JPH04202139A - Vascularization suppressing agent - Google Patents

Abstract

PURPOSE:To obtain vascularization suppressing agent containing interleukin-6 (IL-6) as an active ingredient and capable of suppressing vascularization through the suppressive action on the formation of tubular cavity by vascular endothelial cells. CONSTITUTION:The objective agent containing interleukin-6 (IL-6) as an active ingredient is prepared by such method as to culture a human glioblastoma and obtaining the agent from the culture supernatant, culture a host cell transformed by a vector containing a gene coding IL-6, or purify from body fluids using IL-6 receptor or anti-IL-6 antibody specifically binding to IL-6, etc. The suppressing agent reduces the mutual association of vascular endothelial cells by suppressing motility thereof and, further, suppresses vascularization through the inhibition of elongative growth of endothelial cells, thus causing the suppression of vascular wall formation by their binding to other kinds of cells. As a result, the oncogenic and inflammatory exacerfations are prevented and the symptoms can become moderate.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to interleukin (hereinafter referred to as
This invention relates to an angiogenesis inhibitor containing IL-6 as an active ingredient.

(Prior art) IL is a soluble substance produced by lymphocytes and monocytes.
There are still many unknowns about its effects. One type of IL-6 is known as a protein factor that acts when antigen-stimulated B cells proliferate and differentiate into antibody-producing cells (Paige, C.J., 5).
chreier, M., 11. , Sidman,
C.L., 1982.

Mediators from cloned T.c.
ell 1ines affect immunity
oblin expression by B cel
l, (Proc, Natl.

Acad, Sci, USA 79:475B, T, K
ishimoto, Blood vol.

74-1. PI-1O, 1989), but subsequent research has revealed that it is a cytotoxic compound with a wide range of physiological activities, including, for example, hematopoietic effects.

In recent years, it has been suggested that IL-6 exhibits various effects in addition to the above-mentioned effects on the immune system. The present inventors also found that I
Focusing on the production of L-6, we conducted research on the effects of IL-6 on vascular endothelial cells, and found that
We have obtained the knowledge that L-6 suppresses the metabolism of vascular endothelial cells, which is indicated by their fugue and other motility, causes their contraction by polymerizing actin, and makes it difficult for cells to bond with each other. As a result of research, it was surprisingly discovered that IL-6 inhibits angiogenesis, leading to the invention of an angiogenesis inhibitor containing IL-6 as an active ingredient. That is, the present invention is an angiogenesis inhibitor that inhibits angiogenesis through the action of IL-6 to inhibit lumen formation by vascular endothelial cells.

(Structure of the Invention) The present invention was completed based on the previously unknown new findings as described above, and is an angiogenesis inhibitor containing IL-6 as an active ingredient. The present invention will be explained in detail below.

IL-6 is a protein produced not only in humans but also in animals such as mice. Therefore, the present invention is an angiogenesis inhibitor for animal species that produce IL-6, including humans and mice.

It is preferable that the active ingredient IL-6 is derived from the animal species to which it is administered, in view of antigen-antibody reactions in the animals to which it is administered.

For example, human IL-6 can be obtained by culturing human glioblastoma and obtaining it from the culture supernatant (JP-A-1-1
32398), a method for producing a vector by preparing a gene encoding IL-6, and culturing host cells transformed with this vector (Japanese Patent Application No. 16255/1983)
No. 6), anti-I that has the property of specifically binding to IL-6
It can be obtained by a method of purifying body fluids using L-6 antibody or IL-6 receptor.

For example, human IL-6 is usually 1
Although it consists of 84 amino acid residues, it does not need to be such a so-called full-length protein. For example, some amino acid sequences are added to the N-terminus or C-terminus, some amino acid residues near the N-terminus, near the C-terminus, or in the middle of the sequence are missing, or even amino acid residues in the sequence are missing. Even if it is a derivative in which some of the natural amino acid residues are substituted with other amino acid residues, it can be used as an angiogenesis inhibitor if the physiological activity of IL-6 itself is not lost. It is possible to do so.

In the present invention, in order to improve the stability within the animal body, IL modified with polyethylene glycol, for example, is used.
There are no restrictions on the use of IL-6 or IL-6 encapsulated in liposomes.

(Action of the Invention) The present invention shows that IL-6 suppresses the metabolism of vascular endothelial cells, which is indicated by fugue and other motility, polymerizes actin, causes its contraction, and makes it difficult for cells to bond with each other. It was completed based on this. IL-6
The angiogenesis inhibitor of the present invention, which contains as an active ingredient, at least suppresses their motility to reduce the chance of vascular endothelial cell association, and further causes vascular endothelial cells to elongate and combine with other cells to form a vascular wall. It has the effect of suppressing angiogenesis by suppressing its formation.

(Examples) Examples will be described below to further explain the present invention in detail, but these examples are merely examples and do not limit the present invention.

In addition, vascular endothelial cells (hereinafter referred to as vascular endothelial cells) used in this example
Cells (simply referred to as cells) were scraped from the lumen of a bovine aorta blood vessel using a scalpel and transferred to a culture dish.
The primary culture was performed in a medium containing EM (manufactured by Gibco, Eagle medium) to which 10% fetal bovine serum (Fe2) was added, followed by subculture.

IL-6 is produced by culturing Escherichia coli transformed with a vector containing a gene encoding IL-6 (IXIOU/mg) according to the method described in Japanese Patent Application No. 162556/1983. It was used. Here, the unit showing the activity of IL-6 used in this example is IU
-Q', 2g, and 1U is an enhancing effect on antibody production of 5KW6-CL-4 cells, a B cell line (
E, C9-50).

Example I BBRCvol, 159 No. 2. p572-578
, it was observed that cell angiogenesis was suppressed by IL-6.

Two types of collagen gels (manufactured by Col Iagen,
Vitrogen 100.95-100% type
I collagen, typeIII cotIa
Add 11 10x MEM solution (Gib
eO Co., Ltd., a solution of MEM powder for 11 dissolved in 100 ml of distilled water) and 11111 for 0. IN NaOH
was added to prepare a gel suspension, and 0.75 ml was dispensed into each hole of a plate (manufactured by Corning, 12-well plate) and left at 37°C to prepare a gel plate.

Cell suspension 21 adjusted to 3.5×10 4 cells/1 with MEM containing 10% FC3 was added to the solidified gel plate, and cultured at 37° C. for 24 hours.

After removing the supernatant from the wells, 0.75 ml of the gel suspension prepared as described above was added and allowed to stand at 37°C to solidify.

followed by O or 500 U/ml IL-6, 10
MEM supplemented with %FC8 was added in 21 cells to each of three wells, cultured at 37° C. for 3 days, and neovascularization in the gel plate was observed. Here, for comparison, I L-
MEM in which 25 μl of rabbit antiserum obtained by administering IL-6 to rabbits as an immunogen was added at the same time as IL-6, and MEM in which 25 μm of this rabbit antiserum and only 10% FC3 were added were also prepared in 3 or 2 wells, respectively. and cultured in the same manner. Here, it was confirmed prior to the experiment that the anti-IL-6 rabbit antiserum had the effect of almost eliminating at least the effect of IL-6 on enhancing antibody production against B cells.

As a result, when a solution containing IL-6 coexists, cell angiogenesis (new generation of microvessel-like structures) is significantly suppressed, but when a solution containing no IL-6 coexists. In some cases, new blood vessels were found to occur smoothly. It was also found that this effect of IL-6 was abolished by anti-IL-6 rabbit antibody, which itself does not affect cell angiogenesis.

This result clearly shows that IL-6 suppresses cellular angiogenesis.

Example 2 A filter with a pore size (8 μm) that allows cells to pass through (
Thickness 10. czms Millipore, Nuc I eo
p.

Experimental apparatus (manufactured by Neuropobe, microchemotaxis chamber, upper chamber volume 50 μm, lower chamber volume 28° 5 μm) separated into upper and lower chambers by re filter)
) was used to conduct the following experiment using various concentrations of IL-
The effect of 6 on cell metabolism was observed through its reduction in migration. Note that this experiment was carried out in BBRCvol, 159. N
o, 2. This was done with reference to p572-578.

First, 0, 5.50 or 500 U/ml of I
28.5 μm I L-6 solution containing L-6 (I
A solution containing L-6 was added to the lower chamber, and the inside of the filter was filled with the solution. Subsequently, MEM containing 2% FC8 in which 1×10 4 cells were suspended was added to the upper chamber separated by a filter.

After being left at 37°C for 210 minutes, the experimental apparatus was disassembled, the filter was removed, and the surface that was in contact with the upper chamber was washed to remove attached cells. Cells that passed through the pores of the filter and reached the lower chamber side were fixed with alcohol, then the cell nuclei were stained with hematoxylin (manufactured by Sigma), and the number of cells was measured using a phase contrast microscope.

The results of the above experiment conducted six times are shown in Table 1, and a graph of the values is shown in FIG.

Table 1 Table 1 and Figure 1 show that at least 500 U/ml of I
It can be seen that L-6 reduces the filter permeability (fugetaxis) of cells.

This result is consistent with the 500 U/ml concentration of I in Example 1.
This shows that the inhibition of cell angiogenesis by L-6 is through inhibition of cell metabolism.

Example 3 In order to confirm that the results of Example 1 were not due to IL-6 decreasing the number of cells, the following experiment was conducted.

For the four types of gel plates used in Example 1, after removing the supernatant, only the gel portion was collected in a plastic tube and centrifuged at 120 Orpm at 4° C. for 5 minutes to collect the precipitate. Next, 0.0 μm of collagen degrading enzyme (Collagenase, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in MEM so that the amount was 10 μm, 1 g, 10 μm. After adding 5 ml and reacting at 37°C for 20 minutes with shaking to degrade collagen, centrifugation was performed at 4°C at 120 Orpm to collect cells in a precipitate.

0.0% to the resulting cell precipitate. Add 5 ml of a solution containing 0.05% trypsin and 0.05% EDTA and incubate at 37°C.
Incubate for 5 minutes to break up cell clumps, then add 10%
The reaction was stopped by adding MEM supplemented with FC8 at 0°51°C, followed by centrifugation at 1200 rpm for 50 minutes at 4°C.
A precipitate of dissociated cells was collected, and the number of cells was measured using a hemocytometer.

As a result, 10% FCS without IL-6
The number of cells obtained from each gel (3 wells) cultured in the presence of MEM supplemented with was 1.2B.

1.35, 1.40 (5 XIOs), whereas the results were obtained from a gel (3 wells) cultured in the presence of MEM containing 500 U/ml IL-6 and 10% FC8. The number of cells obtained was 162, 1.27, and 1.2, respectively.
4 (5 XIO) and 500U/+nl I L
-10% FC3 containing rabbit antiserum of 6 and 25 μm
The number of cells obtained from the gel (3 wells) cultured in the presence of MEM supplemented with was 1.46, 1, 40, and 1, respectively.
．． 39 (×105 cells), and the number of cells obtained from the gel (2 wells) cultured in the presence of MEM supplemented with 10% FC3 containing 25 μm rabbit antiserum was as follows:
1.34, 1.25 (xlO" pieces).

The above results revealed that cell growth was independent of the presence or absence of IL-6.

This result is similar to the results obtained in Example 1 and Example 2.
This clearly shows that the cell number was not reduced by L-6.

Example 4 Proc, Natl, Sci, USA vol, 7B,
1) With reference to the method described in 4498-4502.1979, it was observed that IL-6 made it difficult to form intercellular junctions.

Cells were cultured on coverslips until confluent and cultured for an additional 21 hours in 10% FC3 containing 0 or 500 U/ml of IL-13-6, followed by Hank-s solution (11 Inside, 0.8g N
aCl, 0.4g, KCI, 0. 121
g Na2HP0 ・12H0,0,06gKH2
PO4, 1,00g glucose, 0. 2g Mg
SO4・7H20.0.14gCaCl2.0.35g
solution containing NaHCO) and fixed with formaldehyde.

A fluorescent stain (FITC-phalloi) was applied to the fixative.
din (manufactured by Sigma) to 20 tt g#nl, and after standing at 37 °C for 40 minutes, the cover glass was washed with Hank-s solution and the degree of polymerization of intracellular actin was observed using a fluorescence microscope. did.

As a result, when cultured in the coexistence of IL-6, IL-6
=6 It was found that the cells clearly did not bond with each other compared to the case where they were cultured without coexistence.

(Effects of the Invention) Angiogenesis is not only a physiological phenomenon observed during wound recovery, but also a phenomenon generally observed in adults in pathological conditions such as cancer, arteriosclerosis, and inflammation. .

For example, in solid cancers, blood vessels that develop within the tumor supply oxygen and nutrients to cancer cells, which maintains their almost limitless proliferation, causing tumor growth and deterioration of the disease state. Furthermore, in cases of chronic inflammation such as chronic rheumatism, new blood vessels are observed in the patient's joint cavities, indicating that there is a deep relationship between new blood vessel formation and the occurrence and deterioration of inflammation.

Therefore, if the angiogenesis inhibitor of the present invention is administered to a site where cancer or inflammation has occurred, angiogenesis at the site will be suppressed, thereby preventing these conditions from worsening and reducing symptoms. It becomes possible.

[Brief explanation of the drawing]

FIG. 1 shows the results of Example 2 of the present invention. Each column shows the value when the target (IL-6 concentration is 0) is taken as 100%, and the solid line on the column shows the standard value when statistical processing is performed on each result.
This indicates an error (SE).

Claims (1)

[Claims] (1) Angiogenesis inhibitor containing interleukin-6 as an active ingredient