JPH04117331A - Remedy for hepatitis - Google Patents

Abstract

PURPOSE:To obtain a remedy for hepatitis useful for the prevention and/or remedy of fulminant hepatitis by using urinastatin as an active compound. CONSTITUTION:The objective agent contains, as an active component, urinastatin which is an acidic glycopeptide originated from human urine, having a molecular weight of about 67,000, inhibiting various enzymes such as trypsin, alpha- chymotrypsin, hyaluronidase, granulocyte elastase and plasmin and stabilizing lysosome membrane. Urinastatin is effective in inhibiting the production and secretion of a hepatocyte-injuring factor and suppressing the necrosis of hepatocyte and acute hepatic insufficiency.

Description

[Detailed Description of the Invention] <Prior Art> Fulminant hepatitis is caused by widespread necrosis of the liver that rapidly occurs.
Hepatitis is a type of hepatitis in which symptoms of liver failure rapidly appear, accompanied by liver atrophy, progressive jaundice, and some neuropsychiatric symptoms (such as hepatic coma), and is an intractable disease with a poor prognosis and a high mortality rate.

The Ministry of Health and Welfare has designated it as a specific disease, and the fulminant hepatitis subcommittee of the Intractable Hepatitis Investigation and Research Group is conducting an investigation into its actual situation, and examining its developmental mechanism, pathophysiology, and treatment. However, in the report of this research team, no effective treatment method has been found, and no difference in survival rate etc. has been observed between the group treated with plasma exchange, interferon, etc. and the non-treated group. Hepatitis Research Group 1937
9th Research Report, 1986, pH6).

Furthermore, various treatments such as conventional plasmapheresis therapy, steroid therapy, glucagon-insulin therapy, and specially formulated amino acid infusion therapy have not improved the mortality rate of Honjo, and the survival rate is only 10 to 10%. 3
Currently, it is only 0%.

<Problems to be Solved by the Invention> In the current situation where there is no effective therapeutic agent for fulminant hepatitis, it is clinically extremely useful to develop a new effective therapeutic agent.

An object of the present invention is to provide a new therapeutic agent effective against fulminant hepatitis.

Means for Solving the Problems> In view of the current situation, the present inventor conducted various studies in order to find an effective means for treating fulminant hepatitis, which is clinically extremely serious and has a high mortality rate.

As a result, it was discovered that uriastatin (human urinary trypsin inhibitor) has the effect of suppressing hepatocyte necrosis and liver dysfunction in fulminant hepatitis and increasing the survival rate, thereby completing the present invention.

Uriastatin is a human-derived substance with extremely low toxicity and antigenicity (and is a highly safe therapeutic drug).

Uriastatin is derived from human urine and has a molecular weight of approximately 67,000.
It is an acidic glycoprotein that has inhibitory effects on various enzymes such as trypsin, α-chymotrypsin, hyaluronidase, granulocyte elastase, and plasmin, and has a stabilizing effect on lysosomal membranes. Based on these effects, it is clinically used as a treatment for acute circulatory failure (hemorrhagic shock, bacterial shock, traumatic shock, and scald shock) and acute pancreatitis (Medical History, 129.
70 (1984), History of Medicine, 129.730
(1984)).

As mentioned above, uriastatin is already known to improve liver function and deterioration of liver energy metabolism after acute circulatory failure or surgical operations such as liver resection.
This mechanism of action is explained as a secondary effect of uriastatin by improving systemic circulation insufficiency or hepatic blood flow, etc. (Clinical and research studies,
66, 2985 (1989), Modern Medicine, 42.21
90 (1987), Anesthesia, 35.1495 (1986
)).

On the other hand, the symptoms of acute liver failure in fulminant hepatitis are based on rapid widespread hepatocyte necrosis, which is caused by hepatocyte-damaging factors (hebatocytes) produced and secreted by liver tissue-infiltrating cells (macrophage cells). Toxic factor) is thought to be one of the causes, and inhibiting the production and secretion of this hepatocyte-damaging factor is thought to lead to the prevention and treatment of fulminant hepatitis, and even to an improvement in the mortality rate. The present inventors discovered for the first time that uriastatin inhibits the production and secretion of hepatocyte-damaging factors, suppresses hepatocyte necrosis, and furthermore suppresses acute liver failure. The mechanism of action of uriastatin on this fulminant hepatitis is completely different from the previously known mechanism described above, and is a finding that could not be predicted from previous reports.

Uriastatin can be prepared, for example, by the method of Prokshe (J, Lab, C11n,
Med, 79, 491 (1971)) i: According to
Uriastatin used in the present invention can be produced in the following manner, but is not limited to these production methods.

That is, after concentrating Healthy Adult Law 650f2 and dialyzing it against deionized water, the pH was adjusted with IN sodium hydroxide solution.
7,8 and then passed through a DEAE cellulose column (20×80 cm) equilibrated with 0.05 M Tris-HCl buffer (pH 7,8) to adsorb uriastatin. After washing this column with the same buffer solution 4oρ, using the same buffer solution containing 0.3M sodium chloride,
The adsorbed uriastatin was eluted, and the eluate was heated at 60° C. for 20 minutes to deactivate the contaminating protease, yielding 16 g of crude uriastatin. This crude uriastatin was adsorbed onto a DEAE cellulose column (8 x 60 cm) equilibrated with 0.02 M glycine-hydrochloric acid buffer (pH 3, 4). After sequentially washing this column with the same buffer solution 1042 and then with the same buffer solution 10ρ containing 0.2M sodium chloride,
Uriastatin was eluted with the same buffer 8β containing sodium chloride.

After concentrating the eluate by ultraconcentration, Sephadex G-1
Gel chromatography was performed using a column (10 x 95 cm) packed with 00 and physiological saline as the developing solvent.
Purified uriastatin was obtained.

Uriastatin thus obtained has a molecular weight of approximately 67.0
00, is an acidic glycoprotein with an isoelectric point pH of 2 to 3 and a sugar content of 5 to 12%, and its specific activity is about 2500 units/mg. In addition, the activity of uriastatin is determined by 2 μg of trypsin.
The amount of uriastatin that inhibits the activity of 50% is expressed as 1 unit.

Next, the effect of uriastatin on fulminant hepatitis will be explained using experimental examples.

Propionibacterium acnes (Pro) in mice
When heat-killed bacteria of ionibacterium acnes (anaerobic bacterium, hereinafter abbreviated as P, aCneS) is intravenously injected, remarkable cell infiltration is induced in the liver tissue. Additional intravenous injection of lipopolysaccharide (hereinafter abbreviated as LPS) to such mice induces extensive hepatocyte necrosis, and most mice die within 24 hours. This method is considered a good experimental model for human fulminant hepatitis because it causes acute liver failure similar to fulminant hepatitis.

Furthermore, it is believed that the mechanism of hepatitis becoming more severe cannot be fully explained by a single theory, and that various factors are involved in a complex manner. As an acute liver failure model, the mechanism of extensive hepatocyte necrosis has already been investigated in detail, and it is also used to determine the therapeutic effects of drugs (Hepatobiliary Pancreas, 17.475 (19
88) ).

This time, we used this method to examine the effects of uriastatin.

Experimental Example 1 Animal Experiment Experimental Method 1mg of P and heat-killed acne bacteria were intravenously injected into BALB/c male mice (approximately 20g of age 6), and after 7 days, 1ag of LPS was observed in the liver tissue. Acute liver failure was created by additional intravenous infusion of . Uriastatin was administered at 1,000 units/dose at the same time as LPS administration.
Mouse (experimental example n=10), 100 units/mouse (n=
20) and mice administered intravenously with 50 units/mouse (n=20) and physiological saline (control group n=40)
I compared it with.

Efficacy was determined by survival rate, serum transaminase (glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT))
, based on pathological findings of liver tissue.

Test result 1, survival rate is uriastatin 1,000 units/mouse,
In the 100 units/mouse administration group, it was significantly higher than in the control group (see Figure 1).

2. The increase in serum transaminases (GOT, GPT) was significantly suppressed by administration of uriastatin at 1,000 units/mouse (see Table 1).

3. As pathological findings of liver tissue, Figures 2a and 2
Figure b shows the results of microscopic observation (magnification: 100).

Figure 2a is a liver tissue micrograph of a group without uriastatin administration, and Figure 2b is a liver tissue micrograph of a group treated with 1,000 units of uriastatin/
It is a microscopic photograph of liver tissue of a mouse administration group.

Intense and widespread hepatocyte necrosis was observed in the non-administered group (see Figure 2a), but almost no hepatocyte necrosis was observed in the uriastatin 1,000 units/mouse administered group (see Figure 2b).

Experimental Example 2 In vitro experiment Experimental method BALB/c male mice (6 exercise 20g) were given P,
Heat-killed P. acnes bacteria were intravenously injected, and 7 days later, infiltrated cells were collected from the liver. Adding LPS to the infiltrating cells,
After culturing for 48 hours, the culture supernatant was collected. This supernatant was allowed to act on isolated normal mouse hepatocytes, and further 3H-
Leucine was added and 3H-leucine uptake was measured.

It is known that inhibition of its uptake, that is, inhibition of protein synthesis ability, is due to hepatocyte-damaging factors produced and secreted into the culture supernatant.

This hepatocyte-damaging factor is a proteinaceous substance with a molecular weight of 10 to 40 Daltons, and is unstable to heat, so it is clearly different from TNF, a known cell-damaging factor. 83.1161 (1986)).

In this measurement system, uriastatin 5 was added when LPS was added.
0 units/m1 (n=10) or 100 units/m1 (n
= 10) was added to examine the effect of uriastatin on the production and secretion of hepatocyte-damaging factors. The results are shown in Figure 3.

In the uriastatin non-added group (n=10), LP
The protein synthesis ability of hepatocytes was significantly suppressed by hepatocyte-damaging factors produced and secreted by the addition of S. On the other hand, in the uriastatin addition group, this suppression was markedly improved by the addition of uriastatin (see Figure 3).

The above results indicate that uriastatin suppressed the production and secretion of hepatocyte-damaging factors by LPS from liver tissue-infiltrating cells 7 days after administration of heat-killed P. acnes bacteria. In other words, the acute liver failure-improving effect of uriastatin is thought to be due to suppression of production and secretion of hepatocyte-damaging factors.

There is a report that hepatocyte-damaging factors are inactivated by trypsin (Nikko Shi, 83.1161 (1986)).
As shown in Experimental Example 1, Lika and uriastatin are effective against fulminant hepatitis.

Experimental Example 3 Acute Toxicity 1 group of 10 ddY male mice (weight 20-22 g)
Uriastatin 4 g/kg was administered intravenously or intraperitoneally, and symptoms and weight changes were observed for 1 week. Body weight changes during the observation period were similar to those in the control group, and no deaths were observed.

Uriastatin is recommended from the time when hepatitis is suspected to become fulminant.
Administer daily until liver function such as prothrombin time improves.

The appropriate daily dosage of uriastatin is 150,000 to 5,000,000 units, preferably 30,000 to 3,000,000 units.

Uriastatin may be used as a prophylactic agent for fulminant hepatitis. In this case, it may be used when the onset of fulminant hepatitis is predicted.

Uriastatin is administered after being dissolved in an appropriate infusion solution.

Further, the dosage can be increased or decreased as appropriate depending on the symptoms or usage.

The therapeutic agent for hepatitis of the present invention is preferably administered intravenously as an injection or infusion.

As an injection, it is preferable to use a lyophilized preparation that is dissolved before use.

These preparations can be prepared by conventional methods using conventional pharmaceutical carriers or excipients.

Example 1 40 g of lyophilized Uriastatin was dissolved in 2000 ml of physiological saline and aseptically filtered using a membrane filter. The filtrate was filled into sterilized glass containers at 1 mρ portions and freeze-dried by a conventional method to obtain a freeze-dried preparation.

[Brief explanation of drawings]

FIG. 1 is a graph showing the effect of uriastatin on the mortality rate of acute liver failure model mice. Figures 2a and 2b are photographs substituted for drawings showing the morphology of organisms, and are microscopic photographs showing the effect on pathological findings of liver tissue of acute liver failure model mice. FIG. 3 is a graph showing the inhibitory effect of uriastatin on the production of hepatocyte-damaging factors.

Claims (1)

[Claims] (1) A hepatitis therapeutic agent containing uriastatin as an active ingredient and used for the prevention and/or treatment of fulminant hepatitis.