JPH10218791A - Therapeutic agent of chronic rheumatoid arthritis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject therapeutic agent, capable of suppressing an induction of a vascular penetrating property and a vascular formation, and also a vascularization inducing a pannus formation by using an antagonist for a vascular epithelial cell growth factor/a vascular penetration factor as an active ingredient. SOLUTION: This therapeutic agent of chronic rheumatoid arthritis, contains an antagonist for a vascular epithelial cell growth factor(VEGF)/a vascular penetrating factor(VPF) [e.g.; a mouse/human chimeric antibody or humanized antibody affecting the VEGF/VPF (more concretely, IgG type, TgA type, etc.), or an inactive VEGF/VPF capable of inhibiting the action of the VEGF/VPF] as an active ingredient. Preferably, the therapeutic agent is administered non orally by injecting intravenously, intramuscularly, intraperitoneally or subcutabeously and systemically or locally in a range of 100-5000mg/patient divided by <=10 times in the case of human.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a vascular permeability factor (V
PF) [also called vascular endothelial cell growth factor (VEGF),
In this specification, they are collectively referred to as "VEGF / VPF".] The present invention relates to a therapeutic agent for rheumatoid arthritis or a diagnostic composition comprising an antagonist as an active ingredient.

[0002]

2. Description of the Related Art Rheumatoid arthritis (hereinafter referred to as RA)
Is a chronic inflammatory disease of unknown cause mainly involving the joint synovium, and the lesion site is not limited to the joint site, and it is not rare that the disease extends to the whole body. The inflammation that develops in the synovium of the joints eventually causes cartilage and bone destruction, resulting in the destruction of joint tissue,
RA patients are not only severely restricted both socially and at home, but also have a considerable financial burden. RA
Patients are 0.1-0.3% of the population and there is no regional difference.
Including surrounding diseases, the number of patients seems to have reached about 10 times the above. In the future, this number of patients is expected to increase further as the aging of society progresses.

At present, treatment of inflammatory diseases such as RA includes:
It is common to use steroids and non-steroidal anti-inflammatory drugs. Steroids significantly improve the symptoms of various inflammatory diseases, but the problem is that with prolonged administration, resistance may occur, gastrointestinal disorders, skin disorders, nephritis, etc. There may be certain side effects. Non-steroidal anti-inflammatory drugs, even if they temporarily suppress inflammatory symptoms, do not fundamentally treat inflammatory diseases.

On the other hand, with recent advances in immunology,
It has been found that the mechanism of RA joint destruction is related to infiltration of immunocompetent cells such as CD4-positive T cells into the synovium and abnormal expression of cell adhesion factors and cytokines. Therefore, therapy that administers antibodies targeting these cells and molecules is expected as a specific treatment that can suppress the joint lesions of RA. Already, anti-CD4 antibodies, anti-CD52 antibodies, anti-I
CAM-1 antibody, anti-TNF-α antibody, anti-CD5 antibody, anti-CD7 antibody, anti-I
The treatment of RA with L-2 receptor (IL-2R) antibody, anti-IL-6 antibody, and anti-IL-6 receptor (IL-6R) antibody has been reported. (H. Takahashi, et al, Current Therapy, 14 (6) 10
57-1061 (1996) and JP-A-8-208514).

[0005] However, the pathogenesis of RA is unknown at present, and the treatment of RA has a strong empirical component, and its effects cannot be understood unless many antirheumatic drugs are used for patients. There are many. In addition, rheumatism treatment often becomes ineffective after several years, even if the drug is initially well-recognized, which necessitates the use of a different antirheumatic drug. In view of the above, the emergence of a rheumatic drug having a different mechanism of action from the conventional ones is desired.

It is believed that VEGF / VPF not only stimulates vascular endothelial cell proliferation, but also induces vascular permeability and angiogenesis, resulting in chronic rheumatism with pannus formation characterized by angiogenesis. High levels are found in synovial fluid in patients (M. Nagashima, et.
al, J Rheumatol., 22 (9) 1624-1630 (1995) and RAFava
et al, J. Exp. Med., 180 (1) 341-346 (1994)). When a similar collagen-induced arthritis model with pannus formation was treated with AGM-1470 and cyclosporin, VEGF
Decreased concentrations have been reported (SJOliver et
al, Cell Immunol., 166 (2) 196-206 (1995)). Furthermore, when the chemotaxis of human umbilical cord vascular endothelial cells (HUVEC) in rheumatoid arthritis synovial fluid was measured in a Boyden chamber, anti-hVEGF antibody (A4.6.
It has been reported that the addition of 1) suppresses the chemotaxis of HUVEC (Japanese Patent Laid-Open No. 8-502514,
See Example 6.)

[0007]

SUMMARY OF THE INVENTION
The inventors of the present invention have thought that if angiogenesis in chronic rheumatoid arthritis is suppressed, a therapeutic agent for rheumatism having a new mechanism of action can be obtained, and as a result of intensive studies, the present inventors have completed the present invention.

[0008]

That is, the present invention relates to
The present invention relates to a therapeutic agent for rheumatoid arthritis containing an EGF / VPF antagonist as an active ingredient. Also, this VE
By labeling the GF / VPF antagonist with an appropriate label, a composition for diagnosing rheumatoid arthritis can also be obtained. As the label, a radioisotope label, a fluorescent label, an enzyme label, or the like can be used.

In the present invention, VEGF / VPF antagonist refers to VEGF / V having a specific cell growth promoting activity on vascular endothelial cells or a vascular permeability promoting activity.
It has a function of inhibiting the function of the PF, and may have any form as long as it has the function.
Most commonly, an antibody that acts on VEGF / VPF, or a portion thereof, including, but not limited to, an inactive VEGF / VPF that inhibits the action of VEGF / VPF, or a portion thereof. , VEGF
/ VPF, which impairs the function of the receptor, for example, an antibody against a vascular permeability factor receptor, or a part thereof, and an agent that suppresses VEGF / VPF production itself. Examples of the antibody include a mouse antibody and the like, and a mouse / human chimeric antibody or a humanized antibody is preferred. Examples of the chimeric antibody or humanized antibody include an IgG type or an IgA type, and examples of the IgG isotype include IgG1 and IgG.
2, IgG3 or IgG4.

The therapeutic agent for rheumatoid arthritis of the present invention can be administered preferably parenterally, for example, systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like. In addition, it can take the form of a kit of pharmaceutical compositions together with at least one pharmaceutical carrier or diluent. The dose of the therapeutic agent for rheumatoid arthritis of the present invention to humans varies depending on the disease state, age and administration method of the patient, but it is necessary to select an appropriate amount as appropriate. For example, no more than 10 divided doses can be selected in the range of approximately 100-5000 mg / patient. However, the therapeutic agent for rheumatoid arthritis of the present invention is not limited to these doses.

The remedy for rheumatoid arthritis of the present invention can be formulated according to a conventional method. For example, an injectable preparation may be prepared by dissolving a purified VEGF / VPF antagonist in a solvent such as a physiological saline solution, a buffer, and the like, and adding an anti-adsorption agent such as Tween 80, gelatin, human serum albumin (HSA) and the like. It may be added or lyophilized for reconstitution before use. As an excipient for lyophilization, for example, sugar alcohols such as mannitol and glucose and sugars can be used.

According to the present invention, the action of VEGF / VPF, which induces vascular permeability and angiogenesis, is suppressed by the action of a VEGF / VPF antagonist.
us) by suppressing the angiogenesis that causes the formation
It is believed that RA has an effect of treating or preventing inflammation.

[0013]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Reference Examples and Examples, but the present invention is not limited thereto.

Reference Example 1 (Preparation of GST-VEGF / VPF and preparation of anti-VEGF / VPF rabbit polyclonal antibody) Human cervical cancer cell HeLa / v5 (deposited with the Research Institute of Microbial Industry: Deposit No. No. 13185 "), the cDNA of VEGF / VPF was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), and the obtained protein was used as an antigen to immunize rabbits in a conventional manner. IgG was fractionated from the serum with the increased antibody titer by DEAE chromatography.

In order to confirm that the obtained antibody reacts with active VEGF / VPF, a protein was taken out from the medium in which HeLa / v5 was cultured by salting out with ammonium sulfate, and developed by 12% SDS-PAGE, followed by gelation. Was transferred to a nylon membrane, and the antibody obtained by the above immunization was reacted and subjected to Western blot. VEGF / V
Specific cross-reaction was observed at a position corresponding to the molecular weight of PF, confirming that the prepared antibody recognized active VEGF / VPF molecules.

Example 1 (Effect of Inhibition of Onset by Anti-VEGF / VPF Antibody on Collagen-Induced Arthritis Model Mouse) (1) Preparation of Arthritis Model Mice 8 mg of bovine type II collagen (Elastin Product) was
Stir at 4 ° C. overnight with 0.05 ml of 0.05N acetic acid aqueous solution to dissolve to give a colorless, transparent and viscous liquid. The same volume of complete Freund's adjuvant (Difco) was added to this solution to completely emulsify, and 0.1 m was injected into the tail base skin of 9-week-old DBA / 1JNCrJ mouse (Charles River Japan).
1 / animal. The same treatment was repeated 21 days after this administration.

(2) Treatment with Anti-VEGF / VPF Antibody Anti-VEGF / VPF rabbit polyclonal antibody was treated with 200 μg / rabbit 12 times every other day from the day of the second collagen sensitization.
Subcutaneous administration was performed in mice (dosage volume was 0.2 m
l). As a control group, 0.2 ml of physiological saline or 200 μg of an immunoglobulin fraction obtained from rabbit serum / mouse (DAKO X903, administration amount: 0.2 ml) was similarly subcutaneously administered.

(Evaluation method) Untreated control group without collagen treatment (group A), collagen-induced arthritis (CIA) treated / untreated (administered saline) control group (group B), CIA
Treatment / rabbit serum immunoglobulin fraction administration group (group C),
The following evaluation was performed for the CIA-treated / anti-VEGF / VPF rabbit polyclonal antibody administration group (Group D), and the anti-VEGF / V
The effect of treatment (suppression of onset) by the PF antibody was measured:

The number of joints From the day of the boost, every other day, the limbs of the mice were measured according to the following criteria to evaluate the therapeutic effect. The results are shown in FIG. 0: normal 1: slight swelling and / or erythema 2: marked edema swelling 3: joint stiffness

The thickness of the limbs (Paw thickness) of both hind limbs was measured using a constant-pressure dial gauge on alternate days from the day of boosting. The results are shown in FIG.

From the results of FIGS. 1 and 2, the anti-VEGF / VPF
The rabbit polyclonal antibody administration group significantly suppressed the onset of CIA, which is a human rheumatoid model, as is clear from comparison with the non-treatment group and the rabbit serum immunoglobulin administration group.

Example 2 (Treatment of Arthritis Model Mice with Anti-VEGF / VPF Antibody (Suppression of Exacerbation)) (1) Preparation of Arthritis Model Mice 8 mg of bovine type II collagen (Elastin Product) was
Stir at 4 ° C. overnight with 0.05 ml of 0.05N acetic acid aqueous solution to dissolve to give a colorless, transparent and viscous liquid. The same volume of complete Freund's adjuvant (Difco) was added to this solution to completely emulsify, and 0.1 m was injected into the tail base skin of 9-week-old DBA / 1JNCrJ mouse (Charles River Japan).
1 / animal. The same treatment was repeated 21 days after this administration.

(2) Treatment with Anti-VEGF / VPF Antibody 200 μg / mouse of anti-VEGF / VPF rabbit polyclonal antibody was administered to mice that developed arthritis with a joint score of 1 or more in any of the limbs in Example 1 for 9 days every other day. One subcutaneous administration was performed (the dose was 0.2 ml).
As a control group, 0.2 ml of physiological saline or 200 μg of an immunoglobulin fraction obtained from rabbit serum (DAKO X
903, the administration volume was 0.2 ml).

(Evaluation method) Collagen-induced arthritis (CI
A) treated / untreated (administered saline) control group (group E),
CIA treatment / rabbit serum immunoglobulin fraction administration group (F
Group), CIA treatment / anti-VEGF / VPF rabbit polyclonal antibody administration group (group G)
The therapeutic effect of the F / VPF antibody was measured.

Joint scores The limbs of the mice were measured every other day from the day of onset according to the following criteria, and the therapeutic effect was evaluated. The results are shown in FIG. 0: normal 1: slight swelling and / or erythema 2: marked edema swelling 3: joint stiffness

The thickness of the limbs (Paw thickness) of both hind limbs was measured using a constant-pressure dial gauge every other day from the onset date.
It was shown to.

From the results of FIGS. 3 and 4, the anti-VEGF / VPF
The rabbit polyclonal antibody group significantly suppressed the exacerbation of collagen-induced arthritis (CIA), a human rheumatoid model after onset, as is clear from the comparison between the untreated group and the rabbit serum immunoglobulin group. Was.

[0028]

Industrial Applicability According to the present invention, there is provided a therapeutic agent for rheumatoid arthritis having a new action mechanism of inhibiting the angiogenesis and angiogenesis by VEGF / VPF and suppressing the progression of RA accompanying angiogenesis. Is done.

[Brief description of the drawings]

FIG. 1. Anti-activity against a collagen-induced arthritis model mouse
It is a figure which shows the onset suppression effect by VEGF / VPF antibody.

[FIG. 2] Antibody against collagen-induced arthritis model mouse
It is a figure which shows the onset suppression effect by VEGF / VPF antibody.

[FIG. 3] Anti-antibody against collagen-induced arthritis model mouse
FIG. 3 is a view showing a therapeutic effect of a VEGF / VPF antibody.

[FIG. 4] Antibody against collagen-induced arthritis model mouse
FIG. 3 is a view showing a therapeutic effect of a VEGF / VPF antibody.

Claims (3)

[Claims] 1. A therapeutic agent for rheumatoid arthritis comprising a VEGF / VPF antagonist as an active ingredient. 2. The therapeutic agent for rheumatoid arthritis according to claim 1, wherein the VEGF / VPF antagonist is an antibody against VEGF / VPF. 3. A composition for diagnosing rheumatoid arthritis, comprising a labeled VEGF / VPF antagonist.