WO1993024140A1 - Drug for depressing serum got and gpt activities and hepatic disease curative composition - Google Patents

Abstract

A drug containing an effective dose of calcitonin for depressing the elevated activities of serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase, both being hepatic leaking enzymes, and a novel hepatic disease curative composition containing calcitonin as the active ingredient and having extremely reduced side effects. The above drug containing calcitonin can effectively depress the elevated activities of the leaking enzymes serving as an index of hepatic injury in hepatic diseases. Therefore, a drug composition containing calcitonin as the active ingredient in a dose effective against hepatic diseases can be utilized for treating various hepatic diseases such as drug-induced hepatitis, alcoholic hepatitis and autoimmune hepatitis.

Description

 'Specification

Drugs for reducing serum G〇T and GPT activity, and for treating liver diseases

Technical field

 The present invention relates to a novel use of calcitonins. More specifically, the present invention relates to serum glutamate oxa oral transaminase (hereinafter often referred to as "GOT") and glutamin containing calcitonin as an active ingredient. Acid pyruvate transdermal kinase (hereinafter often referred to as "GPT"). Furthermore, the present invention relates to a therapeutic composition for a liver disease containing calcitonin as an active ingredient.

 In the present invention, amino acid sequences and peptides are represented using abbreviations adopted by the IUPAC-IUB Biochemical Nomenclature Committee (CNB). When amino isomers and other optical isomers are present, they represent the L-isomer unless otherwise specified, and the left and right ends of the amino acid sequence of the peptide are N-end and C-end.

 Background art

The liver is the largest single organ in the living body, and plays a central role in glucose metabolism, protein metabolism, and lipid metabolism. In addition, drug metabolism and detoxification are also important functions of the liver, and are therefore essential organs for maintaining homeostasis and maintaining life. These functions include liver parenchymal cells, Kupffer cells, and sinusoidal endothelial cells that make up the liver. It is understood physiologically as the result of the action or interaction of any of a variety of cells, but among them the role of liver parenchymal cells is so extensive that viruses, drugs, toxicants, alcohols, etc. If liver parenchymal cells are damaged, toxic or infectious hepatitis, liver abscess, cirrhosis, fatty liver, hepatocellular carcinoma, etc. can be extremely serious.

 Numerous hepatic drugs are used for liver disease based on these various causes, but there is still no definitive treatment. This is described in detail in The Journa1ofPracctiCalPharmAccy, Vo1.41, No.11. For example, many amino acid transfusions and vitamins of a specific composition have the meaning of adjuvant therapy to replace essential nutrients, vitamins, etc., which were lacking due to the decrease in hepatic metabolism. However, the effectiveness of antidote, such as SH compounds such as glutathione and chopin, is questionable.

In addition, anti-inflammatory or immunomodulatory agents such as adrenocortical hormone, glycyrrhizin, and azathioprine, protein synthesis promoters represented by malotilate, and interflus having an antiviral effect. Drugs that have a therapeutic effect on liver diseases such as lon have also been known to have serious side effects such as hepatic dysfunction, hypoaldosteronism, agranosis, jaundice, and cardiomyopathy. Thus, drugs currently used for the treatment of liver disease have shown sufficient efficacy. This did not fully satisfy clinicians, including concerns about serious side effects.

 Under such circumstances, it has been desired to develop a small drug that is effective for the treatment of liver disease and that has a small amount of side effects.

Disclosure of the invention

 In order to meet the above-mentioned demand, the present inventors first reduced the activity of GOT and GPT, and in some cases, the activity of hepatic escape enzymes such as γ-GTP, which are observed in serum as indicators of liver damage. We worked diligently to obtain a material that can be used. As a result, calcitonins, hormones with serum calcium-lowering activity discovered by C0ρρ et al. In 1962, are not GOT, GPT or γ-GTP activity inhibitors. Nevertheless, surprisingly, serum GOT and GPT, which are general indicators of liver disease (when serum γ-GTP activity is increased due to alcoholic hepatitis, etc.) ) Was found to be extremely effective in lowering the activity value. In addition, they found that calcitonins increased their survival rate in acute liver failure models.

As described above, calcitonins are serum calcium-lowering hormones discovered by Copp et al. In 1962 [Endocrinology, Vol. 70, 638 (1966). 3)] Then, it has been extracted and purified from various animals including humans, and its amino acid structure has been elucidated as described below. These calcitonins are associated with bone paget disease [Journal of the Japan Society for the Practice of Shape, Vol. 58, 93 (1977)], osteoporosis.

 [(Comprehensive Clinic, Vol. 39, 3623 (1990))], hypercanoresidemia [J. Bone Mineral Metab., Vol. 6, 93 (1988) )] It is used clinically as a therapeutic agent, etc. In addition, it promotes the excretion of electrolytes such as sodium and phosphorus on the kidney [Am.J.Physiol., Vol. 2 4 6, 9 2 7 (1984)], a gastric secretion inhibitory effect on the stomach [Acta Endocrinol. (S uppi), Vol. 15 5, 2 16 (197 1) And an exocrine-suppressing effect on the kidney [Gut, Vol. 18, 615 (1977)].

Calcium concentration or content increasing effect on normal liver [Horm.Metab.Res., Vol.20, 22 (1988)]; Horm.etab.Res., V Jpn., Vol. 27, 38 1 (1980); Endocrinol. Jpn., Vol. 27, ol. Endocrino 1. Jpn .; Vo 1.26, 65 (1977); Chem. Pharma. Bull., Vol. Biophys.Acta., Vol. 852, 169 (198.6); Endocrino 1.Jn., Vol. 28, 709 (19981); 96, 104 (1975)] is known, and its mechanism of action, such as calcium ATPase inhibitory action, is known [Endocrino 1. 27, 381 (1980); Endocrinol. Jpn., Vol. 27, 273 (1980); Endocrinol. Jpn., Vol. 26, 605 (19779); Biochem. J., 266, 817 (1990)].

In addition, gluconeogenesis and glycogenolysis promoting action [Horm.Metab.Res., Vo1.16,650 (1994); Horm. 15, 176 (1983); Endocrinol. J pn., Vol. 29, 55 3 (1982); Endocrinol. J Jpn., Vol. 28, 51 (1 981)], fatty acid synthesis promoting action [Horm. , Vol. 18, 378 (1966); Horm. Metab. Res., Vol. 18, 22 (1996); Horm. 17, 34 6 (1985)], mitochondrial function activation [Chem. Pharm. Bull., Vol. Chem. Pharm. Bull., Vol. 35, 425 (1998); Horm. Metab. Re. , Vol. 19, 15 2 (198 7); Horm. Metab. Res., Vol. 16, 47 8 (198 4); Endocrinol. , Vol. 28, 709 (19981)] is known.

 However, all of the above reports on the effects of calcitonins on the liver are directed to normal liver, and not to liver failure. In general, it is known that when a large amount of calcium flows into various tissues, cell necrosis is caused. Hepatocytes are no exception, and hepatotoxic substances such as carbon tetrachloride and menadione increase the calcium concentration in hepatocytes [J. Biol. Chem., Vol. 130 (1960); Metabolism, Vol. 26, 1107 (11989)].

 In addition, calcium ionophore increases the intracellular calcium concentration in liver cells, causing liver damage [Science, Vo1.206, 700 (19779)]. Disorders are inhibited by calcium inhibitors [Liver, Vol. 30, 850 (19989)]. From this perspective, the use of calcitonins, which have been reported to increase hepatocyte calcium during liver injury with increased liver cell calcium concentration, further exacerbates liver injury It is expected that.

Considering the above-mentioned reports on the unfavorable effects of calcitonins on normal hepatocytes as described above, patients with liver disease Administration of calcitonin to the liver decreases the elevated levels of GOT and GPT, which are liver deviating enzymes observed in serum, which are indicators of liver damage. The finding of a therapeutic effect on the model is quite surprising.

 More specifically, in a liver injury model using cultured hepatocytes, calcitonins prevent hepatic cell death due to, for example, carbon tetrachloride, and also serve as an indicator of the injury in the culture supernatant. It shows a marked decrease in liver hepatic enzyme levels, and calcitonin enhances survival in an immune-related acute liver failure model. In addition, intravenous infusion of calcitonin into patients with liver diseases, including patients with hepatitis and cirrhosis, shows a decrease in serum liver deviating enzyme level, which is an indicator of liver damage.

 The inhibitory effect of this substance on hepatotoxicity is attributed to the isolation of hepatocytes from mature rats and cultivation with hepatotoxic substances such as carbon tetrachloride. It is also evident from the fact that, as well as the clinical findings, the remarkable suppression of the disorder is apparent from the simultaneous addition or pre-addition, and it is effective for the treatment of liver diseases including the scope of prevention. It was found that

 The present invention has been made based on the above findings.

Accordingly, one object of the present invention is to provide a novel agent for reducing the elevated activity of liver deviating enzymes such as GOT and GPT observed in serum, which is an indicator of liver disease. . Another object of the present invention is to provide a novel therapeutic composition for a liver disease which is effective in treating a liver disease and has very few side effects.

 That is, according to one embodiment of the present invention, the increased activity of glutamate oxa mouth transacetate and glutamate pyruvate transamine is reduced in serum. And a reducing agent comprising an effective amount of calcitonin. According to another aspect of the present invention, there is provided a therapeutic composition for liver disease, comprising an effective amount of anti-hepatic disease in the form of lucitonin and a pharmaceutically acceptable carrier, diluent or excipient. You.

 As used herein, the term "calcitonin" refers to natural calcitonins known as peptides having serum calcium-lowering activity and their analogs having serum calcium-lowering activity. It means at least one kind of calcitonin selected from the group consisting of mouth peptides.

 Natural calcitonins are peptides consisting of 32 amino acids, which have cystine residues at positions 1 and 7 and the two cystine residues are disulfide bonds. To form a ring structure, and the C-terminal is proline amide. That is, natural force nolestonin is represented by the following formula (I):

 — S— S——:

 Cys X Cys Y P ro N H (I)

(Where X represents 5 amino acids and Y represents 24 amino acids. Shown).

 The structure of formula (I) is a structure very specific to calcitonin, and by having this structure, it shows a so-called calcitonin activity called serum calcium-lowering activity only when it has this structure. Or a compound having no structure similar thereto (for example, or a structure of the calvatonin calcitonin of the formula (π) of an analog peptide described later), the calcitonin activity is significantly reduced, and the present invention It is not preferred as an active ingredient for.

Examples of natural calcitonin include porcine calcitonin (Japanese Patent Publication No. 48-10848 号), ゥ calcitonin, natural calcitonin, and human calcitonin. 4 8 — 4 3 9 18), rat calcitonin, salmon calcitonin I (He 1 V. Chim. Acta, 1969, ^ l, 1789), salmon calcitonin II, salmon calcitonin Nitto, Penagi calcitonin (Japanese Patent Publication No. 53-186), Nitrika calcitonin (Japanese Patent Application Laid-Open No. 61-47498), and the like. it can. The amino acid sequences of these calcitonins are shown in Sequence Listing 1 below.

Sequence list (Natural calcitonin)

I s S- ¹

 ') N Cy sSc rAsnLcuSe rTli rCysVa l LcuSc rA l aTy rTr L sAspLcuAsnAsnTyrl! I sAr gPhcSc rG l M

 '— ~ -S S'

 "Di CysSc rAs nLcuSc rTh rCysVa I LcuSe rA! AT rTr pLysAs pLcuAsnAsnT rll i sAr gTy rSc rG I yM

 L -s s- t CysG l yAs nLcuSc rTli rCysMc l LcuG l Th rTy rTh rG l nAs pPhcAsnLysPhcll i sTh r PhcP f oG l nT

S—— ~ ¹

 r; y \ CysG l yAs nl.cuSc rTlirCysMe t LcuG l yTh rTy rTh rGl nAs pLcuAsnLys Lcu! Ii sT r hcProGlnT

I ss ¹

f device I CysSc rAs nLcuSc rTli rCysVa l Lc uG l yLys LcuSc rG l nG l uLcu! li sLysLcuG l nTh rTy rP r oAr gT

I s— ~ s ¹

Salmon D CysSe rAs nLcuSc rTh rCysVa l LeuG l, "l, cuSe rG l nAs pLcuin sLysLeuG l nTh rPheProA [gT

¹ SS ¹

 ') C CysSc r As nLc iiSe rTli rCy "lc I LeuG l, y s l, cuSc rG I nAs pLeull i sL" LcuG I nTli r PheP r oAr gT

1 SS ¹

Eel Cy sSc r As nl.cuSc rTh r Cy sVa 1 LeuG I yLys Lc uSc rG 1 nC I uLcu! Li sLys LcuG! nTh rTy r Pr oAr gT

1 SS ¹

Two CysA l aSe r LeuSc rTh [CysVal l LeuG sl, euSe rG l nG l uLculli sL LcuG l nTh rT "P [oAr gTh

L- SS ¹

These calcitonins are based on the amino acid sequence shown in Sequence Listing 1 above, and have the ability to be obtained by a conventional peptide synthesis method, or using the DNA of the desired calcitonin. After being obtained by a conventional method of gene manipulation, it can be purified and recovered by various types of column chromatography.

 The calcitonin analog peptide of the present invention is, for example,

(A): at least some of the amino acid structures of X and Y in formula (I)

One obtained by substituting one amino acid residue with another amino acid residue and having a serum calcium-lowering activity (hereinafter often referred to as "calcitonin activity");

 (B): No cystine residue at position 1 of the natural calcitonin of the formula (I) is present, and a cystine residue at position 7 is replaced by a-L-anosulin represented by the following formula:

 C H C H C H C H

C H 2 C O O H H 2 N- C H C〇 O H

 * ω position power

Wherein the carboxyl group at the ω-position condenses with the amino group of the amino acid residue at the 2-position of the formula (I) to form a disulfide bond at the 1- or 7-position of the formula (I). Is a peptide consisting of 31 amino acids, which has a cyclic structure formed by the pentamethylene bond of amino amino acid instead of amino acids, and has calcitonin activity. (Hereinafter often referred to as “leba-type calcitonin”) (Also, this leuva-type calcitonin is compared with that of the formula (I) in the first place of the formula (I)) The amino acid residue corresponding to the cystine residue has been deleted, but the amino acid number hereafter will for convenience follow that of formula (I));

 (C): at least one of the amino acid residues Nos. 2 to 6 and amino acids Nos. 8 to 30 of the lactate calcitonin of the above (B) is at least one other amino acid residue; Those substituted with an amino acid residue and having calcitonin activity,

Is included.

 The forceba type calcitonin of the above (B) is represented by the following formula (Π)

. "(CH ₂₎ one

COXNHCOYPro NH ₂ (Π) In the formula, X and 通 are as defined in formula (I).

The above (beta) of the force resolver type calcium DOO Nin, [indicate with a prefix of A su ¹ · ^7] (e.g., [A su ^{1 '7]} human mosquito Noreshi DOO Nin carbamate type human Calcitonin).

Is an example of a force resolver type calcium Tonin'ana port Gupepuchi de, (A su ^7] Unagikarushi preparative Nin (generic name: erucic phosphatonin) (GB 1 5 1 6 9 4 7 No.), [A su ^{1 '7} :) Sakekanore collected by two down 1 (GB 1 5 1 6 9 4 7 No.), [A su ^{1' 7} :) d Wa Application Benefits calcium DOO two emissions, (JP 5 1 - 1 2 8 9 9 3), [A su ^{1. 7]} human calcium preparative two emissions (GB 1 5 9 0 6 4 No. 5), [A su ¹ + ^7] Butakanoreshi DOO two emissions, [A su ¹ · ^7] © Shikarushi DOO two down, [A su ¹ · ^7] hits Jikarushi door two down, ί A su ¹ · ^7] rats Kanoreshi door two down, [A su ^{1. 7]} Sakeka Noreshi door Nin Π, [A su ^{1. 7} Salmon calcitonin! E and so on.

The amino acid structure of these specific Luba-type calcitonins is shown in Sequence Listing 2 below.

Rooster system Table 2 (Power Luba type calcitonin)

In the above-mentioned calvaton-type calcitonin, the cysteine residue at the i-position of the amino acid sequence of the corresponding natural calcitonin is deleted, and the cystine residue at the 7-position is removed from the amino acid sequence. When a peptide is synthesized by a conventional peptide synthesis method based on a sequence substituted with an acid residue, for example, the cystine residue at the 7-position is replaced with an amino-suberic acid residue According to the method described in, for example, British Patent No. 1,516,947, the carboxyl group of an aminosuberic acid residue is replaced with the amino group of an amino acid residue at the 2-position. and I Li cyclic structure and this condensation, the resulting [a su ^{1 '7]} calcium preparative Nin purified Ri by the various Ca La Solid Loma preparative grayed Rafi one' Ru can and this recovering.

 In addition, at least one amino acid residue of the amino acid sequence of the 2nd to 6th and 8th to 31st amino acids in the above-mentioned Luba type calcitonin is replaced by another amino acid residue. A substituted product having the calcitonin activity [the above (C)] can also be synthesized in the same manner as described above.

 The calcitonin that can be used in the present invention is not limited to the above, as long as the calcitonin has an activity to reduce bone strength calcium.

According to the present invention, a human having an increased activity of glutamate oxaloacetate transaminase (GOP) and glutamate pyruvate transaminase (GPT) in serum. Or animals are provided with natural calcitonins having serum-calcium-lowering activity and their analogs having serum-calcium-lowering activity. 24140

 1 6

The administration of an effective amount of at least one calcitonin selected from the group consisting of log peptides leads to an increase in blood levels.

GOP and GPT activity levels are reduced.

 The activity value of the liver deviating enzyme present in the serum of healthy humans is generally as follows in terms of international units (IU), and is the same for animals.

 G O T About 8 to about 45 I U / β

GPT about 5 to about 40 IU / β γ — GTP about 0 to about 50.1 U / β LD Η about 50 to about 450 IU / β where GOT, GPT and y — GTP activity It is known that the value increases due to liver disease and increases from 100 OOI UZ fi or more to 300 I UZ £ or more. The hepatic departure enzyme activity in serum thus increased can be effectively reduced by administering an effective amount of calcitonin according to the present invention.

 Further, according to the present invention, at least one selected from the group consisting of natural calcitonins having serum calcium lowering activity and analogs thereof having serum calcium lowering activity in patients with liver disease. Administering an effective amount of one calcitonin can treat liver disease.

In the calcitonin used in the present invention, the karuba type Naginica nolesitin (hereinafter often referred to as "elcatonin") is used. According to the International Unit of Natural Salmon Calcitonin specified in the Medical Research Counci 1 (MRC) and WHO in the United Kingdom, lmg is set to 600,000 units, and other power On the basis of the serum calcium lowering activity (calcitonin activity) of el-pottonin, the amount of calcitonin activity equivalent to the calcitonin activity of a certain number of units of phenol-tonin is defined as that of elcatonin. Expressed as the same number of units. Therefore, the unit used for defining the dose range of calcitonin in the present invention, which will be described later, is also a unit corresponding to the calcitonin activity unit of L-potency.

 The above-mentioned calcitonins are extremely low-toxic and highly toxic.For example, L-potency is administered to mice and rats by intravenous, intramuscular, subcutaneous, or oral routes of 135,000 or 7400 units / No fatal toxicity was observed even after administration of kg (body weight). '

 In the present invention, the form of the agent for decreasing the activity of liver deviating enzyme and the therapeutic agent for treating liver disease include, for example, injections, rectal absorbents, vaginal absorbents, nasal absorbents, transdermal absorbents, pulmonary absorptions Agents, oral absorption agents, oral administration agents, etc., but the administration form of these is not particularly limited.

It is preferably used for intramuscular or intravenous administration as an injection, and rectal and vaginal absorbents are generally used in the form of suppositories, nasal absorbent and transdermal absorption The drug is used in the form of a formulation to which an appropriate absorption enhancer has been added. It is used in the form of an aerosol composition containing a suitable dispersant or water and a propellant. Oral absorbents are used in the form of, for example, sublingual tablets with the addition of an appropriate absorption enhancer, and oral dosage forms are used in the form of liposomes, microcapsules, etc. You.

 As described above, the hepatic prolapsing enzyme lowering agent and the hepatic remedy composition of the present invention are prepared into dosage forms suitable for each administration form. The carrier, diluent, or excipient used is not particularly limited.

—Generally known ones can be used. Specific examples are listed below.

 For injection, calcitonin is dissolved in distilled water for injection in which buffer, isotonicity agent and PH regulator have been dissolved in appropriate amounts, and sterilized through a sterilization filter, and then dispensed into ampoules. And can be prepared by:

 Rectal absorbers and vaginal absorbers can be used to reduce calcitonin, such as sodium pectate and sodium alginate, which have chelating ability, sodium chloride and glucose. It is prepared as a rectal / vaginal injection suppository or suppository by dissolving or dispersing it in distilled water or an oily vehicle, using a hypertonic agent as appropriate (UK Patent No. 2,920,200). No. 2 and No. 20099594).

Nasal absorbent is a liquid formulation containing calcitonin and an absorption enhancer such as water-soluble organic acids such as dalonic acid, succinic acid, and tartaric acid. Alternatively, it is prepared as a powder (JP-A-63-243303, JP-A-63-316737, JP-A-1-230530) Japanese Patent Application Laid-Open No. HEI 2-111, Japanese Patent Application Laid-Open No. H02-10431). Further, a nasal absorbent can be obtained by adding an appropriate emulsion to calcitonin (see JP-A-4-97729).

 Percutaneous absorption agents can enhance absorption through the skin by adding an absorption enhancer such as azone (Azone) to calcitonin. (Summary of the 2nd Annual Meeting of the Pharmaceutical Society of Japan, Vol. 507, 32 (1998, 878), and the method using iontophoresis [Ann.N.Y.A.cad.Sci., Vol. )] Can also be used to obtain a transdermal absorbent of calcitonin. The pulmonary absorbent comprises calcitonin, which is pulverized and ground with a dispersing agent such as Arlacel or Span 80 to obtain a homogeneous paste, and then the paste is cooled to Freon 11 or Freon 11. It can be obtained by dispersing in a propellant such as Leon 12 and filling it in a container equipped with a valve (see Japanese Patent Application Laid-Open No. 60-169224).

Oral absorbents include calcitonin, e.g., ascorbic acid, acidic amino acid, citric acid, unsaturated fatty acid, salicylic acid, etc., alone or in combination of two or more. It can be obtained as troches, sublingual tablets, powders, etc. by adding excipients such as menthol and flavoring agents such as menthol. (Showa 56-140 9 24).

 For oral administration, calcitonin is prepared, for example, by a method using WZ〇 W emulsion (see Endocrinol. Japan; Vo 1.23, 4993 (19776)). Alternatively, calcitonin can be prepared by a liposome preparation method (see Hormone Res .; Vo 1.16, 249 (1989)).

 The agent for reducing liver deviating enzyme activity and the therapeutic agent for liver disease of the present invention is administered in a single dose of 0.5 to 500,000 units of calcitonin, preferably 1 to 400 units. Is a unit. The frequency of administration may be once or twice a day, or daily or once to three times a week. Furthermore, an appropriate amount of calcitonin can be dissolved in an appropriate infusion such as Solita T-3 (manufactured by Shimizu Pharmaceutical Co., Ltd. in Japan), and the solution can be administered by intravenous drip over 1 to several hours, for example. The amount of calcitonin in the drug is determined as appropriate.

 Calcitonin as an active ingredient of the composition for reducing the activity of liver hepatic enzyme and the therapeutic agent for liver disease according to the present invention is characterized by the increased activity of liver hepatic enzyme observed in serum as shown in the following Examples. It effectively lowers the value and improves and maintains liver function disorders very well.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in further detail with reference to Examples. The present invention is not limited to these. 1

 The meanings of the abbreviations in the tables and attached figures are as follows.

 C C 1 c a b 0 n c h 1 o d e

GOT; glutamic ox aloac e t i c t r a ns a mi a n a se (gunoletamate oxaloacetate transaminase)

 G P T g 1 u aam Ρ y u v a n s a m a n a s e (glutamate pinolevate transaminase)

 L D H; l a c t a t e d e h y d r o g e n a se

 (Lactate dehydrogenase)

 γ — G T P; g a mmm a — g 1 u t a m y 1 a n s

 Pepda ses (γ—Gnoletamine minoletranspeptidase) Example 1

Liver parenchymal cells were isolated from a F344 / DuCrj-based rat (body weight: about 200 g) by collagenase perfusion, and the cells were incubated with 10% fetal serum-supplemented Williams E medium ( W illiam 's M edium E; below, "WE medium" and Les, were suspended in U), it was seeded in tissue culture di Tsu shoe at a density of 50,000 cell number / cm ^2. After 24 hours, replace with 5 mM or 7 mM carbon tetrachloride-dissolved WE medium (10% fetal serum added) and continue for 24 hours. After culturing, the level of the enzyme that escaped the liver in the supernatant was measured.

At this time, experimental groups were prepared in which L-tonin was added at various concentrations simultaneously with the addition of carbon tetrachloride or continuously 4 hours before the addition of carbon tetrachloride and after the addition of carbon tetrachloride. Phosphate-buffered saline supplemented with 0.2% serum albumin was used for dissolving and diluting L-tonin. The result was as shown in Fig. 1. In carbon tetrachloride 5 m M medium supplemented maximum inhibition of El forces Toni emissions containing solution 1 0 ^{1 1} molar in ward was simultaneously併added (0.0 0 2 units equivalent) liver departing enzyme values in the above range An effect was observed. Below this concentration, the inhibitory effect gradually diminished.

 Fig. 3 shows a micrograph (X400) showing the appearance of liver liver cells 24 hours after culture with the addition of 7 mM carbon tetrachloride.

(Without addition of L-tonin) and Fig. 4 (with L-tonin added). As a control, FIG. 2 shows a micrograph (400) showing the appearance of liver parenchymal cells cultured for 24 hours without addition of carbon tetrachloride or elcatonin. Compared to the homogenous monolayer morphology of hepatic parenchymal cells seen in the group without carbon tetrachloride (Fig. 2), the culture dish in the group with 7 mM carbon tetrachloride (without elcatonin) was compared. The cells were remarkably detached and shed (Fig. 3). And mosquitoes article al, in Gu added four after carbon tetrachloride added subsequently and yet the El force phosphatonin 1 0- ⁷ M before carbon tetrachloride added 4 hours, spalling of Itoda cells is rather suppressed authored, single The layer morphology was strongly maintained (Fig. 4). This means that L-potatonin directly acts on liver parenchymal cells to obtain resistance to hepatotoxicity and survive, and this is why elcatonin is a therapeutic agent for liver diseases. It clearly shows that it is at least a mechanism that acts as

Example 2

Liver parenchymal cells were isolated from the F344 / DuCrj-based rat (body weight: about 200 g) by collagenase perfusion and suspended in WE medium supplemented with 10% fetal calf serum. Then, the cells were seeded on a tissue culture dish at a density of 50,000 cells / cm ² . 24 hours later, carbon tetrachloride

The medium was replaced with a WE medium (containing 10% fetal bovine serum) in which 5 mM was dissolved, and the cells were further cultured for 24 hours, and the liver-leaching enzyme value in the supernatant was measured.

At this time, an experimental zone was added in which various concentrations of natural salmon calcitonin (obtained from UCB—Bioproducts S.A., Belgium; 600 units Zmg) were added simultaneously with the addition of carbon tetrachloride. . For dissolution and dilution of natural salmon calcitonin, phosphate buffered saline supplemented with 0.2% serum albumin was used. The result was as shown in Fig. 5. The effect of salmon calcitonin on the hepatic protruding enzyme level was maximal in the range of ^{10 to 11} molar concentrations or more, and the inhibitory effect gradually decreased at concentrations below this concentration.

Example 3 Distilled water for injection containing 0.1 M sodium acetate and 154 mM NaCl (adjusted to pH 5.5 by adding a small amount of hydrochloric acid). 0 0 units _/ ^/ 111) were dissolved 4 0 mg, then after through the sterilization filter one, 1 ampoule lml not a one filled, El and melt sealed a sample force preparative double down 4 0 units a sample Was obtained. Thereafter, the present injection was used in the following Examples, but the present invention is not limited thereto. Example 4

 An acute liver failure model was created by partially modifying the method of Mizoguchi et al. (Inflammation, Vol. 10, 11 5 (1990)). In other words, lmg of Propionibacterium bacterium overheat killed (manufactured by Asahi Kasei Kogyo Co., Ltd.) dissolved in physiological saline was intravenously administered to an ICR male mouse (6 to 8 weeks old). One day later, 1 μg of endotoxin (derived from a large intestinal bacterium, manufactured by Difco, USA) was administered intravenously to the mice, thereby causing acute liver failure involving immunity. The degree of acute liver failure was determined by the mortality at 6 hours and 24 hours after endotoxin administration.

At this time, an experimental group (Elcatonin group i, π and m) in which a predetermined amount of each elutonin dissolved in 0.2% serum albumin was administered immediately after administration of endotoxin, An experimental group (control group) in which 0.2% ゥ serum albumin solution was intravenously administered instead of L-Kertinin was provided. Table 1 shows the results. Table 1.Effects of intravenous administration of nirukatonin on mouse acute liver failure model

Note) *: Human conversion was calculated assuming a body weight of 50 kg.

As shown in Table 1 above, in the artificially produced mouse acute liver failure model, when 40 units g of el-pottonin was administered, the mortality rate decreased 6 hours and 24 hours after administration. A reduction effect was observed. At 80 hours after administration, 80% of the control group's mortality was 0% compared to 50% at 6 hours after administration, and 40% compared to 100% of the control group's mortality at 24 hours after administration. Even in time, a significant reduction in mortality was observed with El Powertonin. When L-unitin was administered at 120 units kg, the mortality reduction effect after 24 hours was not as pronounced as at 80 units / kg, and was particularly effective at 80 units kg. Recognized, 100 units Z kg (equivalent to 500 units in human) Could be determined to be valid.

Example 5

Elcatonin is administered once a week to three patients with NT, ET, and SO who have been diagnosed with liver disease and whose liver deviating enzyme level is higher than the normal level. Treatment was given. That is, 40 units of elkatonin was dissolved in Solita Ding-3 (manufactured by Shimizu Pharmaceutical Co., Ltd., Japan), and the solution was intravenously administered over 1 to 2 hours. After 24 hours, 48 hours, 72 hours, and one week as appropriate, serum biochemical values were measured and liver function tests were performed. As a result, Table 2 below shows the effect on patient NT.

140

2 7

 Table 2 Effect of intravenous infusion of elkatonin on NT for patients with liver disease

1

 II T 1 year old 601 sex

 !

Liver disease name Hepatitis (HBs antigen 'antibody negative / HBeS ^' antibody !! / HCV antibody negative) *

Blood biochemistry! GOT GPT LDH -GTP

Ryu (just before administration) 106 110 219 22

24 hours after administration 52 57 192 20 48 hours after administration 43 43 225 19 72 hours after administration 40 34 197 18 1 week after administration 34 31 181 17

 1

(Biochemical value unit; Uβ) Note) *: Unknown hepatitis that is neither type I nor type C viral hepatitis. As shown in Table 2, the GOT and GPT before administration of elkatonin were both 106 units / β and 110 units / が, respectively. Unit / £, 31 unit / £ improved very well. 140

 2 8

 In addition, Table 3 below shows the effects of ET on patients. Table 3 Effects of intravenous elcatonin infusion on patients with liver disease

血精中生化学 GOT GPT LDH 值（投与直前） 151 304 314 1 207 投与 24時間後 120 297 327 199

Serum biochemistry GOT GPT LDH 值 (immediately before administration) 151 304 314 1 207 24 hours after administration 120 297 327 199

 Administration 48 hours 102 272 293 197

 72 hours after administration 22 104 246 164

 Administration 1 stroke 24 80 208 145

(Biochemical value unit; U Ζ β) Note) *: Unknown hepatitis that is neither type I nor type C viral hepatitis. As shown in Table 3 above, G〇 〇 and The values of GTP and GTP were both 151 units / pound and 304 units / β, but after one week of elcatonin administration, they improved to 24 units β and 80 units β, respectively, which were very good.

 In addition, Table 4 below shows the effect in patient SΟ.

 Table 4.Effects of L-tonin intravenous drip infusion on SO in patients with liver disease Patients SO age 721 gender Male liver disease name alcoholic cirrhosis i seminal biochemistry 中 GOT GPT LDH r-GTP (immediate administration) 98 133 397 188 24 hours later 61 85 335 181

 48 hours after administration 65 81 356 202

 72 hours after administration 59 64 349 117

(Biochemical value unit; U / β) As shown in Table 4 above, GOT, GPT and γ-GTP before administration of elcatonin were all 98 units Ζ β, 133 units

/ £, 1 8 8 units, but EL power Tonin administration 7 2:00 After a while, they improved very well to 59 units / £, 64 units / β, and 117 units / β, respectively.

 Thus, after appropriate administration of elcatonin, serum biochemical values of various liver-departing enzymes were significantly improved in all cases.

BRIEF DESCRIPTION OF THE FIGURES

 Figure 1 is a graph showing the inhibitory effect of elcatonin on extracellular enzyme leakage in primary cultured rat hepatocytes damaged by carbon tetrachloride (5 mM).

 Fig. 2 is a micrograph (X400) showing the appearance of primary culture rat liver parenchymal cells that had been incubated for 24 hours without addition of carbon tetrachloride or elcatonin. is there.

 FIG. 3 is a micrograph (X400) showing the appearance of primary cultured rat hepatocytes that had been incubated with carbon tetrachloride (7 mM) for 24 hours.

Fig. 4 shows that elutin tonine 10 to ⁷ M was added in advance for 4 hours, and elutonin was continuously added after addition of carbon tetrachloride (7 mM).

Was added to 1 0- ⁷ M is 2 4 hours b Nkyube you encountered a primary cultured rat micrograph showing the appearance form of hepatocytes (X 4 0 0).

Fig. 5 is a graph showing the inhibitory effect of salmon calcitonin on extracellular enzyme leakage of primary cultured rat hepatocytes damaged by carbon tetrachloride (5 mM). Industrial applicability

 A novel use of calcitonin disclosed by the present invention is an agent for decreasing the elevated activity value of hepatic escape enzymes such as GOT and GP in serum, which is an indicator of liver damage in liver disease. Therefore, a pharmaceutical composition containing an effective amount of calcitonin for an anti-liver disease is useful for treating various liver diseases such as drug-induced hepatitis, alcoholic hepatitis, and autoimmune hepatitis.

Claims

The scope of the claims 1. Contains an effective amount of calcitonin to reduce the increased activity of glutamate oxa mouth acetic acid transaminase and glutamate pyruvate transaminase in blood. Become a reducing agent.  2. The laxative according to claim 1, which is a composition comprising said calcitonin and a pharmaceutically acceptable carrier, diluent or excipient.  3. The calcitonin is at least one kind of calcitonin selected from the group consisting of natural calcitonins having serum calcium lowering activity and analogs thereof having blood calcium lowering activity. Item 7. The reducing agent according to Item 1 or 2. 4. The reducing agent according to any one of claims 1 to 3, which is formulated so that the dose per administration is 0.5 to 5000 units as calcitonin. .  5. A therapeutic composition for liver disease, comprising an effective amount of calcitonin for anti-liver disease and a pharmaceutically acceptable carrier, diluent or excipient.  6. The calcitonin is at least one kind of calcitonin selected from the group consisting of natural calcitonins having serum calcium lowering activity and analogs thereof having serum calcium lowering activity. 6. The composition according to 5. 7. The dose per dose is 0.5 to 5 as calcitonin. The composition according to claim 5 or 6, wherein the composition is formulated so as to be 0.000 units. > 8. The method according to any one of claims 5 to 7, which is in the form of an injection, a rectal suppository, a vaginal suppository, a nasal absorbent, a transdermal absorbent, a pulmonary absorbent, an intraoral absorbent, or an oral administration preparation. Composition.  9. Administer an effective amount of calcitonin to the human body in a manner that reduces the elevated activity of serum glutamate oxa mouth transaminase and glutamate pyruvate transaminase. And a method comprising:  10. A method for treating liver disease, comprising administering an effective amount of calcitonin to a patient with liver disease.