JPH05194256A - Acute stage protein inducing agent containing il-6 as active ingredient - Google Patents

Abstract

PURPOSE:To obtain a subject agent containing interleukin-6 as an active ingredient, capable of inducing production of an acute stage protein to livestock, especially cow and useful for treatment of cancer or infectious disease. CONSTITUTION:The objective agent contains a human interleukin-6(IL-6) as an active ingredient. The agent is preferably administrated by subcutaneous injection, intravenous injection or sustained release administration carried out by embedding an osmotic pressure pump into skin and the dose is 1-100mug/kg livestock. The acute stage protein is a protein increasing in blood according to inflammatory change including infection and includes fibrinogen, etc., and has inhibiting action of protease, hemagglutination inhibiting action, action for recovering lowering of activity of helper T cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a livestock acute-phase protein inducer containing IL-6 as an active ingredient, and more particularly to an acute-phase protein inducer for cattle.

[0002]

2. Description of the Related Art IL-6 (interleukin-6) is
It is a cytokine that plays an important role in biological defense systems such as immunity, hematopoiesis, and inflammation, and is widely involved in the proliferation and differentiation of various cells in the body. IL-6 has been found in various mammals, and the amino acid primary structure of human, mouse, and rat IL-6 has already been determined (Hirano, Inte.
rnational Journal of Cell Cloning, 9, p166, 1991
Year).

It has been known that human IL-6 has an action of enhancing antibody production, an action of increasing platelets, an action of inducing protein in the acute phase, and the like in humans. Drugs for treating infectious diseases have been proposed.

Such therapeutic agents for infectious diseases are not only effective for humans, but also for livestock such as cattle, which have conventionally been protected against infectious diseases by mixing antibiotics in feed. Is also considered effective.

[0005]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
Whether L-6 is present is unknown, and IL-6 is isolated and purified to determine its structure or to determine the recombinant form due to the delay of biochemical or immunological studies using the cells or tissues. Is very difficult to manufacture.

[0006]

Means for Solving the Problems The present inventors have surprisingly found that human IL-6 has a property of inducing the production of bovine acute phase protein, and completed the present invention. ..

That is, the present invention is based on the fact that human IL-6 has the property of inducing the production of acute phase proteins in livestock such as cattle. Acute veterinary animals containing human IL-6 as an active ingredient. It is a phase protein inducer. The present invention will be described in detail below.

IL-6 may be naturally-occurring naturally-occurring IL-6 as long as it is human IL-6, or genetically engineered recombinant human IL-6. Hereinafter, in the present specification, human IL-6 is simply referred to as IL-6.

Native IL-6 is more specifically a polypeptide composed of 184 amino acid residues. In the present invention, in addition to natural or recombinant IL-6 consisting of the 184 amino acid residues, a part thereof, particularly N
It may be an IL-6 derivative in which an amino acid residue near the terminal or C-terminal is deleted, substituted with another amino acid residue, or a new amino acid residue is inserted.

Particularly in the recombinant IL-6,
Any of those having no sugar chain and those having a sugar chain bound to a different type or different site from natural IL-6 may be used.

As described above, natural or recombinant IL-6 can be used in the present invention, but it is preferable to use the recombinant IL-6 which can be easily mass-produced. Recombinant IL-6 can be produced, for example, according to the method of Yasukawa et al. (K. Yasukawa et al., Biotech. Lett., 12, p419, 1990).

The acute phase protein whose production is induced by the acute phase protein inducer of the present invention is a protein that increases in blood in response to inflammatory changes including infection, and more specifically, C
Examples include RP (C-reactive protein) and fibrinogen. Such acute phase proteins prevent or treat cancer and infectious diseases through various functions such as protease inhibitory action, hemagglutination inhibitory action, helper T cell activity reduction and recovery action. ..

The acute phase protein inducer of the present invention may be administered to livestock so that the active ingredient, IL-6, does not lose its physiological activity. For example, subcutaneous injection, intravenous injection, or sustained release administration in which an osmotic pump is embedded under the skin is preferable. Although the dose varies depending on the type and age of livestock, the size (body weight), and the activity (specific activity) of IL-6 contained as an active ingredient, the findings of the present inventors indicate that the livestock to be administered About 1 μg to 100 μg per kg body weight of
The livestock to be administered are horses, sheep, goats, etc., but administration to cattle is most suitable.

The acute phase protein inducer of the present invention can exert its effect even if it is administered only once, but it is preferably administered continuously.

The acute phase protein inducer of the present invention is IL-6.
Can be dissolved in an ordinary carrier solution for injection such as a physiological saline solution, sterilized by filtration, etc., and then lyophilized if necessary. Just before use, the freeze-dried solution may be redissolved in physiological saline, isotonic solution such as about 5% glucose solution, Ringer's solution or the like before use.

[0016]

EXAMPLES Examples will be described below in order to explain the present invention in more detail, but the present invention is not limited to these examples.

Example 1 Administration of Recombinant IL-6 to Cows Recombinant IL-6-PBS solution (hereinafter simply referred to as IL-6 solution) was administered to 6 cows (Holstein breed, body weight 115-180 kg). ..

Recombinant IL-6 was produced according to the method of Yasukawa et al. (K. Yasukawa et al., Biotech. Lett., 12.p419. 1990) using Escherichia coli as a host. The recombinant IL-6 was administered by intravenous injection into the jugular vein or subcutaneously embedded osmotic pump (manufactured by Alzet). When implanting the osmotic pump, first, 0.15 mg / kg of serratactle (made by Bayern) was administered as a sedative, and 10 ml of procaine (made by Yamaguchi Pharmaceutical Co., Ltd.) was administered as an anesthetic to the relevant local area. went.

The dose of the IL-6 solution is 2 ml of a solution prepared at 1.25 mg / ml with a PBS solution for administration into the jugular vein, and 2 ml of a solution prepared at 1.25 mg / ml with a PBS solution for administration by an osmotic pump. By injecting 1.7 ml into the pump,
0.6 mg IL-6 was administered daily for 7 consecutive days.
On the other hand, in order to confirm the effect of IL-6, the same amount of PBS was used.
Was also administered.

Example 2 Measurement of Fibrinogen Concentration and Haptoglobin Concentration in Blood Before and after administration, blood was collected from cows administered with recombinant IL-6, and the fibrinogen concentration and haptoglobin concentration were measured by the thrombin time method. (Kanai et al., Clinical Laboratory Method Proposal, 28th Edition, 1978) and simple radial immunodiffusion method (Right field et al., Immunological Experimental Procedures, p.
6 years). The blood collection after IL-6 administration is
The first time was performed 4 hours after the start of administration, and the second time and thereafter were performed 1 day after the previous blood collection.

The results of administration of IL-6 are shown in FIGS. 1 and 2.
Shown in. According to FIG. 1, when the IL-6 solution was directly administered to the subject (Cont1,2) and the jugular vein (IV), no increase in fibrinogen in the blood was observed, but the low concentration I
It can be seen that when L-6 was gradually administered (PF-1 to 3), the fibrinogen in the blood significantly increased. Further, from FIG. 2, in the subject (Cont 1, 2), there was no change in the haptoglobin concentration in blood during the experiment.
It can be seen that the haptoglobin concentration increased significantly when -6 was administered (IV, PF-1 to 3). Further, it can be seen that the case where the low concentration IL-6 is gradually administered (PF-1 to 3) is more effective than the case where the IL-6 solution is directly administered to the jugular vein (IV). ..

On the other hand, the immune reaction such as anaphylaxis which could be observed when the protein derived from different species was administered to the animal was not observed during the experimental period.

[0023]

According to the present invention, production of acute phase proteins such as fibrinogen and haptoglobin can be induced in livestock, particularly cattle. It has not been previously known that human IL-6 has such an effect on livestock such as cows, and that cows etc. have IL-6 or cytokines equivalent thereto. unknown. Therefore, the present invention is a new finding on human IL-6 and, at the same time, an effective drug in the fields such as dairy farming.

The acute phase protein is a protein effective for preventing or treating infectious diseases. Therefore, according to the present invention, it is possible to protect or treat livestock such as cattle against infectious diseases. This means that, for example, without mixing antibiotics, etc., which have a problem in bioaccumulation in the feed,
It has been shown that infectious diseases can be prevented or treated by giving IL-6.

IL-6 has a short half-life in blood because it is affected by various enzymes existing in blood. This means that, unlike antibiotics and the like, it has a low bioaccumulative potential and is suitable for administration to meat and livestock such as cattle.

The inducer of the present invention is not effective per se for the prevention and treatment of infectious diseases, but achieves the prevention and treatment of infectious diseases by increasing the resistance and healing power of livestock itself. Is. Therefore, unlike ordinary drugs, it is considered that there is little risk that the physical strength of livestock will decline due to overdose.

[Brief description of drawings]

FIG. 1 shows the change in fibrinogen concentration when human IL-6 was administered to cattle over time in Example 2. The vertical axis represents the fibrinogen concentration (mg / dl) in 100 ml of bovine blood, and the horizontal axis represents the elapsed time. On the horizontal axis, -1d is the result of the day before the administration of human IL-6, 4h is the result 4 hours after the administration of human IL-6, and the following is 1d, 2d, ... 14d. , Human I
The results are shown 1, 2 ... 14 days after L-6 administration.
In the figure, open squares are cont-1, that is, PB containing no IL-6.
The S-solution-administered cow (No. 1) is shown, and the white circle indicates cont-2, that is, the bovine (No. 1) administered with the PBS solution containing no IL-6.
2), the open diamond indicates IV, that is, the cow that was administered the IL-6 solution in the jugular vein, and the black square is PF-1, that is, the cow that was continuously administered the IL-6 solution by the pump (No. 1). ), A black circle represents PF-2, that is, a cow (No. 2) that was continuously administered an IL-6 solution by a pump, and a black triangle represents PF-3, that is, a IL-6 solution by a pump continuously. The cow (No. 3) that was administered is shown.

FIG. 2 shows changes in haptoglobin concentration over time when human IL-6 was administered to cattle in Example 2. The vertical axis shows the haptoglobin concentration (μg / mg) in 1 mg of bovine blood, and the horizontal axis shows the elapsed time. On the horizontal axis, -1d is the result of the day before administration of human IL-6, 4h is the result 4 hours after the administration of human IL-6, and the following are 1d, 2d, ... 14d. , Human IL
-6 shows the results 1, 2, ..., 14 days after administration. In the figure, white squares are cont-1, that is, PBS containing no IL-6.
The cattle administered with the solution (No. 1) are shown, and the white circles are cont-2, that is, the cattle administered with the PBS solution containing no IL-6 (No. 2).
The open diamonds indicate IV, that is, the cow that was administered the IL-6 solution in the jugular vein, and the black squares indicate PF-1, that is, the cow that was continuously administered the IL-6 solution by the pump (No. 1). In the figure, black circles represent PF-2, that is, a cow (No. 2) continuously administered with the IL-6 solution by a pump, and black triangles represent PF-3, that is, continuously administered the IL-6 solution by a pump. The cow (No. 3) is shown.

Claims (2)

[Claims] 1. A livestock acute-phase protein inducer containing human IL-6 as an active ingredient. 2. A bovine acute phase protein inducer containing human IL-6 as an active ingredient.