JPH083065A - Therapeutic agent for hepatopathy - Google Patents

Abstract

PURPOSE:To obtain a therapeutic agent for hepatopathy, containing thrombomodulin as the active component, reduced in side effect and excellent in safety. CONSTITUTION:This therapeutic agent for hepatopathy contains thrombomodulin such as human-derived thrombomodulin which is a protein containing at least an amino acid sequence represented by the formula or its homologous variant as the active component. In addition, this thrombomodulin is preferably a protein having an amino acid sequence of 557 amino acids containing an intracellular domain of human thrombomodulin and the dosage is preferably within a range of 0.001 to 20mg/kg weight per day on active component base.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for liver disorders containing thrombomodulin as an active ingredient.

[0002]

2. Description of the Related Art The liver is the largest single organ in a living body and plays a central role in sugar metabolism, protein metabolism and lipid metabolism. In addition, drug metabolism, detoxification, storage of substances, protein synthesis such as blood coagulation system are important functions of the liver, which are essential organs for homeostasis and life support. These functions are physiologically understood as a result of a single action or an interaction of various cells such as liver parenchymal cells, Kupffer cells and sinusoidal endothelial cells that compose the liver. Therefore, once a virus, alcohol, or the like is invaded from the outside world, it is generally prone to serious symptoms.

The causes of liver damage are diverse, and in the case of toxic substances that directly impair the metabolism and survival of liver parenchymal cells, when they are damaged by active oxygen, lipid peroxides, etc. generated in alcohol and other metabolic processes, In some cases, microcirculatory disorders resulting from localized hypercoagulation may be the main cause of autoimmune mechanism such as hepatitis B.

Although many hepatic drugs have been used for liver damage due to such various causes, the current situation is that there is no definitive therapeutic one. This is the [Pharmacy (The Journal of Practical
Pharmacy); Vol. 41, No. 11 (1
990)].

[0005] For example, many of special composition amino acid infusions and vitamin preparations are essential nutrients lacking due to a decrease in liver metabolism.
The antidote represented by SH compounds such as glutathione and tiopronin is only questioned as an adjunct therapy to supplement vitamins and the like, and its effectiveness is questioned. In addition, drugs having a therapeutic effect on liver disorders such as corticosteroids, anti-inflammatory or immunomodulators such as glycyrrhizin and azathioprine, protein synthesis promoters represented by malotilate, and interferon having an antiviral effect are Serious side effects such as dysfunction, hypoaldosteronism, agranulation, jaundice and cardiomyopathy are known.

[0006] As described above, the drugs currently used for the purpose of treating liver damage do not sufficiently satisfy the clinician because they do not show sufficient effects and there are fears of serious side effects. .

[0007] On the other hand, thrombomodulin is a substance that specifically binds to thrombin to inhibit the procoagulant activity of thrombin and, at the same time, significantly enhances the ability of thrombin to activate protein C, and has a strong blood coagulation inhibitory effect. It has been known. Thrombomodulin has been proved by animal experiments to be effective in the treatment and prevention of diseases associated with hypercoagulability such as thrombosis and DIC [K. Gomi et al Blood 75.1396-13.
99 (1990)].

It was initially confirmed that thrombomodulin is a glycoprotein present on the vascular endothelial cell membrane,
In recent years, it has been produced using genetic engineering technology. Although the cloning of the human thrombomodulin cDNA has already been clarified (S. Ya
Mamoto et al. International Publication Number WO88 / 05033),
Thrombomodulin has N-terminal domain 1 (N-terminal domain), domain 2 (EGF domain), domain 3
(O-glycosylation siterich
Domain), domain 4 (transmembrane domain), domain 5 (intracellular domain), and the minimum active unit of thrombomodulin is the EGF domains 4, 5 and 6 in domain 2. Therefore, partial peptides of thrombomodulin are also considered to be effective in treating diseases associated with hypercoagulability such as thrombosis and DIC.

[0009]

However, no application of thrombomodulin to diseases other than diseases associated with hypercoagulability such as thrombosis and DIC has been reported yet.

[0010]

As a result of intensive studies on the treatment of liver disorders, the present inventors have surprisingly found that thrombomodulin is effective for the treatment of liver disorders. That is, when thrombomodulin was administered to a rat liver injury model in which LPS (lipopolysaccharide) was administered after experimentally excising a part of the liver, the serum hepatic deviation enzyme level, which is an index of liver injury, was marked. Significant improvement was recognized. Also, the effect was recognized in the histology.

Furthermore, when thrombomodulin was administered to a mouse liver injury model experimentally administered with heat killed bacteria and LPS, a significant improvement in survival rate was observed. These models are said to be reproducible models of the clinical picture of liver damage [Bioclinica, 9
(4), 238-242 (1994) and the Japanese Society of Gastroenterology, 83 (6), 1161-1167 (1986).
], The invention of the present therapeutic agent as an effective therapeutic agent for liver disorders was completed.

That is, the present invention is a therapeutic agent for liver disorders containing thrombomodulin as an active ingredient. In the present invention, thrombomodulin means one having an action of binding to thrombin to promote activation of protein C by thrombin, and may be, for example, human-derived thrombomodulin or a fragment thereof. A preferred example is a protein containing at least the amino acid sequence represented by SEQ ID NO: 1 or a homologous variant thereof.

Specifically, the protein consisting of the amino acid sequence of SEQ ID NO: 1 is mentioned as a preferable example, and the protein consisting of the amino acid sequence of SEQ ID NO: 2 is also mentioned as a preferable example. These proteins are soluble and are particularly preferable in the production and use of the therapeutic agent of the present invention, but if soluble, other proteins having the action of thrombomodulin are also mentioned as preferable examples.

Thrombomodulin having an amino acid sequence having no transmembrane domain (hydrophobic domain), such as SEQ ID NOs: 1 and 2, is soluble in an aqueous medium without treatment with a surfactant or the like. is there. Therefore, it is particularly preferable to use thrombomodulin having the amino acid sequence of SEQ ID NO: 1 or 2 because the operation is easy in producing and using the therapeutic agent of the present invention.

The term "soluble" as used herein means that at least the property of being soluble in a detectable concentration without the presence of a surfactant or the like when measuring the action of promoting the activation of protein C by thrombin, for example, 0 Thrombin and protein C were present in 20 mM Tris-HCl buffer of pH 7.4 containing 1 M sodium chloride, 2.5 mM calcium chloride, and 1 mg / ml serum albumin, and a sample sample was added to the thrombin to activate protein C activity. In measuring the activity of promoting activation, under these conditions, a concentration at which protein C activation is detected and the amount of thrombomodulin can be detected, for example, at least 10 ng
The property of being dissolved in a concentration of / ml or more is exemplified.

In addition, a protein having a 557 amino acid sequence including the intracellular domain of human thrombomodulin is also mentioned as a preferred example. The thrombomodulin in the present invention is not particularly limited as long as it has an action of binding to thrombin and promoting activation of protein C by thrombin, and particularly includes homologous variants having homology with the above amino acid sequence.

The homologous variant is one in which a part of the above amino acid sequence is mutated by substitution, deletion, etc. The homology of the homologous variant is usually 60% or more, preferably 80%. Above, particularly preferably 90% or more. In addition, even if these proteins contain sugar chains,
It may or may not be included, and particularly may or may not include a chondroitin sulfate sugar chain.

Thrombomodulin can be prepared by a known method, for example, [S. Yamamoto et al., International Publication Number WO88 / 0
5033] or [M. Zushi
Et al., J. Biol. Chem. 266, 19886-1
9889 (1991)], or [C. T. Esmon et al. Biol. Chem. 25
7, 859-864 (1982)].

In the present invention, liver damage is a phenomenon such as degeneration and necrosis of liver parenchymal cells as a result of damage due to some factor, and mobilization of Kupffer cells, leukocytes, etc., infiltration, activation, fibrosis and the like as a biological reaction thereto. Are collectively referred to as processes that occur individually or in combination. Further, from the course of symptoms of liver damage, it is classified into acute, subacute, chronic, fulminant, etc., but the therapeutic agent of the present invention is not particularly limited to these, but a particularly preferable therapeutic target is fulminant hepatitis. To be Fulminant hepatitis is a hepatitis that causes liver failure symptoms such as consciousness disorder due to extensive hepatocyte necrosis during the course of acute hepatitis, and is known to cause various complications.

When the therapeutic agent for liver damage of the present invention is produced, it may be prepared by mixing an effective amount of thrombomodulin with a pharmaceutically usable carrier. That is, an amount of thrombomodulin effective for treating the above-mentioned diseases can be mixed with a known appropriate amount of carrier to prepare a pharmaceutical composition suitable for effective administration to a patient. For example, when the therapeutic agent of the present invention is used as an injection, sucrose, glycerin, methylcellulose,
It can be prepared by adding a thickening agent such as carboxymethyl cellulose and a pH adjusting agent for various inorganic salts as additives.

The pharmaceutical composition of the present invention is generally used for peptide drugs, that is, parenteral administration, for example,
It is desirable to administer by intravenous administration, intramuscular administration, subcutaneous administration and the like. Oral administration, rectal administration, intranasal administration, sublingual administration and the like are also possible.

The dose of the pharmaceutical composition of the present invention varies depending on the age, body weight, degree of disease, administration route, etc. of the patient, but is generally in the range of 0.001 to 20 mg / kg, and the daily dose is 1 Dosage once or several times as needed.

[0023]

The present invention will be described in more detail with reference to the following examples and reference examples, but the present invention is not limited thereto.

Example 1 Method of Mochida et al. (Gastro
enterology 99, p771-777 (19
90)), 70% of the liver of SD rat (male) was excised, and LPS (lipopolysaccharide; D
E.F. manufactured by ifco. Coli026: B6) 200 μg /
kg and 0.4 ml of the test solution were simultaneously administered through the tail vein. The test liquid was S. Yamamoto et al., 1 mg / k of the protein produced in Example 10 and Example 20 of International Publication No. W088 / 05033 (hereinafter abbreviated as TMD123; the protein consisting of SEQ ID NO: 2 as an amino acid sequence).
g, and 3 mg / kg equivalent amount, physiological saline 0.4 ml
It was prepared by dissolving in.

Blood was collected from the lower leg vein 5 hours after the administration, and the GPT value (Glutamic Pyruv) in serum was collected.
ic Transaminese) was measured. In addition,
A group was prepared in which only saline was administered. TMD123
Regarding the 3 mg / kg administration group and the physiological saline administration group,
Liver tissue staining was performed using hematoxylin and eosin. The results are shown in Table 1.

[0026]

[Table 1]

As shown in Table 1, the rats in the TMD123-administered group were compared with those in the physiological saline-administered group in GP.
The T value was remarkably low. Further, as shown in Comparative Example 1 described below, there was no difference in GPT value between the group administered with heparin and the group administered with physiological saline. Furthermore, the results of tissue staining showed that in the physiological saline-administered group (Fig. 1), TMD12 showed liver damage accompanied by extensive necrosis of hepatocytes.
In the 3 administration groups (FIG. 2), no hepatocyte necrosis was observed and the histology was almost the same as that of normal liver. From these results,
It was shown that TMD123 is extremely useful as a therapeutic agent for liver disorders. Heparin was ineffective.

Example 2 The procedure of Example 1 was repeated, except that the test liquid was replaced with the test liquid described below. As the test liquid, M.
Zushi et al. Biol. Chem. 266,19
1 mg / kg and 3 mg / k of the protein (hereinafter abbreviated as TME456; a protein consisting of SEQ ID NO: 1 as an amino acid sequence) produced by 866-19889 (1991).
It was prepared by dissolving an equivalent amount of g in 0.4 ml of physiological saline.
The results are shown in Table 2.

[0029]

[Table 2]

As shown in Table 2, the rats in the TME456-administered group were compared with the rats in the physiological saline-administered group in GP.
The T value was remarkably low, indicating that TME456 is useful as a therapeutic agent for liver disorders.

Example 3 Method of Mizoguchi et al. [Inflammation; VOL
10, 115-118 (1990)] was partially modified and an acute liver failure lethal model was prepared as follows. That is, 1 mg dry weight of ICR male mice was used to produce Propionibacterium acnes.
Heat killed bacteria of ium acnes, ATCC 11827) was dissolved in 0.2 ml of physiological saline and injected into the tail vein.

After 7 days, LPS (lipopolysaccharide,
Manufactured by Difco, E.I. Coli 0127: BB) 1μ
What was dissolved in 0.1 ml of physiological saline and 0.1 ml of the test solution (TMD123, 3 mg / kg equivalent was dissolved in 0.1 ml physiological saline) were simultaneously injected into the tail vein for 6 hours. Then, the survival rate after 24 hours was observed.
A group in which only physiological saline was administered instead of the test liquid was provided as a control group. The results are shown in Table 3.

[0033]

[Table 3]

As shown in Table 3, the survival rate of the TMD123-administered group of mice was significantly higher than that of the physiological saline-administered group, demonstrating that TMD123 is useful as a therapeutic agent for liver disorders. It was

[Example 4] TME456, 3 m
The same procedure as in Example 3 was carried out except that the amount of g / kg equivalent was replaced with the test solution dissolved in 0.1 ml physiological saline.
The results are shown in Table 4.

[0036]

[Table 4]

As shown in Table 4, the survival rate of the TME456-administered group was significantly higher than that of the saline-administered group, demonstrating that TME456 is useful as a therapeutic agent for liver disorders. It was

Example 5 TMD123 and TME45
Six single dose toxicity studies were performed in rats (n = 5). Even at 180 mg / kg, neither male nor female died.

Comparative Example 1 Instead of TMD123, heparin, which is well known to have a blood coagulation inhibiting action, was dissolved in physiological saline (150 U / kg),
Using saline as a control, substantially the same operation as in Example 1 was performed. The results are shown in Table 5.

[0040]

[Table 5]

As shown in Table 5, there was no significant difference in GPT value between the heparin administration group and the physiological saline administration group.

[0042]

INDUSTRIAL APPLICABILITY According to the present invention, since the therapeutic agent contains thrombomodulin as an active ingredient, it can be provided as a useful therapeutic agent for liver disorders.

[0043]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 114 Sequence type: Protein sequence Asp Pro Cys Phe Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn 1 5 10 15 Gln Thr Ser Tyr Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro 20 25 30 His Glu Pro His Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro 35 40 45 Ala Asp Cys Asp Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly 50 55 60 Tyr Ile Leu Asp Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu 65 70 75 80 Asn Gly Gly Phe Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe 85 90 95 Glu Cys Ile Cys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr 100 105 110 Asp Cys

[0044]

[Sequence list]

 SEQ ID NO: 2 Sequence length: 498 Sequence type: Protein sequence Ala Pro Ala Glu Pro Gln Pro Gly Gly Ser Gln Cys Val Glu His Asp 1 5 10 15 Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln 20 25 30 Ile Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45 Ala Ala Asp Val Ile Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp Ile Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp 65 70 75 80 Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gln Trp Val Thr Gly Asp 85 90 95 Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110 Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125 Val Pro Ser Glu Pro Ile Trp Glu Glu Gln Gln Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160 Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly 165 170 175 Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro Val Gly 180 185 190 Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys Thr Ala 195 200 205 Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220 Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln 245 250 255 Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp 260 265 270 Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr 275 280 285 Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gln His Arg 290 295 300 Cys Glu Asp Val Asp Asp Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln 305 310 315 320 Arg Cys Val Asn Thr Gln Gly Gly Phe Glu Cys His Cys Tyr Pro Asn 325 330 335 Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350 Arg Ala Asn Cys Glu Tyr Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr 355 360 365 Leu Cys Val Cys Ala Glu Gly Phe Ala Pro Ile Pro His Glu Pro His 370 375 380 Arg Cys Gln Met Phe Cys Asn Gln Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400 Pro Asn Thr Gln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp 405 410 415 Asp Gly Phe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430 Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys 435 440 445 Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys Asp Ser 450 455 460 GLy Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser 465 470 475 480 Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu Val His 485 490 495 Ser Gly

[Brief description of drawings]

FIG. 1 is a micrograph of a tissue image of a physiological saline-administered group.

FIG. 2 is a micrograph of a TMD123 3 mg / kg administration group.

Claims (9)

[Claims] 1. A therapeutic agent for liver damage, which comprises thrombomodulin as an active ingredient. 2. The therapeutic agent according to claim 1, wherein thrombomodulin is a protein containing at least the amino acid sequence represented by the sequence of SEQ ID NO: 1 or a homologous variant thereof. 3. The therapeutic agent according to claim 1, wherein thrombomodulin is a protein consisting of the sequence of SEQ ID NO: 1 (the protein may contain a sugar chain). 4. The therapeutic agent according to claim 1, wherein thrombomodulin is a protein having the sequence of SEQ ID NO: 2 (the protein may contain a sugar chain). 5. The therapeutic agent according to claim 1, wherein the thrombomodulin is human-derived thrombomodulin. 6. The therapeutic agent according to claim 1, wherein the thrombomodulin is a soluble fragment of human-derived thrombomodulin, which is a peptide having an action of thrombomodulin. 7. The therapeutic agent according to claim 1, wherein thrombomodulin is a protein having an amino acid sequence of 557 including the intracellular domain of human thrombomodulin. 8. The therapeutic agent according to claim 1, which is a parenteral administration agent. 9. The therapeutic agent according to claim 1, which is a parenteral administration agent containing an effective amount of thrombomodulin and a pharmaceutically usable carrier.