Preventive and therapeutic agent against liver disorder

A new preventive and therapeutic agent given by oral administration containing carnosine zinc salt as the effective component and, effective against alcoholic liver disorder, viral hepatitis or liver disorders caused by drugs, toxicants, radiation, etc.

TECHNICAL FIELD 
The present invention relates to a new preventive and therapeutic agent 
against liver disorder (hepatopathy), which is particularly effective for 
the prevention and treatment against alcoholic liver disorder, viral 
hepatitis and liver disorders caused by drugs, toxic substances, 
radiation, etc. 
BACKGROUND ART 
The liver is the largest organ in human body and fulfills the most 
important functions such as metabolism and storage of nutritional 
substances, detoxicating various substances, etc. The liver maintains 
circulatory dynamics under normal conditions as a circulatory system next 
to systemic circulation and pulmonary circulation. Therefore, when hepatic 
cells are adversely affected either acutely or chronically by causes such 
as toxic substances, drugs, alcohol, viruses, etc. passing through the 
liver or by malnutrition, radiation, cholestasia, etc., the entire body is 
adversely affected. 
In liver disorder, characteristic changes are observed from clinical, 
biochemical and histological viewpoints. 
Liver disorder is characterized by increase of hepatic enzymes such as 
glutamic pyruvic transaminase ("GPT"), glutamic oxaloacetic transaminase 
("GOT"), alkaline phosphatase ("AL-P"), sorbitol dehydrogenase ("SDH"), 
etc. in blood or by increase of serum bilirubin. 
Measurements of activity of hepatic enzyme and bilirubin value in blood are 
utilized for characterization and judgment of degrees of liver disorder, 
and these methods are generally used for clinical examination. For 
example, increase of serum AL-P activity suggests physical blocking of 
extrahepatic bile duct, early development of liver cirrhosis or arrest of 
choleresis by drug. Increases of activity of serum GPT, GOT and SDH are 
common to all types of liver disorder and indicate damage of hepatic cells 
(Merk Manual, 13th edition, Chapter 8, p. 836, 1977). 
Liver disorder is associated with necrosis of hepatic cells, and this 
necrosis is histologically identifiable and provides excellent indices to 
show degree of liver disorder. 
Since the degree of liver disorder can be judged by conditions of hepatic 
enzymes in blood, serum bilirubin and necrosis of hepatic cells, these 
indices are generally used in search of preventive and therapeutic agents 
against liver disorder. Various models of hepatitis have been developed 
for experimental evaluation of hepatic disorder. Of these models, the 
liver disorder caused by hepatic toxicants such as D-galactosamine, carbon 
tetrachloride, etc. are most akin to the viral hepatitis or the hepatitis 
caused by drug, toxicants, etc. as actually seen. 
In the hepatic disorder caused by carbon tetrachloride, the linkage of 
carbon tetrachloride is severed in liver by cytochrome P-450, thereby 
generating the highly toxic free radical (.CCl.sub.3), and it is generally 
believed that this free radical causes the disorder by combining with 
thiol group of protein in hepatic cell membrane or by accelerating 
peroxidation reaction of the lipids in cell membrane. (Biochem. 
pharmacol., Vol. 25, p. 2163, 1976, and Biochem. pharmacol., Vol. 21, p. 
49, 1972). As the result, it biochemically induces suppression of protein 
synthesis in liver and escape of hepatic enzymes such as GPT, GOT and SDH 
into blood. Histologically, it causes coagulation necrosis, edematous 
degeneration and fat formation, etc. 
It is said that the models with disorder caused by carbon tetrachloride and 
D-galactosamine are akin to the liver disorder due to chronic alcoholism. 
On the models with carbon tetrachloride, various descriptions have been 
given in literature: Amer. J. Path., Vol. 79, p. 579, 1975; Virchowa Arch, 
B. Cell. Path., Vol. 26, p. 331, 1978; and Semirars in Liver Disease, Vol. 
1, p. 143, 1981. It is described that this model is most akin to the liver 
disorder caused by viruses, drugs, toxicants, etc. 
As liver disorders, there are acute hepatitis, chronic hepatitis, liver 
cirrhosis, fatty liver, etc., and the causes are diverse such as 
toxicants, drugs, alcohol, viruses, malnutrition, radiation, cholestasia, 
etc. Various preventive and therapeutic agents have been conceived against 
these liver disorders. Most of these drugs were the drugs to prevent and 
treat the metabolic anomaly, which secondarily occurs when hepatic cells 
are affected, and they are disadvantageous in that the therapeutic effects 
against liver disorders caused by specific causes such as viruses are 
rather weak. 
The present invention offers preventative and therapeutic agents against 
various types of liver disorders. 
DISCLOSURE OF THE INVENTION 
The present inventors have found through experiments with model animals 
having hepatitis experimentally induced by carbon tetrachloride that, when 
carnosine zinc salt was given before liver disorder occurs, the increase 
of hepatic enzymes in blood was effectively prevented, that it was 
effective to prevent liver disorder before it occured, that the same 
preventive effect was obtained when carnosine zinc salt was given after 
liver disorder had actually occurred, and that carnosine zinc salt is 
effective in prevention and treatment of liver disorder. 
Specifically, the present invention relates to a preventive and therapeutic 
agent against liver disorder, containing carnosine zinc salt as effective 
component, and the improvement of liver disorder by carnosine zinc salt is 
revealed by significant decline in the increase of GPT and GOT in blood 
and by the reduction of the degree of necrosis of hepatic cells. 
Carnosine zinc salt of the present invention is a drug known for the 
treatment of peptic ulcer, and its application and manufacturing method 
are described in the Japanese provisional patent publication No. 59-33270. 
147 g of L-carnosine dissolved in 441 ml of pure water was added with a 
solution of 88.6 g of zinc chloride in 177 ml of pure water. 325 ml of 4N 
sodium hydroxide aqeous solution was dropped into said reaction solution 
while stirring up for 30 min., and the reaction was completed. After the 
reaction, the deposited precipitate was taken by filtration and was rinsed 
well with water until the washing solution was turned to neutral. When 
dried at 40.degree. C. for 2 days, it gave 175 g of colorless powder of 
L-carnosine zinc salt. 
The analysis result of this substance is as follows: 
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Loss on drying (1 g; dried under reduced 
pressure at 60.degree. C. for hours) 
7.63% 
Zinc content (weight analysis) 
23.20% 
Carnosine content (weight analysis) 
76.81% 
Melting point 300.degree. C. or more 
I.R. spectrum (KBr, cm.sup.-1) 
3280, 1620, 
1480, 1385, 1260, 1120, 
1050, 1000, 980 
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PREVENTION OF EXPERIMENTAL LIVER DISORDER IN RAT 
EXPERIMENTAL METHOD 
Carnosine zinc salt was suspended in 0.5% sodium carboxymethyl cellulose 
(CMC) solution to prepare 60 mg/ml and 20 mg/ml suspensions. This was 
given to rats orally every day for 8 days at the rate of 0.5 ml/100 g so 
that the dosage was to be 300 mg/kg and 100 mg/kg, respectively. To the 
control group, the same volume of 0.5% CMC was given. 
To prepare the model animals with liver disorder caused by carbon 
tetrachloride, carbon tetrachloride mixed in olive oil to have a 10% (V/V) 
mixture was given orally at the rate of 4 ml/kg (0.4 ml/kg as carbon 
tetrachloride) one hour after the final administration of carnosine zinc 
salt or 0.5% CMC. The animals were anesthetized by intraperitoneal 
injection of pentobarbital at 24 hours after carbon tetrachloride was 
given, and blood was collected from carotid arteries. The blood was 
centrifuged at 3000 rpm for 15 minutes to fractionate serum, and GOT and 
GPT were determined (Reitman Frankel and Momose modification). 
From the isolated liver, the paraffined sections were prepared, stained by 
H-E staining and were examined by histopathological examination. 
For the experiment, male Wistar rats were used. With 10 rats in each group, 
they were divided into three groups: the group given with carbon 
tetrachloride (CCl.sub.4) and 0.5% CMC only (CCl.sub.4 +CMC), the group 
given with carbon tetrachloride and 300 mg/kg or 100 mg/kg of carnosine 
zinc salt (CCl.sub.4 +carnosine zinc salt) and the group given with 0.5% 
CMC only (CMC). 
The results are as shown in the table below: 
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Items of measurement 
Specimen GOT (KU) GPT (KU) 
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CCl.sub.4 + CMC 
2115.9 .+-. 260.2 
2243.8 .+-. 333.0 
CCl.sub.4 + CAZ 
(100 mg/kg) 2167.7 .+-. 777.5 
1917.7 .+-. 748.6 
(300 mg/kg) 629.7 .+-. 121.2 
427.7 .+-. 69.4 
CMC 89.1 .+-. 3.2 
42.5 .+-. 1.3 
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(Note)- 
CAZ represents carnosine zinc salt. 
KU means KARMEN unit. 
The result of this experiment suggests the effect of carnosine zinc salt to 
prevent the experimental liver disorder caused by carbon tetrachloride. 
In the acute toxicity test, female and male Wistar rats, each with body 
weight 150-200 g, were divided into the groups with 10 rats each, and 10 
g/kg each of carnosine zinc salt was given orally. By the observation for 
7 days, there was no case of death, showing very weak toxicity of this 
compound. 
THE BEST WAY TO EXECUTE THE INVENTION 
Adequate method to give carnosine zinc salt is oral administration. Dosage 
form may be any of suspensions, tablets, pills, capsules or powder. Though 
dosage of carnosine zinc salt for prevention and treatment of liver 
disorder may differ according to degree of liver disorder, the effective 
daily dosage is 0.3 mg-30 mg/kg, preferably 1.5 mg-15 mg/kg. 
CAPABILITY OF EXPLOITATION IN INDUSTRY 
As described above, carnosine zinc salt according to the present invention 
suppresses increase of GPT and GOT in blood, depending upon the dosage. In 
the group with 300 mg/kg administration, it was significantly decreased at 
risk rate of 1%. 
Also, in the histopathological observation, the suppression of necrosis of 
hepatic cells and fat formation were observed in the group given with 
carnosine zinc salt. 
Further, it was confirmed that acute toxicity of carnosine zinc salt of 
this invention is extremely weak. 
Therefore, it is clear that carnosine zinc salt of this invention is 
effective to the model animals with liver disorder caused by carbon 
tetrachloride. Since these model animals with liver disorder due to carbon 
tetrachloride are evaluated as good models with actual liver disorder 
caused by viruses, drugs, toxicants, etc., it follows therefore that 
carnosine zinc salt of this invention is very useful as the preventive and 
therapeutic agent by oral administration against liver disorders.