WO1998024467A1

WO1998024467A1 - Remedies for fulminant hepatitis

Info

Publication number: WO1998024467A1
Application number: PCT/JP1997/004478
Authority: WO
Inventors: Toshikazu Nakamura; Kunio Matsumoto; Kenichiro Kosai
Original assignee: Toshikazu Nakamura; Kunio Matsumoto; Kenichiro Kosai
Priority date: 1996-12-05
Filing date: 1997-12-05
Publication date: 1998-06-11
Also published as: JPH10167982A; JP4463885B2

Abstract

Remedies for fulminant hepatitis which contain as the active ingredient HGF (Hepatocyte Growth Factor). HGF makes liver parenchyma cells to grow and inhibits apoptotic liver disorders induced by Fas antibodies. Thus, the above remedies are useful in treating and preventing fulminant hepatitis.

Description

― Description Remedy for fulminant hepatitis disease Technical field

The present invention relates to a therapeutic agent for fulminant hepatitis disease. More specifically, the present invention relates to a therapeutic agent for fulminant hepatitis disease, which contains hepatocyte growth factor (HGF) as an active ingredient and is useful for treating and preventing fulminant hepatitis. Background art

The liver is essential for living organisms with many physiological functions, such as gluconeogenesis, amino acid metabolism, lipid metabolism, plasma protein synthesis, secretion, bile production and secretion, detoxification, storage of energy sugars, and storage of vitamins. Is an organ. This organ develops hepatitis due to viral infections and prolonged alcohol consumption.

Fulminant hepatitis has a high fatality rate due to severe hepatopathy resulting from widespread necrosis and loss of hepatocytes caused by hepatitis virus and drugs. According to the diagnostic criteria of the 1st and 2nd Inuyama Symposium (August 1989, August), severe hepatitis is a hepatic coma caused by severe liver dysfunction within about 8 weeks after the onset of hepatitis. The prothrombin time is assumed to be 40% or less, including an acute type in which encephalopathy develops within 10 days after the onset and a subacute type which develops after 11 days. In the clinical picture, jaundice becomes severe within a few days after the appearance of hepatic inflammation, and consciousness is rapidly accompanied. Hemorrhagic tendency is seen from an early stage, and the liver turbid sound field is reduced. Serum total pyrilvin is often more than 1 O mg / dl, and serum transaminases show a remarkably high level at the beginning, but fall significantly within a few days. The prothrombin value shows a remarkable decrease of less than 40%. It is often associated with cerebral edema, gastrointestinal bleeding, renal failure, intravascular coagulation, and systemic infections, and these complications often cause death. The prognosis is very poor, with a survival rate of 20--30% in Japan. There is no established treatment, and systemic management is required at present. Plasma exchange, transfusion transfusion, artificial liver assist devices, and glucagon / insulin therapy are provided. Thus, it is no exaggeration to say that there is no therapeutic agent to treat this fulminant hepatitis. Plasma exchange is performed at about 80% in Japan, but there is almost no difference in the survival rate between the treated group and the non-treated group. Glucagon ♦ insulin therapy has also been administered in 90 patients, but there is no difference in the overall survival rate between the treated group and the non-treated group. Specialized amino acids are also administered in most cases, but there is little difference in survival between the two groups. In other words, 80% of patients undergoing plasma exchange, glucagon / insulin therapy, or specialty amino acid therapy, which are typical treatments in Japan during this period, are almost completely treated before such treatment is performed. This means that the survival rate has not improved, and the effect has to be very skeptical.

Fulminant hepatitis, according to the diagnostic criteria of the Inuyama Symposium of 1981, showed that hepatitis within 8 weeks of coma and prothrombin time (PT) based on severe liver dysfunction within 8 weeks Defined as cases falling to 40%. The liver is an organ with a large reserve, so if about 30% remain, it is usually well compensated. However, below that level, liver failure occurs, called liver failure.

Specifically, the functions of the liver can be broadly divided into two major functions: the function of synthesizing substances and the function of metabolizing substances. Due to the dysfunction of the function, PT is reduced as an expression of reduced synthetic ability. PT is a test that measures the function of all five clotting factors, I, II, V, VI I, and X, all of which are made in the liver. Among them, factor VII, in particular, has a short half-life, halving in approximately a few hours. That is, when the synthesis ability decreases, the factor VII decreases from the beginning, and the PT time including the factor decreases. Second, coma occurs due to a decrease in detoxification metabolic function. The reason for setting the degree to I or higher was that the degree of I or higher was significant because the subjective judgment of the attending physician was entered in the case of I degree.

Hepatitis is the predominant disease in Japan. Hepatitis includes both viral and drug-induced hepatitis. In countries other than Japan, fulminant hepatitis is considered to be a disease in the category of acute hepatitis, but in Japan, there are many HBV carriers, and Fulminant cases are rarely seen. In the case of carriers, the time of onset of hepatitis is unknown, and chronic hepatitis can occur as long as it has the virus, and it can become fulminant.

On the other hand, in countries other than Japan, such as the United Kingdom, the virus is slightly less than half and slightly more than half drug-active. Especially in the UK, fulminant hepatitis is often caused by taking the paracetamol, or acetaminophen, an analgesic and antipyretic for suicide. Usually, clinically experienced drug-induced liver injury is rather allergic hepatitis, in which case the dose is small enough to cause liver injury, and even if the causative drug is removed, the inflammation maintenance mechanism is already activated. However, liver damage cannot be terminated immediately.

It is said that there are two conditions for the mechanism by which hepatitis becomes fulminant. One is that many hepatocytes are infected. Another is that there is a very strong cytotoxic T lymphocyte immune response and vigorous hepatocyte destruction. In other words, fulminant hepatitis will not occur without two factors: many hepatocytes are infected, and the immune response is severe and hepatocyte destruction is severe. Based on this theory, the hepatitis A virus is cleared rapidly, so the elimination reaction takes place as the infection spreads to the point where it spreads, and it is eliminated without fulminating. In the case of the hepatitis C virus, infection progresses to many hepatocytes, and it does not evoke a strong host immune response. It is understood that hepatitis B is the most susceptible to fulminant disease because of the intermediate spread of the virus and a fairly strong immune response.

In 1972, a pathologist Kerr et al. Described a form of cell death characterized by "nucleus aggregation and fragmentation" and called it apoptosis. Since then, cell death has been divided into two categories: necrosis (necrosis) and apoptosis (apoptosis). Necrosis is caused by pathological and non-physiological factors such as viruses, ischemia, and toxic substances, and morphologically shows the breakdown of the cell membrane after expansion of the cytoplasm, nucleus, and mitochondria. Apoptosis, on the other hand, is a physiological death, which is thought to be caused by the cell's comprehensive judgment of information by itself and the activation of a self-death device built into the cell. And nuclear It exhibits the morphological characteristics of enrichment and fragmentation and the biochemical characteristics of DNA fragmentation. Put another way, gene-controlled death is apoptosis, and non-genetically controlled death is necrosis.

In recent years, advances in molecular biology have led to advances in the analysis of reapoptosis at the gene level, and its relationship with apoptosis diseases has also attracted attention. In the liver, the acidophilic body often seen in viral hepatitis is actually an apoptosis of hepatocytes, and the typical fragmentation of cell death in viral hepatitis, such as the piecemeal necrosis. In fact, it was shown that apoptosis is not the main component of necrosis, and that apoptosis is involved in many liver diseases. In particular, the Fas / Fas ligand system has been studied the most to induce apoptosis.In 1993, Nagata et al. Reported that intraperitoneal administration of anti-Fas antibody to mice caused extensive hepatocellular death. The authors suggested that the Fas system could cause fulminant hepatitis (Nature, 364, 806, 1993).

In any case, more than 90% of fulminant hepatitis in Japan is viral and acute, with a fatality rate of more than 50% in the acute form and more than 90% in the subacute form. Moreover, it is a disease with a poor prognosis, and elucidation of the causes and development of effective treatments are urgently needed.

From such a viewpoint, the present inventors have found that HGF has a suppressive, ameliorating or preventive effect on liver damage due to apoptosis, that is, Fas-induced apoptosis and the accompanying liver damage, and that HGF prevents fulminant hepatitis disease. It has been found useful in treatment.

The above-mentioned HGF is a protein found as a factor for growing hepatocytes in vitro (Biochem. Biophys. Res. Commun., 122, 1450, 1984, Proc. Natl. Acad. Scad. USA, 83 , 6489, 1986, FEBS Letter, 22, 31

1, 1987, Nature, 342.440, 1989, Proc. Natl. Acad. Sci. USA, 87, 3200, 1990). HGF, which was discovered as a factor that specifically promotes hepatocyte proliferation, has shown various activities in vivo, such as tissue injury healing, based on recent research results by many researchers including the present inventors. Not only as a research target but also as a therapeutic drug for humans and animals. Expectations are gathering.

It has been elucidated that HGF is mainly produced by mesenchymal cells, and it has been revealed that a so-called paracrine mechanism has been established in which HGF is supplied from neighboring cells as needed. . However, when liver and kidneys are injured, HGF production is also increased in uninjured organs such as the lungs, so it is considered that HGF is also supplied by the so-called endocrine mechanism.

Recent studies on the receptor for HGF have confirmed that the c-Met proto-oncogene encodes the HGF receptor (Bottaro et al., Science 251, 802-804). , 1991; Naldini et al., Oncogene 6, 501-

504, 1991). Disclosure of the invention

Although a great deal of knowledge has been obtained on HGF as described above, it is a new finding that HGF can suppress, improve, or prevent liver damage due to apoptosis. The present invention has been made based on such findings, and an object of the present invention is to provide a novel therapeutic agent for fulminant hepatitis disease.

That is, the present invention relates to a therapeutic agent for fulminant hepatitis disease, which comprises HGF as an active ingredient, and particularly for a fulminant hepatitis disease against fulminant hepatitis disease mainly caused by apoptosis. is there.

Another aspect of the present invention is a method for treating fulminant hepatitis disease, which comprises administering an effective amount of HGF; and use of HGF for producing a therapeutic agent for fulminant hepatitis disease. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an example of a micrograph of a liver histopathological specimen of the mouse of Test Example 1. In FIG. 1, A and B are micrographs at a magnification of 100 times, and C is a micrograph at a magnification of 200 times.

Fig. 2 shows the liver histopathological specimen of the HGF-administered group in Test Example 2. It is a microscope picture. In FIG. 2, A is a micrograph (200 × magnification) of a pathological tissue specimen of the liver of the mouse of Test No. 16, and B is a micrograph of a pathological tissue specimen of the liver of the mouse of Test No. 19 (× 1 magnification). 00 times).

FIG. 3 is a photomicrograph of a liver histopathological specimen of a saline-administered group (control) mouse (Test No. 14 mouse) in Test Example 2. In FIG. 3, A is 100 times magnification and B is 200 times magnification. BEST MODE FOR CARRYING OUT THE INVENTION

As the HGF used in the present invention, those prepared by various methods can be used as long as they are purified to the extent that they can be used as pharmaceuticals.

Various methods are known for preparing HGF. For example, extraction and purification from mammalian liver, spleen, lung, bone marrow, organs such as bone marrow, brain, kidney, placenta, blood cells such as platelets, leukocytes, plasma, serum, etc. (See FEBS Letters, 224, 311, 1987, Proc. Natl. Acad. Sci. USA, 86, 5844, 1989, etc.).

In addition, HGF can be obtained by culturing primary cultured cells or cell lines that produce HGF, and separating and purifying them from cultures (culture supernatants, cultured cells, etc.). Alternatively, a gene encoding HGF is inserted into an appropriate vector by a genetic engineering technique, inserted into an appropriate host, transformed, and the desired recombinant is obtained from the culture supernatant of the transformant. HGF can be obtained (see, for example, Nature, 342, 440, 1989, Japanese Patent Application Laid-Open No. 5-111383, Biochem. Biophys. Res, Commun., 163, 967, 1989). The above host cells are not particularly limited, and various host cells conventionally used in genetic engineering techniques, for example, Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant or animal cells, and the like can be used. You.

More specifically, as a method of extracting and purifying HGF from living tissue, for example, rats are intraperitoneally administered with carbon tetrachloride, and the liver of a rat in a hepatitis state is extracted, excised, pulverized, and crushed. Purify by standard protein purification methods such as gel column chromatography such as Sepharose and Heparin Sepharose and HPLC. be able to.

In addition, by using a gene recombination method, a gene encoding the amino acid of human HGF is incorporated into a vector such as sipper lipovirus virus DNA or the like, and the expression vector is used to express animal cells such as Chinese hamster ovary (CH0) cells. Cells, mouse C127 cells, monkey COS cells, etc. can be transformed and obtained from the culture supernatant.

In the HGF thus obtained, a part of the amino acid sequence may be deleted or replaced with another amino acid, or another amino acid sequence may be partially inserted, as long as it is substantially equivalent to HGF. One or more amino acids may be bound to the N-terminal and / or the C-terminal, or the sugar chain may be similarly deleted, substituted and / or added.

The therapeutic agent of the present invention contains the above-mentioned HGF as an active ingredient, and the HGF suppresses, improves or prevents HGF against liver damage due to apoptosis by proliferating hepatic parenchymal cells as shown in the test examples described later. Has the effect of Furthermore, since HGF does not act on unaffected tissues but acts only on impaired tissues, it has the advantage that it is less likely to cause side effects. Therefore, the therapeutic agent of the present invention is effective for the treatment and prevention of fulminant hepatitis disease, particularly a lesion mainly composed of hepatic parenchymal cell proliferation.

The therapeutic agent of the present invention is useful for the treatment and prevention of various diseases caused by liver damage in various mammals (for example, mice, pigs, pigs, sheep, dogs, cats, etc.) in addition to humans. Applied.

The therapeutic agent of the present invention can be in various formulation forms (eg, liquid, solid, capsule, etc.), but generally, the active ingredient HGF alone or an injection and an inhalant, a suppository together with a conventional carrier are used. Or oral preparation. The injection can be prepared by a conventional method. For example, after dissolving HGF in an appropriate solvent (for example, sterilized water, buffer solution, physiological saline, etc.), sterilize by filtering through a filter or the like. Then, it can be prepared by filling in a sterile container. The HGF content in the injection is usually adjusted to about 0.0002-0.2 (w / v%), preferably about 0.001-0. L (w / v%). Oral drugs include, for example, tablets, granules, powders, soft or hard capsules, solutions, emulsions, suspensions, It is formulated into a dosage form such as a lip-drop preparation, and these preparations can be prepared according to a conventional method of formulation. Suppositories can also be prepared by a conventional method using a conventional base (for example, cacao butter, laurin butter, glycerinated gelatin, macrogol, witepsol, etc.). Inhalants can also be prepared according to the conventional procedures for preparations.

The HGF content in the preparation can be appropriately adjusted depending on the dosage form, the disease to be applied, and the like.

At the time of formulation, a stabilizer is preferably added, and examples of the stabilizer include albumin, globulin, gelatin, glycine, mannitol, glucose, dextran, sorbitol, ethylene glycol and the like. Further, the preparation of the present invention may contain additives necessary for preparation, for example, excipients, dissolution aids, antioxidants, soothing agents, tonicity agents and the like. In the case of a liquid preparation, it is desirable to store it after freezing or freezing it to remove water. The freeze-dried preparation is used after reconstitution by adding distilled water for injection at the time of use.

The therapeutic agent of the present invention can be administered by an appropriate administration route according to the formulation. For example, it can be administered in the form of an injection to a vein, artery, subcutaneous, intramuscular, or the like. The dosage is adjusted appropriately according to the patient's condition, age, weight, etc., but is usually 0.05 mg-500 mg, preferably lmg-10 Onig as HGF, which is divided into one or several times a day. Or continuous administration. Industrial applicability

In the present invention, HGF, which is an active ingredient, has an inhibitory, ameliorating, or preventing action on liver damage due to apoptosis, which is a cause of fulminant hepatitis, ie, Fas-induced apoptosis and liver damage accompanying the same. Therefore, the therapeutic agent of the present invention is effective in treating or preventing the above-mentioned fulminant hepatitis disease. Furthermore, since HGF acts only on the affected tissue, it is possible to obtain a drug with few side effects. Example

Hereinafter, the present invention will be described in more detail based on experimental examples and examples, but the present invention is not limited to these examples.

Test example 1

Basic condition setting for apoptotic liver injury

In tests of liver cell death by the considered apo Bok one cis by Nagata et al., Using BALB / C mice 3-5 weeks old, Jo- 2 antibody (Fas antibody) 100! ^ And _10/6 ^ a It is administered intraperitoneally (Nature, 364, 806, 1993). In this study, the optimal antibody dose was set to determine the inhibitory effect of HGF administration on apoptotic liver injury, and basic conditions were set to minimize individual variation in apoptotic liver injury. did.

As materials, Jo-2 antibody (Fas antibody) and BALB / C mouse male (8 weeks old) were used. The method is as follows.

Jo-2 antibody dose per mouse

g intraperitoneally (ip) (2 mice), Jo-2 antibody (2 mice), 2 (3 mice), and lg (3 mice) in the tail vein (iv), and control Was administered iv (one mouse). After administration of Jo-2 antibody or saline, blood was collected from the tail vein 6 hours later, necropsy 24 hours later, and tissues were collected. GPT, TP, ALB, and GOT were measured as liver function markers, a part of the liver was fixed in 10% formalin, and liver damage was determined by H & E staining under a microscope. In addition, evaluation of apoptosis was performed. In addition, a part of the liver was frozen, DNA was extracted, and DNA fragmentation analysis was performed by electrophoresis. The analysis of DNA extraction and DNA fragmentation is as follows.

1) 20-30 mg of frozen liver B was homogenized with 2nd cell lysis buffer (320 mM Saccharose, 5 mM MgCl ₂ , 10 mM Tris-HCl, 1% Triton X-100, pH 7.5).

2) 100 µl of 20 mg / ml protease was added and incubated at 50 ° C for 2 hours. 3) RNase A was added to 200 g / ml and incubated at 50 ° C for 1 hour.

4) 500 μl was taken, precipitated with isopropanol, washed with ethanol, and dissolved in 20 μl buffer. 5) The whole volume (20 μΐ) was electrophoresed on agarose containing ethidium mouth mouth, and DΝΑ fragmentation was observed.

The results are shown in Table 1 and FIG. 1 (micrograph of pathological tissue specimen).

As shown in Table 1, administration of 5 tg i.v. or ip was optimal as a result of setting basal conditions.

Figure 1 shows an example of a micrograph of a histopathological specimen of the liver. In the figures, A and B are micrographs at a magnification of 100 times, and C is a micrograph at a magnification of 200 times. As shown in the figure, in the liver histopathology, apoptosis induced by Jo-2 antibody administration occurs from hepatocytes around the Zone U portal vein, and increases in the amount of Jo-2 antibody. Apoptosis extended from Zone 1 to Zone 2 and Zone 3 (hepatocytes around the central vein), and the degree of necrosis progressed. It was found that almost all hepatocytes in a wide area and diffusely fell into apoptotic necrosis.

table 1

J o--2 Liver tissue D Ν fragment G Ρ

(mg) * Liver damage * Atro-cis 6 h r 24 η r

1 5 i .P. + + 1 082

2 5 i • P. 9 Bad organization 1 34 256

»3 5 i. V. + + tens + + + tens extensive hepatocyte necrosis n. D. 63 8 80

4 5 i.v. Almost normal 23

5 2 i. V. +++ Relatively widespread hepatocyte necrosis 10? 28830 1 2592

6 2 i. V. Almost normal 60 1 34

7 2 i.. + + Zone 1 hepatocyte apoptosis

8 1 i.V. + + +10 Zone 1 Hepatocyte eosinophilic change 1 64 6

9 1 i.V. +/- Ten Zone 1 Hepatocyte eosinophilic change 54 1

10 1 i.V. + + + + + focal apoptotic area, macroph + 240 Evaluation of liver damage and apoptosis

Ten: Minor, Ten +: Moderate, Ten +: Serious, Ten + 10: Extremely severe

** 3 Die after 6 hours of blood collection, tissue collection.

Test example 2 —

HGF administration suppresses apoptosis liver injury

The ability to suppress liver damage caused by HGF-ca-induced apoptosis was examined in vivo.

Materials used were Jo-2 antibody (Fas antibody), BALB / C mouse male (8 weeks old) and recombinant human HGF (human recombinant HGF). The method is as follows.

Jo - As 2 antibody dose, and the mouse per 5 [mu] _beta was administered intraperitoneally (ip) (nine mice). Six hours before administration of the Jo-2 antibody, HGF (four mice) or saline (five mice) was administered ip as a control. Furthermore, ip administration of HGF or saline was continuously performed at the time of antibody administration, 6 hours after antibody administration, and 12 hours after antibody administration. Blood was collected from the tail vein 12 hours after administration of the Jo-2 antibody, and necropsy was performed 24 hours later to collect tissues. GPT was measured as a liver function marker, a part of the liver was fixed in 10% formalin, liver damage was determined by H & E staining under a microscope, and apoptosis was evaluated.

The results are shown in Table 2 and FIGS. 2 and 3 (micrographs of pathological tissue specimens). FIG. 2 is a micrograph of a histopathological specimen of the liver of a mouse in the HGF administration group. A is a micrograph of a histopathological specimen of the mouse of Test No. 16 (magnification: 200 times); 1 is a photomicrograph (magnification: × 100) of a liver histopathological specimen of the mouse of Test No. 19. Fig. 3 is a photomicrograph of a liver histopathological specimen of a mouse in the saline administration group (control) (mice in Test No. 14). is there.

As shown in Table 2, all four mice treated with HGF showed low liver function GPT. In addition, as shown in Fig. 2, liver damage due to apoptosis was clearly suppressed. On the other hand, one mouse that received saline instead of HGF died 8 hours after the administration of Jo-2 antibody, but its liver tissue was diffuse and most of the hepatocytes exhibited necrosis due to apoptosis. And died of liver failure. The remaining 4 patients also showed high liver function GPT. Furthermore, as shown in Fig. 3, from the area around the portal vein (Zone 1) to the intermediate zone (Zone 2), the hepatopathological group of marked apoptosis of hepatocytes and the resulting cell death was observed. It was accompanied by severe hepatic injury with a woven image.

From the above results, HGF was found to suppress apoptotic liver damage induced by Fas antibody.

Table 2

Test No.GPT

1 2 h r 24 h r

1 1 Saltwater death

1 2 Administration group 1 0 1 1 0 246 0

1 3 (3 nt 1 27 7 0 35 1 0

1 4 roll) 7 9 02 26 2 3

1 5 4030 1 67 7

1 6 33 4 6

1 7 HGF 30 30 3

1 8 Administration group 1 60 7 2

1 9 <1 5 1 3 8Example 1

A solution containing 1 mg of HGF, 1 g of mannitol and 10 mg of polysorbate 80 in 100 ml of physiological saline was aseptically prepared, dispensed into vials 1 ml at a time, and freeze-dried. A lyophilized preparation was obtained by sealing. Example 2

0.02 M phosphate buffer (0.15 M NaC1 and 0.01% polysorbate 80, pH 7.4) 100 mg HGF 1 mg and human serum albumin 1 in 100 ml An aqueous solution containing 00 mg was aseptically prepared, dispensed into vials 1 ml at a time, lyophilized and sealed to obtain a lyophilized formulation _:

Claims

- The scope of the claims

1. A therapeutic agent for fulminant hepatitis disease, comprising hepatocyte growth factor as an active ingredient.

2. The therapeutic agent for fulminant hepatitis disease according to claim 1, wherein the fulminant hepatitis disease is fulminant hepatitis disease mainly caused by liver damage due to apoptosis.

3. The therapeutic agent for fulminant hepatitis disease _{c according to} claim 1, wherein the HGF is human recombinant HGF.

4. A method for treating fulminant hepatitis disease, comprising administering an effective amount of HGF.

5. The method for treating fulminant hepatitis disease according to claim 4, wherein the HGF is human recombinant HGF.

6. Use of HGF for producing a therapeutic agent for fulminant hepatitis disease.

7. Use of HGF according to claim 6, wherein the HGF is human recombinant HGF.