WO2002072131A1

WO2002072131A1 - Remedies for liver diseases

Info

Publication number: WO2002072131A1
Application number: PCT/JP2002/001216
Authority: WO
Inventors: Sotaro Mushiake; Hiroki Kondou
Original assignee: Ajinomoto Co., Inc.
Priority date: 2001-03-12
Filing date: 2002-02-14
Publication date: 2002-09-19
Also published as: JPWO2002072131A1

Abstract

LSKL (Leu-Ser-Lys-Leu) or its salt are used as the active ingredient of drugs efficacious in, for example, treating, ameliorating or preventing liver diseases. These compounds are particularly useful as drugs whereby liver parenchymal cells can be prevented from damages and fibrosis. A method of treating damages in the above liver diseases, etc. is also provided.

Description

Specification

Technical field

The present invention relates to a novel liver disease drug containing LSKL (Leu-Ser-Lys-Leu) or a salt thereof as an active ingredient. It can be used as a drug for treatment, improvement, prevention of progress and prevention of various liver diseases (liver disease). Furthermore, the present invention also relates to treatment methods such as treatment, improvement, prevention of progress and prevention of liver diseases, and the use of specific active ingredients (the above-mentioned active ingredients) as drugs for liver diseases. Background art

There are various causes of hepatic diseases such as viral hepatitis, alcoholic hepatitis, non-alcoholic non-viral hepatitis, and other inflammatory liver diseases, and biliary atresia such as biliary atresia. Hepatic parenchymal cells are damaged, causing liver fibrosis, resulting in portal vein hypertension, which leads to cirrhosis and liver failure.

At present, there are no selective drugs that can suppress hepatic parenchymal cell damage and fibrosis. Regarding fibrosis, it has been shown that TGF plays an important role in inducing fibrosis. TGF] 3 is secreted as inactive latent TGFβ and accumulated in the extracellular matrix. Therefore, it is considered that the regulation of TGF activity is mainly in the process of conversion from inactive latent TGF | 3 to active form. Latent TGF iS is known to be converted to the active form by proteolytic enzymes such as plasmin, active oxygen, integrins, thrombospondin-1 (thr.mb. spndin-1; TSP-1), etc. However, the activation molecule under physiological conditions has not been determined.

KRFK (Lys-Arg-Phe-Lys), a partial structure of TSP-1, interacts with LSKL of the N-terminal precursor peptide (LAP), which is a part of latent TGF] 3. As a result, the structural change of LAP occurs and is cleaved from latent TGF] 3 to become activated TGF / 3. It has also been shown that LSKL, a partial structure of LAP, competes with TSP-1 and inhibits conversion of latent TGF iS to activated TGF. Crawford et al. Using TGF) 81 and TSP-1 knockout mice, that TSP-1 is an important TGF j3 activator in vivo It has been shown to produce histological changes in the kidney and bronchus similar to those in quat mice (see Cell, 93, 1159-1170, 1998).

In contrast, Abdelouahed et al. Show that TSP-1 is not an activator of TGF) 3 in platelets (see J, Bilo. Chem., 275, 17933-17936, 2000), and TSP-1. It has been controversial whether the regulation of TGF / 3 by physiology is universal in all organs.

Under such circumstances, development of an excellent drug for liver disease is required. Problems of the Invention

The problem to be solved by the present invention is to provide an excellent drug (including a prophylactic agent) applicable to various liver diseases, in particular, hepatic fibrosis or hepatic parenchymal cell disorder that leads to such hepatic fibrosis. The aim is to develop drugs that can be controlled. Disclosure of the invention

The present inventors have proposed that the above-mentioned tetrapeptide LSKL (hereinafter also referred to as “LSKL peptide”) has a fibrosis-suppressing action in the liver as well as a suppression of hepatic parenchymal cell degeneration and death, ie, an inhibitory action and a protective action. The inventors have found that the present invention has been completed. That is, the present invention is directed to a liver disease drug, preferably a drug for suppressing hepatic parenchymal cell damage, more preferably a drug for suppressing hepatic fibrosis, which is characterized by containing the LSKL peptide or a salt thereof as an active ingredient. As the above-mentioned active ingredient, a free form of LSKL and a salt can be used in combination, and this content is naturally included in the present invention.

LSKL stands for Leucyl-Seryl-Lysyl-Leu (Leu-Ser-Lys-Leu) and can be used in the present invention in the form of its salt (a pharmaceutically acceptable salt). The amino acid constituting this tetrapeptide is preferably an IL-form which can be used for both L-form and D-form, and more preferably LSKL which is composed entirely of L-form amino acids. .

LSKL or its salt is used as an active ingredient in Honmei, but examples of salt include Examples of such salts include salts with organic or inorganic acids such as hydrochloric acid, sulfuric acid, and oxalic acid, and salts with pharmaceutically acceptable salts such as salts with organic and inorganic bases such as triethylammonium salt and sodium salt.

In the present invention, a liver disease drug is a liver disease or a drug used to prevent a liver disease, and more specifically, a drug used to treat, improve, prevent progression, prevent, or prevent liver disease. Is included.

Another aspect of the present invention is a method for treating a liver disease, which is characterized by administering LSKL or a salt thereof to a living body, such as treatment, amelioration, prevention of progression, and prevention of a liver disease. Method of treatment, improvement or prevention of "." Furthermore, the present invention also provides a method for suppressing hepatic parenchymal cell damage or a method for suppressing hepatic fibrosis characterized by administering LSKL or a salt thereof to a living body.

Similarly, a free form of LSKL and a salt can be administered in combination, and this content is, of course, included in these inventions.

In the above-mentioned administration form, the above-mentioned liver disease drug (including the above-mentioned hepatic parenchymal cell damage inhibitor and hepatic fibrosis inhibitor) can be adopted.

The present invention also includes, as yet another form, the use of LSKL or a salt thereof for a drug for liver disease.

Similarly, a free form of LSKL and a salt thereof can be used in combination, and this content is naturally included in the present invention. The drug for the liver disease (including the drug for inhibiting parenchymal cell damage and the drug for suppressing liver fibrosis) is as described above. BRIEF DESCRIPTION OF THE FIGURES

[Figure 1 ]

3 illustrates the effect of inhibiting hepatocyte degeneration and death obtained in the examples. The left side shows the results of the control group (physiological saline), and the right side shows the results of the LSKL-administered group (p <0.01).

DMN: dimethylnitrosamine; Saline: saline.

[Figure 2 ]

3 illustrates the effect of suppressing fibrosis obtained in the examples. The control group (physiological saline) is shown on the left side of the figure, and the LSKL administration group is shown on the right side.

D 丽: dimethylnitrosamine; Saline: physiological saline.

[Fig. 3]

1 is a diagram illustrating a change in body weight of an experimental animal SD rat obtained in an example. ■: LSKL; Hata: Saline.

The vertical axis: body weight (BW) (g); the horizontal axis: the number of days passed from the start of drug administration (Day).

[Fig. 4]

Fig. 2 shows a survival curve of an experimental animal SD rat obtained in the example.騸: LSKL; ·: Saline.

The vertical axis: survival rate (%); the horizontal axis: days elapsed from the drug administration start date (Day). Embodiment

Hereinafter, embodiments of the present invention will be described.

The subject to which the liver disease drug of the present invention is administered is not particularly limited as long as it seeks treatment, improvement, progress prevention, prevention, etc. of the liver disease, but is applicable to mammals, usually humans (patients). Is done.

There is no particular limitation on the type of liver disease to which the agent of the present invention is applied. Representative liver diseases include viral hepatitis, alcoholic hepatitis, non-alcoholic nonviral hepatitis, inflammatory liver diseases such as autoimmune hepatitis, biliary atresia and other cholestatic liver diseases, cirrhosis, and liver failure And the like. In particular, hepatic parenchymal cells are damaged for various reasons, resulting in and without cause fibrosis of the liver, resulting in portal vein pressure injuries, formation of varicose veins, cirrhosis, and liver failure It can be preferably applied to a liver disease that is likely to be transferred.

The LSKL peptide used in the present invention is known (see WO95 / 05191, etc.), and can be easily prepared based on a known method. For example, the LSKL peptide is prepared based on a production example described later. You can also.

The administration form of the drug of the present invention is not particularly limited. Therefore, various administration forms such as oral administration and parenteral administration (such as intravenous administration) can be adopted. The components are obtained as described above, but parenteral administration such as injection is preferred. The dosage of the drug of the present invention is appropriately selected according to the type of liver disease, the degree of symptoms, the form of the preparation, and the like. You. For example, when the active ingredient LSKL peptide is administered by injection, the dose of the LSKL peptide to a patient per day is about 0.01 to 1 OOO mg, more preferably about 0.05 to 50 mg. More preferably, 0.1 to: about L 0 O mg can be used. In severe cases, the dose can be increased further. The number and timing of administration can be once every few days or once a day, but it is usually several times a day, for example, divided into 2 to 4 times, or mixed and administered by infusion, etc. Can be administered.

On the other hand, in the case of oral administration, it can be selected and administered on the basis of a dosage about 10 to 20 times that of the above injection administration.

In the present invention, it can be used, for example, in combination or in combination with other drug components (pharmaceutically active substances). In such a case, the active ingredient intended in the present invention is contained and Any drug exhibiting pharmacological activity is included in the liver disease drug of the present invention.

In addition, it may also contain various pharmacologically acceptable substances for pharmaceuticals (as adjuvants, etc.). Pharmaceutical substances can be appropriately selected according to the dosage form of the preparation, for example, excipients, diluents, additives, disintegrants, binders, coating agents, lubricants, lubricants, lubricants, Flavoring agents, sweetening agents, solubilizing agents and the like can be mentioned. Furthermore, specific examples of substances for preparation include magnesium carbonate, titanium dioxide, lactose, mannitol and other saccharides, talc, milk protein, gelatin, starch, cellulose and derivatives thereof, animal and vegetable oils, polyethylene glycol / And solvents such as sterile water and monohydric or polyhydric alcohols such as glycerol.

The drug of the present invention can be prepared in various pharmaceutical forms known or developed in the future as described above, for example, various administration forms such as oral administration, intraperitoneal administration, transdermal administration, and inhalation administration. it can. In order to prepare the drug of the present invention in the form of these various pharmaceutical preparations, known or future-developed methods can be appropriately employed.

These various forms of pharmaceutical preparations include, for example, appropriate solid or liquid preparation forms, Examples include granules, powders, coated tablets, tablets, (micro) capsules, suppositories, syrups, juices, suspensions, emulsions, drops, injectable solutions, products which prolong the release of active substances, etc. Can be.

It is natural that the drug of the present invention in the preparation form exemplified above should contain an effective amount of LSL of the above-mentioned component in order to exhibit a medicinal effect. Can be incorporated into the drug.

Even when LSKL salt is used as the active ingredient, or when a free form of LSKL and its salt are used in combination, etc., based on the above description of LSKL, use of known formulation technology Necessary preparations can be prepared according to various dosage forms. As described above, the present invention provides, as another form, a method for treating, ameliorating or preventing a liver disease, which is characterized by administering LSKL or a salt thereof to a living body, a method for suppressing hepatic parenchymal cell damage, or liver fibrosis. The present invention also relates to a method for inhibiting the disease and, as yet another form, the use of LSKL or a salt thereof for a liver disease drug.

All of these inventions are based on the description of the liver disease drug (including the hepatic parenchymal cell damage inhibitor and the hepatic fibrosis inhibitor), and based on the examples described below and the like, if necessary. It can be easily implemented by referring to technology

Preferred embodiment

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited by these Examples.

(Production example) Production of LSKL peptide

(Synthesis of protective peptides)

Using an automated peptide synthesizer (ABI433 model) manufactured by PE Applied Biosystems, the peptide chain was extended from the C-terminal by sequential Fmoc solid phase synthesis according to the program, and the desired protective peptide was synthesized. A resin was synthesized.

As the starting amino acid resin carrier, HMP-resin (lmmol, Wang-resin) was used, and ordinary Fmoc amino acid derivatives such as Leu, Ser (tBu), Lys (Boc) were used. The amino acids used here were all in the L-form. After the construction of the peptide on the resin was completed, the protected peptide resin was dried and subsequently subjected to deprotection.

(Cut and deprotection from solid phase resin)

Deprotection of the resulting protected peptide and cleavage of the peptide from the resin carrier were carried out by trifluoroacetic acid treatment. The crude peptide was precipitated with isopropyl ether, extracted with 0.1% aqueous trifluoroacetic acid, and the resin was removed by filtration to obtain a lyophilized product.

(Purification by RP-HPLC)

Subsequently, the crude peptide was subjected to reversed-phase high-performance chromatography (Shimadzu Corporation, preparative instrument model LC 8A, column used 30X250imn, YMC SH-363-5s-5120A 0DS) to give 0.1% trifluoroacetic acid in acetonitrile. The desired title purified peptide trifluoroacetate was obtained as a lyophilized powder by preparative purification using this system.

Thereafter, the mixture was treated with an acetate-type Muromac, and then converted to an acetate-type by salt exchange to prepare the title LSKL peptide shown in SEQ ID NO: 1 in the sequence listing.

(Example 1)

The LSKL peptide prepared in the above Production Example was evaluated.

Four-week-old 70-80 g SD rats (purchased from Japan SLC) were assigned to physiological saline and LSKL peptide administration groups. 1. /. Dimethylnitrosamine (purchased from Wako Pure Chemical Industries, Ltd.) diluted in physiological saline to obtain a concentration was administered intraperitoneally for 3 days / week for 3 days / week for 4 weeks.

From the first administration of the dimethylnitrosamine, physiological saline and 100 μg of LSKL peptide / rat were intraperitoneally administered for 28 days every day for each group. The body weight and animal condition were observed daily, and on the 28th day after administration, the animals were sacrificed under ether anesthesia, and liver tissues were collected.

After weighing the liver, the liver was fixed in formalin and stained with hematoxylin-eosin and azan. The degree of fibrosis of the organ was examined by microscopic examination of azan-stained tissue sections, indicated by an HM score (hepatitis activity index score), and evaluated based on the following four-grade criteria.

A: No fibrosis, score 0; B: fibrous enlargement of portal zone, score 1;

c: cross-linked fibrosis (pg vein-portal vein or portal vein-central vein), score 3; and

D: cirrhosis, score 4.

The score between the score 1 and score 3 or a transitional type is defined as score 2. Degeneration in the hepatic lobule. ). Specifically, the evaluation was made based on the following four criteria.

A: None (no degeneration, no necrosis), score 0;

B: Mild (1 to 3 lobules or nodules, eosinophils, hepatocellular swelling, sporadic hepatocellular death), score 1;

C: moderate (1/3 of the lobules or nodules to 2/3 of eosinophils, hepatocellular swelling, diffuse hepatocellular necrosis), score 3; and

D: High (eosinophilic, hepatocellular swelling, sporadic hepatocellular necrosis in 2Z3 or more of lobules or nodules), score 4.

In addition, the finding which is considered to be an intermediate or transitional form between the score 1 and the score 3 is defined as the score 2. The above results are shown in FIG. 1 (the effect of suppressing hepatocellular degeneration and death) and FIG.

As is evident from the results in FIG. 1, the LSKL peptide-administered group had a significantly lower hepatocyte degeneration necrosis score than the saline-administered group. (P 0.01). Furthermore, as is clear from the results of FIG. 2, suppression of the liver fibrillation score was observed in the LSKL peptide-administered group.

In the LSKL peptide administration group, an increase in weight gain (see FIG. 3) and an improvement in the survival rate were observed as compared with the physiological saline administration group (see FIG. 4).

Administration of LSKL peptide inhibited the degeneration and necrosis of liver parenchymal cells and fibrosis in a dimethylnitrosamine-induced liver injury fibrosis model. In addition, it is thought that the result led to improvement of general condition (increase in body weight and increase in survival rate). As is clear from the above results, it is understood that LSKL peptide is extremely superior as a drug for liver disease. The invention's effect

According to the present invention, an excellent drug for liver disease using the active ingredient can be provided. In particular, it is useful as a drug capable of suppressing hepatic parenchymal cell damage and fibrosis. Further, the active ingredient is preferably administered in the form of the above-mentioned drug, and the above-mentioned disorder such as liver disease can be treated.

Therefore, the present invention is extremely useful industrially, particularly in the fields of medicine, pharmaceuticals and the like.

Claims

The scope of the claims

1. A liver disease drug characterized by containing LSKL or a salt thereof as an active ingredient

2. The drug for liver disease according to claim 1, which is a drug for suppressing hepatic parenchymal cell damage.

3. The liver disease drug according to claim 1, which is a liver fibrosis inhibitor.

4. The liver disease drug according to claim 1, wherein the amino acids constituting LSKL are all L-forms.

5. The liver disease drug according to any one of claims 1 to 4, which is for parenteral administration.

6. A method for treating, ameliorating, or preventing liver disease, which comprises administering LSKL or a salt thereof to a living body.

7. A method for suppressing hepatic parenchymal cell damage, which comprises administering LSKL or a salt thereof to a living body.

8. A method for suppressing liver fibrosis, which comprises administering LSKL or a salt thereof to a living body.

9. The method according to any one of claims 6 to 8, wherein the administered form is the liver disease drug according to any one of claims 1 to 5.

10. Use of LSKL or a salt thereof for liver disease drugs.

11. The use according to claim 10, wherein the liver disease drug is any one of claims 1 to 5.