch 42
calcium carboxylmethyl cellulose 10
talc 2
magnesium stearate
_ _
200 mg per tablet
Details of the manufacturing method is as given below.
Crystals of isoliquiritigenin were ground, lactose and
starch were added thereto, and they were mixed. 10% of starch
paste was added to the mixture, and they were agitated, thereby
obtaining a granule. After the granule was dried, the grains
--50--
~LZ95~ ~7
were dressed to a grain size of about 850 microns. It was then
mixed with talc and magnesium stearate, and the mixture was
formulated into tablets.
Example 11
Prescription Example t20 mg capsule~
isoliquiritigenin 20
lactose 53
starch 25
ma~nasium stearate _ _ 2
100 mg per capsule
Isoliquiritigenin was well ground, and starch, lactose,
and magnesium stearate were added thereto. After they had been
mixed adequately, the mixture were charged into a capsule.
As described above, the present in~ention provides a
medicine for use in the therapy and prevention of kidney and
liver diseases characterized by containing as the active
ingredient isoliquiritigenin or a pharmaceutically acceptable
salt thereof. According to the present invention, there is
provided a medicine for use in the therapy and prevention of
kidney and liver diseases which exhibits excellent therapeutic
and preventive effects with respect to a wide range of kidney or
liver diseases individually developed or to complications
resulting from diseases of`these organs.
~9Si~7
INDUSTRIAL APPLICATION
The medicine in accordance with the present invention
which contains isoliquiritigenin as the active ingredient has a
broad scope of applications such as a medicine for use in the
therapy and prevention of kidney diseases, a medicine for use in
the therapy and prevention of liver diseases r and a medicine for
use in the therapy and prevention of complications resulting from
kidney and liver diseases. Therefore, the medicine in accordance
with the present invention exhibits excellent therapeutic and
preventive effects with respect to a wide ranye of kidney or
liver diseases individually developed or to complications
resulting from diseases of these organs.
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