WO2011138842A1

WO2011138842A1 - Therapeutic agent for rheumatoid arthritis

Info

Publication number: WO2011138842A1
Application number: PCT/JP2010/070023
Authority: WO
Inventors: 徹八子; 由紀南家; 学川本; 寿山中; 茂小竹
Original assignee: 学校法人東京女子医科大学
Priority date: 2010-05-07
Filing date: 2010-11-10
Publication date: 2011-11-10
Also published as: JP5742061B2; JPWO2011138842A1

Abstract

A therapeutic agent for rheumatoid arthritis, which comprises interleukin-26; and a human osteoclast formation inhibitor, which comprises interleukin-26.

Description

Rheumatoid arthritis treatment

The present invention relates to a therapeutic agent for rheumatoid arthritis and an osteoclast formation inhibitor containing interleukin-26.

Interleukin (hereinafter sometimes referred to as “IL”)-26 is a cytokine belonging to the IL-10 family, and its signal is transmitted through a receptor complex composed of IL-20R1 and IL-10R2. (Non-Patent Documents 1 to 3).
There is a report that IL-26 is produced from Th17 cells by IL-23 stimulation (Non-patent Document 4). However, the detailed action of IL-26 is still unclear. The reason is that no homologue of IL-26 mouse has been identified to date.
The present inventors have so far confirmed that the IL-17 concentration in the joint fluid of rheumatoid arthritis patients is significantly higher than the IL-17 concentration in the joint fluid of osteoarthritis patients (Non-patent Document 5). ), IL-23 has been reported to promote human osteoclast formation via at least the IL-17 pathway (Non-patent Document 6).
However, the effect of IL-26 on human osteoclast formation is unknown.

The present invention aims to provide a new use of IL-26.

As a result of intensive studies by the present inventors, the present inventors have found that human osteoclast formation is suppressed by IL-26. The present invention has been made based on such knowledge.
That is, the present invention provides a therapeutic agent for rheumatoid arthritis containing IL-26.
The present invention also provides use of IL-26 for producing a therapeutic agent for rheumatoid arthritis.
The present invention also provides an osteoclast formation inhibitor containing IL-26.
The present invention also provides the use of IL-26 for producing an osteoclast formation inhibitor.

Since IL-26 can inhibit osteoclast formation and bone resorption, the present invention can provide an excellent rheumatoid arthritis therapeutic agent and human osteoclast formation inhibitor.

It shows human osteoclast formation inhibitory activity of interleukin-26. (A) shows the results of culturing monocytes separated from human peripheral blood mononuclear cells at 37 ° C. for 3 days in the presence of 100 ng / mL of M-CSF. (B) shows the addition of 30 ng / mL of sRANKL to a culture system in which monocytes separated from human peripheral blood mononuclear cells were cultured at 37 ° C. for 3 days in the presence of 100 ng / mL of M-CSF. The result of culturing at 10 ° C. for 10 days is shown. (C) shows a culture system in which monocytes separated from human peripheral blood mononuclear cells were cultured at 37 ° C. for 3 days in the presence of 100 ng / mL M-CSF, and 30 ng / mL sRANKL and 0.1 ng / mL rhIL The results of adding -26 and then culturing at 37 ° C. for 10 days are shown. (D) shows a culture system in which monocytes separated from human peripheral blood mononuclear cells were cultured at 37 ° C. for 3 days in the presence of 100 ng / mL M-CSF, and 30 ng / mL sRANKL and 1.0 ng / mL rhIL were used. The results of adding -26 and then culturing at 37 ° C. for 10 days are shown. (E) shows a culture system in which monocytes separated from human peripheral blood mononuclear cells were cultured at 37 ° C. for 3 days in the presence of 100 ng / mL M-CSF, and 30 ng / mL sRANKL and 10 ng / mL rhIL- The result of adding 26 and then culturing at 37 ° C. for 10 days is shown. (F) shows a culture system in which monocytes separated from human peripheral blood mononuclear cells were cultured at 37 ° C. for 3 days in the presence of 100 ng / mL M-CSF, and 30 ng / mL sRANKL and 100 ng / mL rhIL- The result of adding 26 and then culturing at 37 ° C. for 10 days is shown. The bone resorption ability (bone resorption fossa) of interleukin-26 is shown. (A) shows the results of culturing monocytes separated from human peripheral blood mononuclear cells at 37 ° C. for 3 days on synthetic calcium phosphate in the presence of 100 ng / mL of M-CSF. (B) shows the addition of 50 ng / mL sRANKL to a culture system in which monocytes separated from human peripheral blood mononuclear cells were cultured at 37 ° C. for 3 days on synthetic calcium phosphate in the presence of 100 ng / mL M-CSF. Then, the results of culturing at 37 ° C. for 10 days are shown. (C) shows a culture system in which monocytes separated from human peripheral blood mononuclear cells were cultured at 37 ° C. for 3 days on synthetic calcium phosphate in the presence of 100 ng / mL M-CSF, and 50 ng / mL sRANKL and 10 ng The result of adding / mL rhIL-26 and then culturing at 37 ° C. for 10 days is shown. (D) shows a culture system in which monocytes separated from human peripheral blood mononuclear cells were cultured at 37 ° C. for 3 days on synthetic calcium phosphate in the presence of 100 ng / mL M-CSF, and 50 ng / mL sRANKL and 100 ng The result of adding / mL rhIL-26 and then culturing at 37 ° C. for 10 days is shown. The expression rate of RANK mRNA on human monocytes is shown.

IL-26, which is an active ingredient of the present invention, was previously called AK155 (Knappe A. et al., (2000) J. Virology 74: 3881), a signal peptide consisting of 21 amino acids, six helices, A polypeptide of 171 amino acids in total length containing 4 conserved cysteine sequences. Since IL-26 is a family of cytokines produced in the body, it can be safely administered to the body.
As IL-26 used in the present invention, not only natural products but also artificial products can be used. Artificial objects include recombinant ones. A method is also known in which a part of the amino acid is modified by deletion, substitution or insertion without substantially changing the biological activity inherent in IL by genetic engineering. Therefore, IL-26 used in the present invention includes such modified ones. Human-derived IL-26 (hIL-26) is preferred. Recombinant (rIL-26) is also preferred.

As IL-26 used in the present invention, a commercially available product may be used, or it may be synthesized by a known method. Commercially available products include, for example, “Recombinant” Human26IL-26 / AK155 Monomer ”manufactured by R & D® Systems Inc .;“ Recombinant Human IL-26 / AK155 Monomer ”manufactured by Abnova, and the like. Moreover, it can obtain by isolating and refine | purifying from a lymph node and a synovial tissue by the well-known method using gel filtration, an ion exchange chromatography, a reverse phase chromatography, a mass spectrum etc. IL-26 used in the present invention can also be synthesized by a general amino acid chemical synthesis method such as the Fmoc method, or can be synthesized using a commercially available amino acid synthesizer.

The IL-26 is administered as it is or as various pharmaceutical compositions. Subjects for administration are animals, including humans, in need of rheumatoid arthritis treatment or suppression of osteoclast formation. As the osteoclast, human osteoclast is preferable.
The dosage form of the pharmaceutical composition may be, for example, tablets, powders, pills, granules, capsules, suppositories, solutions, sugar coatings, devoted drugs, or syrups. It can be produced according to a conventional method.
For example, the tablet contains IL-26, which is the active ingredient of the present invention, as a known auxiliary substance, for example, an inert diluent such as lactose, calcium carbonate or calcium phosphate, a binder such as gum arabic, corn starch or gelatin, alginic acid, corn starch or Pre-gelatinized starches, etc., sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry, moisturizers such as magnesium stearate, talc or carboxymethylcellulose, fat, wax, semi Mix with solid and liquid polyols, soft gelatin capsules such as natural or hardened oils and excipients for suppositories, solution excipients such as water, alcohol, glycerol, polyols, sucrose, invert sugar, glucose, vegetable oils Can be obtained.

IL-26 is usually administered at a dose of 3 to 10 μg / kg body weight, preferably 3 to 5 μg / kg body weight, in terms of purified protein, once or multiple times per adult day.
The dosage of the therapeutic agent or inhibitor of the present invention is determined by the administration route, the treatment period, the age and weight of the patient, and the like.

1. IL-26
As IL-26, recombinant human IL-26 (recombinant human IL-26, rhIL-26, manufactured by R & D Systems Inc., "Recombinant Human IL-26 / AK155 Monomer", catalog number: 1375-IL / CF, deposit number Q9NPH9 The predicted molecular weight of 17.7 kDa) was used.
2. Subjects and methods (1) Monocytes were isolated from human peripheral blood mononuclear cells in vitro. The separated monocytes were inoculated into a 48-well plate at 5 × 10 ⁴ / well. The monocytes were cultured at 37 ° C. for 3 days in the presence of macrophage colony-stimulating factor (M-CSF) (100 ng / ml) (FIG. 1 (A)).
On the 4th day after the start of the culture, 30 ng / ml of M-CSF and soluble receptor-activator of NFκB ligand (sRANKL) were added to this culture system and cultured at 37 ° C. for 10 days (FIG. 1 (B)).
On the other hand, rhIL-26 was added at 0.1, 1.0, 10, and 100 ng / ml to the culture system 4 days after the start of the culture, respectively, and cultured at 37 ° C. for 10 days (FIGS. 1 (C) to (F)).
After completion of the culture, osteoclasts were immunostained with an anti-CD51 / 61 antibody, and human osteoclast formation inhibitory activity by rhIL-26 was evaluated.

(2) Using a bone culture system (BD Bioscience, BD BioCoat (TM) Osteologic (TM)), the bone resorption ability of osteoclasts was evaluated in the resorption pits. The culture vessel of the system is coated with a synthetic calcium phosphate thin film as a bone substitute. By culturing osteoclasts on this coat, bone resorption pits are formed on the thin film, and bone resorption ability is evaluated. can do.
First, monocytes were isolated from human peripheral blood mononuclear cells in vitro, and the isolated monocytes were inoculated into a culture vessel of the system so as to be 5 × 10 ⁴ / well, and the monocytes were M-CSF ( In the presence of 100 ng / ml), the cells were cultured at 37 ° C. for 3 days (FIG. 2 (A)).
On the 4th day after the start of the culture, 50 ng / ml of M-CSF and soluble sRANKL were added to this culture system and cultured at 37 ° C. for 10 days (FIG. 2 (B)).
On the other hand, rhIL-26 was added at 10, 100 ng / ml to the culture system 4 days after the start of culture, respectively, and cultured at 37 ° C. for 10 days (FIGS. 2C to 2D).

(3) 5 × 10 ⁴ / well human monocytes were cultured with M-CSF (100 ng / ml) at 37 ° C. for 24 hours. After removing non-adherent cells, M-CSF (100 ng / ml) and rhIL-26 (10 ng / ml) were added to one culture system. Only M-CSF (100 ng / ml) was added to the other culture system. After culturing at 37 ° C. for 24 hours, the mRNA expression rate of receptor-activator of NFκB (RANK) on monocytes was measured by RT-PCR (FIG. 3).

3. Results (1) As shown in FIGS. 1B to 1F, the irregular gray portion surrounding the black spots shows osteoclasts (in fact, stained with anti-CD51 / 61 antibody in pink) Have been). When sRANKL is added to M-CSF, human osteoclasts are induced to differentiate (FIG. 1 (B)). When cultured in the presence of rhIL-26 at the same temperature as in the absence of rhIL-26 in the presence of rhIL-26 (FIGS. 1 (C) to (F)), the area of the gray portion was reduced compared to FIG. 1 (B). . This indicates that the amount of human osteoclasts induced in the presence of rhIL-26 is less than the amount of human osteoclasts induced in the absence of rhIL-26. That is, rhIL-26 suppressed the formation of human osteoclasts. In addition, human osteoclast formation was suppressed as rhIL-26 concentration increased. That is, as shown in FIG. 1, the formation of human osteoclasts was suppressed in a concentration-dependent manner by the addition of rhIL-26.
(2) The white circles shown in FIGS. 2B and 2C indicate bone resorption pits. As shown in FIG. 2, the addition of rhIL-26 decreased the bone resorption pits induced by RANKL in a concentration-dependent manner.
(3) As shown in FIG. 3, the expression of RANK mRNA on monocytes was decreased by the addition of rhIL-26.

As described above, as is clear from the Examples, rhIL-26 suppressed human osteoclast formation and bone resorption. As a mechanism of this inhibitory effect, at least suppression of RANK mRNA expression on monocytes by IL-26 can be considered. In addition, it is suggested that the balance between IL-26, which inhibits osteoclast formation, and IL-23, which promotes osteoclast formation, may regulate human osteoclast formation.
In the future, rhIL-26 is expected to be applied to the treatment of rheumatoid arthritis because it is effective for both osteoclastogenesis inhibition and bone resorption inhibition.

Claims

治療 Rheumatoid arthritis treatment containing interleukin-26.
The therapeutic agent for rheumatoid arthritis according to claim 1, wherein the interleukin-26 is human interleukin-26.
The therapeutic agent for rheumatoid arthritis according to claim 1 or 2, wherein the interleukin-26 is recombinant interleukin-26.
Use of interleukin-26 to produce a therapeutic agent for rheumatoid arthritis.
破 Osteoclast formation inhibitor containing IL-26.
The osteoclast formation inhibitor according to claim 5, wherein the osteoclast is a human osteoclast.
The osteoclast formation inhibitor according to claim 5 or 6, wherein the interleukin-26 is human interleukin-26.
The osteoclast formation inhibitor according to any one of claims 5 to 7, wherein the interleukin-26 is recombinant interleukin-26.
IL Use of IL-26 to produce an osteoclast formation inhibitor.