JP3018186B1 - Anti-inflammatory, monocytic cell growth inhibitor - Google Patents

Abstract

[PROBLEMS] To develop a highly effective and safe anti-inflammatory agent. SOLUTION: In the present invention, it was shown that human adiponectin has a growth inhibitory effect on monocytes and B cells. Human adiponectin having such an action is effective not only as an inhibitor of monocyte and B cell proliferation but also as an anti-inflammatory agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel anti-inflammatory agent utilizing the inhibitory action of human adiponectin on monocyte proliferation.

[0002]

2. Description of the Related Art An inflammatory response is a biological defense reaction that occurs when a tissue is infected or damaged. At the time of inflammation, phenomena such as dilation and perforation of microvessels, leakage of blood components, and migration of leukocytes into the inflamed tissue of the affected area occur. In this case, various chemical mediators are involved. The above-mentioned phenomenon is a reaction that occurs to fight infection, and acts for host defense. Although such an inflammatory response is an important physiological response, an excessive inflammatory response causes great damage to a living body. Therefore, various anti-inflammatory agents have been developed so far.

[0003]

The anti-inflammatory drugs currently used are roughly classified into non-steroidal anti-inflammatory drugs and steroidal anti-inflammatory drugs which are glucocorticoids. These anti-inflammatory agents have different mechanisms of action, and non-steroidal aspirin, phenylbutazone, indomethacin and the like act by inhibiting the biosynthesis of prostaglandin by suppressing the activity of cyclooxygenase. these,
Non-steroidal anti-inflammatory drugs are characterized by having both analgesic and antipyretic effects. Non-steroidal anti-inflammatory drugs are widely used because of their high safety, but their anti-inflammatory effects are not always strong. On the other hand, steroidal anti-inflammatory drugs bind to receptors in the cytoplasm, are taken up into the nucleus, activate specific genes, and induce lipocortin biosynthesis. The lipocortin, inhibition of the path of the cyclooxygenase system due to suppression of phospholipase A _2, inhibition etc. lipoxygenase pathway of accompanying the inhibition of leukotriene B _4, be anti-inflammatory effect is exhibited by inhibiting a variety of chemical mediators Are known. Steroidal anti-inflammatory drugs have a remarkable inhibitory effect on inflammation and are effective for autoimmune diseases and the like, but their strong side effects are problematic. Therefore, development of an anti-inflammatory agent having a strong action and high safety has been demanded.

[0004]

Means for Solving the Problems Human adiponectin according to the present invention is a collagen-like factor specific to human adipose tissue. The gene has been isolated by the inventors (reference: Kazuhisa Maeda et. Al. Bioche
m.Biophys.Res.Communication 221, pp286-289,1996
) Are known to be proteins composed of 244 amino acids. The amino acid sequence of the human adiponectin protein is shown in FIG. In examining the physiological effects of human adiponectin, the present inventors have found that human adiponectin suppresses the growth of monocyte cells and B cell cells, and is effective as a growth inhibitor for these blood cells. Showed things. As described above, a phenomenon in which leukocyte cells such as monocytes, macrophages, and neutrophils proliferate and accumulate during inflammation is observed. Monocytes whose growth is suppressed by adiponectin are cells that play an important role in such cellular defense responses,
Upon maturation, they become macrophages responsible for phagocytosis, while producing inflammatory cytokines such as interleukin-1 and tumer necrosis factor alpha. Monocytes play such an important role in the course of the inflammatory response, and adiponectin can be used as an anti-inflammatory agent by inhibiting the growth of monocytes and preventing their accumulation.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The human adiponectin according to the present invention can be used as various forms of preparations. Since human adiponectin is a polypeptide, parenteral administration is preferred as an administration route. The dose of human adiponectin varies depending on the method of administration, the condition of the patient, the age, etc., but is usually 0.1 to 500 µg / ml, preferably 1 to 50 µg / ml once a day, 1 to 3 times a day. The peptide of the present invention is usually administered in the form of a preparation prepared by mixing with a preparation carrier. As the pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with the human adiponectin of the present invention is used.

[0006] Examples of dosage forms include injections, suspensions, suppositories, ointments, creams, gels, patches, inhalants and the like. These preparations are prepared according to a conventional method. In the case of a liquid preparation, it may be in the form of being dissolved or suspended in water or another suitable solvent at the time of use. In the case of an injection, human adiponectin is prepared by dissolving it in an appropriate solvent, but a buffer or a preservative may be added. These preparations can contain human adiponectin at a rate of 0.1 μg / ml or more, preferably 1 to 50 μg / ml. These formulations may also contain other components of therapeutic value.

[0007]

EXAMPLES (Effect of human adiponectin on colony forming ability) The effect of adiponectin on colony formation necessary for cell growth was examined. Bone marrow cells were collected from the iliac bones of healthy adults from which informed consent was obtained, and mononuclear cells were separated by specific gravity centrifugation (Lymphoprep: Nicomed, Oslo, Norway).
After washing the obtained bone marrow mononuclear cells twice with RPMI-1640 medium, (1) medium alone (2) 10 μg / ml human adiponectin (3) 10 μg used as control
The cells were cultured at a density of 5 × 10 ⁴ cells / ml in a serum-free methylcellulose medium containing 1 / ml human serum albumin, and after 14 to 16 days, the number of colonies of various hematopoietic progenitor cells was measured under an inverted microscope. The composition of the serum-free methylcellulose medium used was IMDM, methylcellulose (0.9%), 2-mercaptoethanol (1
0 ^-4 M), L-glutamine (2 mM), 1% crystallized bovine serum albumin (1%), human transferrin, bovine insulin, rh erythropoietin (3 U / m
l), rh SCF (50 ng / ml), rh GM-
CSF (10 ng / ml), rh G-CSF (10 n / ml)
g / ml), rh IL-3 (10 ng / ml) and r
h Consists of IL-6 (10 ng / ml) (Veritas). The measurement was performed at three points, and the statistically significant difference was tested by the Student's t test. Abbreviations of the various hematopoietic progenitor cells measured are as follows. (1) CFU-GM: granulocyte-mac
rophage colony-forming un
ITS (granulocyte-macrophage colony forming cell and granulocyte-macrophage line) (2) CFU-M: macrophage colon
y-forming units (megakaryocytic colony forming cells and megakaryocytes and platelets) (3) BFU-E: erythroid burst
colony-forming units (erythroid colony group forming cells and erythroid cells) (4) CFU-Mix: mixed erythroi
d-myeloid colony-forming
units (mixed colony forming cells)

As a result, human adiponectin has a
CFU-GM in human normal bone marrow mononuclear cells at a concentration of g / ml
(FIG. 2) and the growth of CFU-M (FIG. 3) were suppressed by about 50%. On the other hand, BFU-E (FIG. 4) and CFU-Mix
No inhibitory action was observed for (FIG. 5).

[0009] Furthermore, from bone marrow mononuclear cells, cluster of difference, a marker of hematopoietic progenitor cells, is used.
CD34-positive cells were separated using magnetic beads (Milteny Biotech), using the expression of ntation (CD) 34 as an index, and cultured in a serum-free methylcellulose medium at a density of 500 cells / ml. As above, (1) Medium only in serum-free methylcellulose medium
(2) Human adiponectin 10 μg / ml (3) Culture under three conditions to which human serum albumin 10 μg / ml was added, and colony formation of hematopoietic progenitor cells was examined. CD34-positive cells are separated from bone marrow mononuclear cells by removing CD3 and CD11b-positive cells using immunomagnetic beads to which anti-human CD3 antibody and anti-human CD11 antibody are bound, and then using anti-human CD34 antibody-bound beads. , CD3
4 positive cells were sorted (Reference: Takafumi Yokota et. Al. Blood 91, pp3263-3272, 199).
8). After purification with CD34, the number of colonies of various hematopoietic progenitor cells was counted under an inverted microscope on days 14 to 16 of the culture. The measurement was performed at three points, and the statistically significant difference was tested by the Student's t test.

As a result, the same results as described above were obtained for a population of human normal hematopoietic progenitor cells purified by CD34. That is, the growth of CFU-GM (FIG. 6) and CFU-M (FIG. 7) was suppressed by 10 μg / ml human adiponectin, but BFU-E (FIG. 8) and CFU-M (FIG. 8).
The growth of FU-Mix (FIG. 9) was not suppressed. From the above results, it was considered that adiponectin suppresses the clonal proliferation of myeloid monocytic progenitor cells at a relatively mature differentiation stage without involving macrophages, lymphocytes, or the like.

Various concentrations (0, 1, 5, 10, 50 μg)
Per ml of human normal bone marrow mononuclear cells in a serum-free methylcellulose medium containing human adiponectin.
0 ⁴ cells / ml density and incubated at the culture 14 -1
On the sixth day, the number of hematopoietic progenitor cell colonies was counted under an inverted microscope. The measurement was performed at three points, and the statistically significant difference was tested by the Student's t test. The inhibitory effect of human adiponectin on colony formation of bone marrow monocyte progenitor cells was observed at an adiponectin concentration of 5 μg / ml or more, and reached an almost steady state at an adiponectin concentration of 10 μg / ml or more (FIG. 10). The number of colonies of myeloid monocyte progenitor cells was determined by the sum of CFU-GM and CFU-M.

(Specificity of the effect of human adiponectin on colony forming ability) In a serum-free methylcellulose medium containing 10 μg / ml of human adiponectin, 30 μg of an anti-adiponectin antibody (isotype IgG1) or its control antibody (purified murine IgG1: cappel) was added. /
ml of human normal bone marrow mononuclear cells by adding 5 × 10 ⁴ cells
After culturing at a density of ls / ml, the number of colonies of hematopoietic progenitor cells was counted under an inverted microscope on days 14 to 16 of culturing. The measurement was performed at three points, and the statistically significant difference was tested by the Student's t test. When the number of myeloid monocyte progenitor cell colonies was determined by the sum of CFU-GM and CFU-M, the growth inhibitory effect of human adiponectin was partially, but not completely, suppressed by monoclonal antibody 9104, which is an anti-adiponectin antibody. Inhibited (Fig. 1
1). The specificity of the action of adiponectin was shown by its inhibition by specific antibodies.

(The growth and survival of various leukemia cell lines
Of human adiponectin) Increase of various leukemia cell lines
The effect of human adiponectin on growth and survival
^Three-H] thymidine incorporation assay and trypan blue
It was examined by a dye exclusion assay. Various types of leukemia
The cell strain was washed twice with RPMI-1640 medium (Gibco).
96 well flat bottom microtiter tap
1 × 10 per well (Iwaki glass)^Four
cells / 100μl cell density in serum free conditioned medium
Resuspend, as human adiponectin or its control
Human serum albumin used (Chemicon International
LU INC. ) Was added at a concentration of 10 μg / ml and cultured.
Was. The serum-free medium used here was insulin-tran
Spherin-sodium selenite additive (10 μg /
ml Boehringer Mannheim), chemically quantified
Lipid concentrate (1% Gibco), bovine serum albumin
(0.2% veritas) and L-glutamine (2 mM
Gibco).

The effect on cell proliferation was determined by adding 0.5 μCi of [ ³ -H] thymidine (specific activity: 5 Ci / mM, Amersham International) to each well at 20 hours of culture and, after 4 hours, culturing the cells with a semi-automatic cell. Harvester (Model 1295; Pharmacia LKB Biotechnology) was used to measure the incorporation into cells with a liquid scintillation counter, and the Stimulation Index (S
I) was calculated and evaluated. The effect on cell viability was evaluated by adding trypan blue at 48 hours of culture, counting the number of living cells and the number of dead cells using a hemocytometer, and calculating the decrease in viability. The measurement was performed at three points, and the statistically significant difference was tested by the Student's t test. The SI and the decrease in the survival rate were calculated by the following formula based on the average value of the three points. SI = (uptake of [ ^3- H] thymidine in the presence of adiponectin) / (uptake of [ ^3- H] thymidine in the presence of human albumin) Decrease in survival rate = (survival rate in the presence of human albumin) −
(Survival rate in the presence of adiponectin)

As a result, human adiponectin contained 10 μm
myeloid cell lines THP1, KU8 at a concentration of g / ml
12, M1, WEHI3 and the proliferation of B cell line, BALL, were significantly suppressed. The obtained SI is 0. THP1.
35, KU812 is 0.85, M1 is 0.70, WEH
I3 was 0.89 and BALL was 0.58. Especially T
HP1, M1 and BALL exhibited a strong growth inhibitory effect, and induction of cell death was also observed. The decrease in survival rate
THP1 was 42.6%, M1 was 31.1%, and BALL was 24.2%. Table 1 summarizes the results for various cells.

[0016]

[Table 1]

(Effect of human adiponectin on mouse myeloid leukemia cell line M1) After washing mouse myeloid leukemia cell line M1 twice with RPMI-1640 medium,
Resuspend in serum-free medium as in Table 1 and place in a 96-well flat bottom microtiter plate at a cell density of 1 × 10 ⁴ cells / 100 μl per well.
(1) Medium only (2) 10 μg / ml human adiponectin (3) Cultivation under three conditions in the presence of 10 μg / ml human serum albumin, the number of viable cells and viability were determined over time (culture 24, 48, 72 At time point), evaluation was performed using the same trypan blue exclusion assay as in Table 1. As a result, at a concentration of 10 μg / ml, human adiponectin significantly suppressed the growth of M1 cells (FIG. 12). The survival rate was reduced to about 50% after 72 hours in the presence of 10 μg / ml adiponectin (FIG. 13).

Further, the morphology of the cells was examined. After 48 hours of culture, the morphology of M1 cells was determined using the May-Grunwald-
Examination by Giemsa staining revealed aggregation and fragmentation of nuclear chromatin (FIG. 14). Thus, flow cytometry and DNA fragmentation were examined on the assumption that the growth suppression shown in FIG. 11 was caused by induction of apoptosis. The nuclear DNA content per cell at 48 hours and 72 hours after the culture was determined using propidium iodide (P
After fluorescent staining of the DNA in I), the DNA was analyzed by flow cytometry. M1 cells adjusted to the same cell density as in the above-described examination of the number of viable cells and the viability were cultured in the presence of human adiponectin or human serum albumin at 10 μg / ml for 24 hours.
The cells were cultured at 1 × 10 ⁵ cells / ml per well in a flat bottom plate of wells. After each culture time, the cells are collected and the cooled phosphate
After washing twice with -buffered saline (PBS) (-), the cells were slowly resuspended in 70% ethanol at -20 ° C and fixed at -20 ° C overnight. After centrifugation,
Resuspend the pellet in 300 μl of PI solution at 37 ° C.
For 30 minutes in the dark and analyzed by flow cytometry. The composition of the PI solution was sodium citrate (0.1%), NP-40 (0.3%).
%), 100 μg of Rnase A per ml of PBS,
And 50 μg of PI per ml of PBS. As a result, an increase in subdiploid (subdiploid) content was observed in the presence of adiponectin (FIG. 15).

The DNA fragmentation after 24 hours and 48 hours of culturing was examined. M1 cells adjusted to the same cell density as in the above-described examination of proliferation of M1 cells were mixed with 10 μg / ml of human adiponectin or human serum albumin.
In the presence, in a 90 × 20 mm dish (Sumilon), 1 × 10 ⁶ cells / 10 per dish
Cultured in ml. Cells are collected after each culture time, and DN
A was extracted (Reference: Hiroyuki Sugahara et.al.Th.
e Journal of Experimental Medicine 179.pp1757-176
6,1994). The obtained DNA was used at 5 μg / lane each.
Electrophoresis was performed in the presence of ethidium bromide, and DNA fragmentation was evaluated by the generation of ladder-like degradation products. As a result, in the presence of adiponectin, remarkable DNA fragmentation was observed, particularly after 48 hours (FIG. 16). From the above results, it was shown that adiponectin suppresses the proliferation of leukemia cell line M1 by inducing apoptosis.

[0020]

According to the present invention, it has been shown that adiponectin has a growth inhibitory effect on monocytes and B cells. Adiponectin having such an effect is
Effective as a monocyte and B cell growth inhibitor,
It can be used as an anti-inflammatory agent because it suppresses the proliferation of monocytes that play an important role in inflammation.

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of human adiponectin.

FIG. 2 shows CFU-GM derived from human bone marrow mononuclear cells.
FIG. 2 is a view showing the effect of human adiponectin on the growth of Adiponectin.

FIG. 3 shows the effect of human adiponectin on the proliferation of CFU-M derived from human bone marrow mononuclear cells.

FIG. 4 is a diagram showing the effect of human adiponectin on the proliferation of BFU-E derived from human bone marrow mononuclear cells.

FIG. 5 shows CFU-Mi derived from human bone marrow mononuclear cells.
FIG. 2 shows the effect of human adiponectin on the growth of x.

FIG. 6 shows the effect of human adiponectin on the proliferation of CFU-GM derived from human bone marrow mononuclear cells purified by expression of CD34.

FIG. 7 shows the effect of human adiponectin on the proliferation of CFU-M derived from human bone marrow mononuclear cells purified by expression of CD34.

FIG. 8 shows the effect of human adiponectin on the proliferation of BFU-E derived from human bone marrow mononuclear cells purified by expression of CD34.

FIG. 9 shows the effect of human adiponectin on the proliferation of CFU-Mix derived from human bone marrow mononuclear cells purified by expression of CD34.

FIG. 10 is a graph showing the concentration dependency of the effect of human adiponectin on the growth of myeloid monocytic progenitor cell colonies (sum of CFU-GM and CFU-M).

FIG. 11 shows the results of examining the specificity of the effect of human adiponectin on the proliferation of colonies of myeloid monocyte precursor cells (sum of CFU-GM and CFU-M) using an anti-human adiponectin antibody. FIG.

FIG. 12 is a graph showing the effect of human adiponectin on the proliferation of leukemia cell line M1.

FIG. 13 is a graph showing the effect of human adiponectin on the survival rate of leukemia cell line M1 during culture.

FIG. 14 is a photograph showing a morphology of a leukemia cell line M1 48 hours after adiponectin treatment.

FIG. 15 is a view showing the results of flow cytometry in which the effect of human adiponectin on the nuclear DNA content of the leukemia cell line M1 was examined.

FIG. 16 is a photograph of electrophoresis showing the effect of human adiponectin on nuclear fragmentation of leukemia cell line M1.

Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12N 5/06 ZNA C12N 5/00 ZNAE

Claims (9)

(57) [Claims] 1. An anti-inflammatory agent comprising an effective amount of human adiponectin to suppress the proliferation of monocyte cells or monocyte precursor cells. 2. The method according to claim 1, wherein the human adiponectin is 5-20 μg /
The anti-inflammatory agent according to claim 1, which is contained at a concentration of ml. 3. A method for inhibiting the growth of monocytic cells or monocyte progenitor cells, which comprises an amount of human adiponectin effective to suppress the growth of monocyte or monocyte progenitor cells. Agent. 4. The method according to claim 1, wherein the human adiponectin is 5-20 μg /
The growth inhibitor according to claim 3, which is contained at a concentration of ml. 5. A B-cell containing a human adiponectin in an amount effective to suppress the proliferation of B cells.
Cell growth inhibitor. 6. An amount of human adiponectin of 5-20 μg /
The growth inhibitor according to claim 5, which is contained at a concentration of ml. 7. A therapeutic agent for leukemia, comprising a human adiponectin in an amount effective to suppress the proliferation of a myelomonocytic leukemia cell line and a B-cell leukemia cell line. 8. A human adiponectin having a concentration of 5-20 μg /
The therapeutic agent for leukemia according to claim 7, which is contained at a concentration of ml. 9. A method for suppressing the growth of myelomonocytic leukemia and B-cell leukemia in vitro by adding human adiponectin.