JPH1149700A - Therapeutic agent for pulmonary disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a prophylactic or therapeutic agent for pulmonary diseases including pulmonary hypertension by formulating a specific inhibitor as an active ingredient. SOLUTION: This prophylactic or therapeutic agent is obtained by formulating a substance that inhibits the acceleration of the monocytes and/or macrophages migration. As a substance promoting the aggregation of monocytes and/or macrophage cells to the pathogenic site, are cited monocyte chemotactic factor having strong monocytes and/or macrophage migratory activity, CC chemokine or CX3C chemokine. As the inhibitory substance, are preferably cited monocyte migratory activating factor inhibitor (particularly monocyte migratory-activating factor-neutralizing antibody is preferred). This therapeutic agent can treat a primary pulmonary hypertension as primary pulmonary hypertension or pulmonary hyper tension conjugated by collagen disease or secondary pulmonary hypertension caused by a variety of other diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a drug for treating or preventing a pulmonary disease, particularly pulmonary hypertension.

[0002]

2. Description of the Related Art Pulmonary hypertension is roughly classified into primary pulmonary hypertension and secondary pulmonary hypertension. Among these, primary pulmonary hypertension includes primary pulmonary hypertension, which causes pulmonary arterial pressure to rise due to unexplained increase in pulmonary vascular resistance and death in several years, and pulmonary hypertension associated with collagen disease. Secondary pulmonary hypertension has a tendency to increase in recent years because pulmonary arterial pressure is increased secondarily due to various causes such as pulmonary embolism and congenital heart disease. In pulmonary hypertension, right heart failure may occur due to increased load on the right heart.

On the other hand, monocyte chemotactic activator (Macrophage C
hemotactic Activating Factor: MCAF)
Is MCP-1 (Monocyte chemoatractant protein-1)
And GDCF (glioma-derived monocyte chemotactic fac
tor), a protein consisting of 76 amino acids, having four cysteine groups, and identification and gene cloning of MCAF have been reported (K. Matsushima et a
l., J. Exp. Med., 169, 1485-1490, 1989.). Hereinafter, in the present invention, MCAF including MCP-1 and GDCF is abbreviated as MCAF.

[0004] In vivo, MCAF is derived from various cells such as fibroblasts, vascular endothelial cells, smooth muscle cells, and various tumor cells in addition to blood cells such as monocytes, macrophages, and lymphocytes. 1, TNF, IFN-γ, L
It is reported that it is produced by stimuli such as PS.

[0005] MCAF is a member of the CC chemokine family, which has a similar number of amino acids and arrangement of cysteine residues. The CC chemokine family is a chemotactic bioactive substance having a feature that two cysteines are linked in the amino acid sequence (N. Mukaida et al., Microbiol. Imm
unol. 36, 773-789, 1992). CC chemokines are also beta
Also called chemokines (Oppenheim et al., Annual R
ev. Immunol., 9, 6171991). The CC chemokine family has about 30% homology with each other at the amino acid level.
Having. In addition to MCAF, other proteins belonging to this family include RANTES, LD78, ACT
2, I-309, MCP-2, MCP-3, JE, MI
P-1α, MIP-1β, TCA-3 and the like are known as structural analogs of MCAF.

Conventionally, vasodilator drugs, anticoagulants, antiplatelet drugs and the like have been used for pulmonary hypertension. In recent years, inhalation therapy of nitric oxide gas having vasodilatory properties has been performed. Inotropic agents and diuretics have also been used to treat right heart failure, which is known to be caused by pulmonary hypertension.

[0007]

However, the effects of these vasodilators, anticoagulants, and antiplatelets are not always sufficient, and it is desired to develop an effective therapeutic agent for pulmonary hypertension. Although no effect has been confirmed so far, it is desired to develop an effective therapeutic agent by inhibiting accumulation of monocytes and / or macrophages in vivo in pulmonary hypertension.

[0008]

SUMMARY OF THE INVENTION It is an object of the present invention to provide prevention and treatment of pulmonary diseases such as pulmonary hypertension, for which existing drugs have not been effective. That is, the present invention is a therapeutic agent for pulmonary disease, comprising as an active ingredient a substance that suppresses accumulation of monocytes and / or macrophages at the local pathology. Enable treatment.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, the therapeutic agent of the present invention is a therapeutic agent for pulmonary diseases containing as an active ingredient a substance that suppresses accumulation of monocytes and / or macrophages at a local pathogen.

[0010] Substances that promote the accumulation of monocytes and / or macrophages in the local pathogen include MCAF, which has a strong monocyte and / or macrophage migration activity,
CC chemokines such as ANTES and MIP-1α and Frac
talkine and other CX3C chemokines.

The substance of the present invention which inhibits the accumulation of monocytes and / or macrophages in the affected area includes inhibiting the production of substances having a migration activity of these monocytes and / or macrophages, and binding to these substances. There are drugs that inhibit the action of these substances or inhibit the action by binding to the receptor of these substances, all of which are included in the present invention.

Representative examples of substances having such an action include antibodies and low molecular weight compounds. Hereinafter, MCAF will be described as a typical monocyte and / or macrophage migrating substance. Adaptable to substances.

The antibody in the present invention includes the above-mentioned MCA
An antibody against an attractant such as F or an antibody against the MCAF receptor may be any antibody capable of inhibiting the binding of the attractant to monocytes or macrophages, and may be a monoclonal antibody or a polyclonal antibody. You may. Moreover, the animal from which it is derived is not particularly limited, and may be derived from any animal such as human, mouse, rat, rabbit, dog, goat and the like. Furthermore, various modified antibodies may be used as long as the binding between the attractant and monocytes or macrophages can be inhibited. For example, an antibody (for example, an antigen-binding fragment such as a Fab fragment or F (ab ') 2 fragment) in which a partial sequence of the antibody has been mutated, substituted, deleted or inserted, or a human antibody It may be a modified antibody (chimeric antibody or humanized antibody) artificially reconstituted to have it, or a modified antibody bound or mixed with various molecules such as polyethylene glycol.
The “antibody” referred to in the claims of the present application also includes these modified antibodies.

The neutralizing activity of the antibody used in the present invention (that is, the inhibitory activity of MCAF) can be confirmed by a competitive inhibition experiment of the action of MCAF. For example, MCAF
Can be confirmed by inhibiting the monocyte migration activity. Alternatively, the neutralizing activity of the antibody can be quantified by the method described in Example 2 below, to the extent that MCAF inhibits binding to human leukemia-derived cell line THP1. That is, isotope-labeled MC
In the AF binding experiment, the relationship between the molar ratio of the neutralizing antibody to the labeled MCAF and the inhibitory ability can be used as an index of the neutralizing activity. The antibody of the present invention is not particularly limited as long as it achieves the purpose of effectively inhibiting the action of MCAF in vivo. However, for example, in the above-described experiment for inhibiting the binding to THP1 cells, a compound that effectively inhibits the binding to MCAF at a molar ratio of 1 to 1000 times is used. Desirably, a compound that effectively inhibits the reaction at a molar ratio of 1 to 100 is used. Here, “effectively inhibits” refers to those having an inhibition rate of 30% or more, preferably 50% or more, more preferably 80% or more in the above experiment.

[0015] Among the antibodies that can be used in the present invention,
For example, an antibody against MCAF can be obtained by immunizing a mammal using MCAF as an immunogen and, if it is a polyclonal antibody, from the antiserum.

A monoclonal antibody can be obtained as follows. That is, a mammal is immunized using MCAF as an immunogen, and antibody-producing cells such as spleen cells and lymphocytes obtained from the immunized animal are fused with myeloma to obtain an antibody-producing hybridoma, and MCAF is selected from the obtained hybridomas. A clone producing an antibody that specifically binds to is selected using a technique such as ELISA. In order to provide a therapeutic agent with a high therapeutic effect, it is important at this stage to select an antibody that has as high an affinity as possible for the antigen. Next, the obtained hybridoma is cultured, and the monoclonal antibody can be purified using the culture supernatant as a material or ascites obtained by inoculating the hybridoma into an appropriate animal as a material. Purification can be performed by a conventional method, for example, an affinity carrier, an ion exchange carrier,
Purification can be carried out by an appropriate purification means in combination with column chromatography such as a hydrophobic carrier or a carrier carrying an antibody.

Since the obtained monoclonal antibody does not always have the activity of neutralizing MCAF, the neutralizing activity is examined and an antibody having neutralizing activity is selected.
The monoclonal antibody thus selected is used for the therapeutic agent of the present invention.

The above-mentioned modified antibody can be obtained by chemically modifying the obtained antibody, or the amino acid sequence of the antibody is modified as in a chimeric antibody. Such cells can be obtained from the cells obtained by culturing and growing recombinant cells into which the gene encoding the modified antibody has been introduced by the same means as described above. These methods are already established in this field.

The production of monoclonal antibodies against MCAF already has several examples (T. Yoshimura et al.,
J. Immunol., 147, 2229, 1991;
67986). However, none of these examples indicate that a monoclonal antibody with neutralizing activity is useful for pulmonary hypertension. Of course, there is no example that has demonstrated the possibility of treating these animal models by administering these antibodies to animals.

As the neutralizing antibody for MCAF, either a polyclonal or monoclonal antibody may be used, and a human monoclonal antibody is preferably used. For example, the monoclonal antibodies ME120 and MI-466 exhibiting human MCAF neutralizing activity described in JP-A-6-205690 and the monoclonal antibody disclosed in JP-A-5-276986 can be mentioned.

The present inventors have shown that these antibodies are useful as a therapeutic agent for pulmonary diseases such as pulmonary hypertension by actually administering the antibodies thus obtained to a living body. Completed the invention. That is, the antibody of the present invention exhibits the activity of neutralizing the action of MCAF. Therefore, when the antibody of the present invention is administered to a living body, it neutralizes MCAF in the living body and treats a lung disease caused by MCAF.

The diseases that can be treated according to the present invention include primary pulmonary hypertension such as primary pulmonary hypertension and pulmonary hypertension associated with collagen disease, and secondary pulmonary hypertension caused by various diseases. Including pulmonary hypertension. In addition, pulmonary hypertension includes pulmonary heart and right heart failure, which are thought to be involved in its onset or exacerbation.

Among these, secondary pulmonary hypertension is a disease that temporarily impairs air passage through the airways and alveoli, chronic bronchitis, bronchial asthma, emphysema, pulmonary fibrosis, various types of pneumonia,
Includes pulmonary infections, malignancies, alveolar microlithiasis, post-pulmonary resection, severe hypoxia such as altitude sickness, and pulmonary hypertension due to chronic obstructive diseases of the upper respiratory tract. In addition, after thoracoplasty, which is a disease that temporarily injures thoracic movement, chronic neuromuscular diseases such as pleural bench and polio, obesity with alveolar hypoventilation, and lungs with idiopathic alveolar hypoventilation Hypertension, and even
Pulmonary thrombosis, a disease that primarily impairs the pulmonary vascular bed, pulmonary embolism, collagen disease, various arteritis, pulmonary hypertension due to pulmonary vein occlusion, mitral valve, a disease that temporarily impairs the left heart system Pulmonary hypertension due to stenosis, left heart failure, Eisenmenger syndrome, ventricular septal defect, and congenital heart disease, atrial septal defect, pulmonary hypertension due to patent ductus arteriosus, and adult respiratory distress Pulmonary hypertension due to a disease that causes hypoxemia, such as syndrome (ARDS), is included in secondary pulmonary hypertension.

Further, administration of the agent of the present invention may be accompanied by pulmonary hypertension or diseases causing pulmonary hypertension, for example, chronic bronchitis, bronchial asthma, emphysema, pulmonary fibrosis, various pneumonia, lung infection, malignant tumor Alveolar microlithiasis, obesity with alveolar hypoventilation, idiopathic alveolar hypoventilation, pulmonary thrombosis pulmonary embolism, various pulmonary arteritis, pulmonary vein obstruction, Eisenmenger syndrome, adult respiratory distress syndrome (ARDS) It is also effective for treatment.

The present invention also relates to vasodilators, nitric oxide, prostaglandin preparations, anticoagulants conventionally used for the treatment of pulmonary hypertension, and digitalis used for the treatment of right heart failure associated with pulmonary hypertension. The preparation may contain a drug that assists in the treatment of this disease, such as a cardiotonic or diuretic.

[0026] Low molecular compounds having MCAF inhibitory activity include phenylethanolamine and its derivatives and homopiperazine derivatives. Phenylethanolamine and its derivatives include catecholamine and its derivatives.
757. The homopiperazine derivative is a compound having a homopiperazine skeleton, for example, Brt.pat. 1,107,470, US Pat. No. 3,532,685, JP-A-3-2144, JP-A-7-1450
No. 60, including those cited in JP-A-60. Furthermore, JP-A-7-16
No. 5585, JP-A-8-208600, JP-A-8-310956, and the compounds described in JP-A-7-188025.

The therapeutic agent of the present invention contains the above-mentioned compound and / or antibody as an active ingredient, may contain only the above-mentioned antibody alone, or may contain a pharmaceutically acceptable carrier or additive. May also be included. Examples of such carriers and pharmaceutical additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, aqueous solutions Dextran, sodium carboxymethyl starch, pectin,
Methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, polyethylene glycol, petrolatum,
Examples include paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, and surfactants that are acceptable as pharmaceutical additives. The actual additive is appropriately or in combination selected from the above depending on the dosage form of the therapeutic agent of the present invention, but is not limited to these.

The administration method is not particularly limited. It is appropriately selected from local administration and systemic administration for the purpose of administration. As a local administration form, an external preparation, a local injection and the like can be suitably carried out. The external preparation is selected from coating agents such as ointments, gels, creams, emulsions and solutions, patches such as tapes and patches, and sprays such as sprays and powders. The agent for systemic administration is appropriately selected from injections and sustained-release preparations using various carriers.

An example of a specific composition of a preparation that can be used as an injection includes 50 mg of a drug and 10 ml of a phosphate buffer, but is not particularly limited thereto.

The effective dose is 1 kg body weight per day.
It is selected in the range of 0.001 mg to 1000 mg per g. Preferably from 0.01 mg / kg to 10 mg / kg
It is selected in the range of mg. The above-mentioned dose varies depending on the condition, and is not limited to these values. The frequency of administration is usually selected within the range of once or twice a day, or once every two to several days, but is not limited thereto.

[0031]

The present invention will be described more specifically below with reference to examples.

Example 1 Preparation of polyclonal antibody against rat MCAF: A goat anti-rat IgG antibody was prepared according to the method described in Japanese Patent Application No. 7-182548. Neutralizing activity of the polyclonal antibody was confirmed by an experiment of inhibiting calcium influx induced by MCAF in human monocyte-derived cell line THP-1 cells and a test of inhibiting migration of rabbit peripheral blood lymphocytes.

Example 2 Effect of Goat Anti-Rat Polyclonal Antibody on Rat Experimental Pulmonary Hypertension Model: Rat (SD rat) was used as a pulmonary hypertension model, and 60 mg / kg MC was used as an MCT group.
T was injected subcutaneously, and sterile physiological saline was injected as a control group.

In the present pulmonary hypertension model rat, macrophages in the lung tissue increased as MCAF expression in the tissue increased, and MCA in bronchoalveolar lavage fluid was observed.
Increased alveolar macrophages were observed as F expression increased. This indicates that the expression of MCAF plays an important role in the migration and activation of macrophages.

In this model, the ratio of right ventricular weight / (left ventricular weight + ventricular septum weight) ratio (hereinafter RV / (LV + S)) was calculated as an index of the progression of pulmonary hypertension. RV / (LV + S)
Was 0.267 ± 0.01 in the saline-administered group, while 0.685 ± 0 in the control group, MCT + control IgG antibody-pretreated group.
05. On the other hand, in the MCT + anti-MCAF antibody pretreatment group, 0.4
It was 77 ± 0.076, which was significantly suppressed as compared with the control IgG administration group. Furthermore, the right ventricular systolic pressure was measured to be 22.7 ± 0.021 mmHg in the saline-administered group, whereas it was 72.5 ± 0.05 mmH in the MCT + control IgG antibody-pretreated group of the control group.
g. On the other hand, 53.5 in the MCT + anti-MCAF antibody pretreatment group
± 8.8 mmHg, which was significantly suppressed as compared to the control group.

[0036]

The above results indicate that administration of a substance that inhibits the activity of MCAF is effective in treating and preventing pulmonary hypertension.

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Masanobu Narito 2-2-1 Nihonbashi Muromachi, Chuo-ku, Tokyo Toray Industries, Inc. Tokyo Office

Claims (4)

[Claims] [1] A therapeutic or preventive agent for pulmonary diseases, comprising an inhibitor of a substance which promotes the migration of monocytes and / or macrophages as an active ingredient. 2. The monocyte and / or macrophage migration promoter inhibitor is a monocyte chemotactic activator inhibitor.
A therapeutic or prophylactic agent for the pulmonary disease according to the above. 3. The method according to claim 2, wherein the monocyte chemotactic activator inhibitor is a monocyte chemotactic activator neutralizing antibody. 4. The pulmonary disease is pulmonary hypertension.
A therapeutic or prophylactic agent for the pulmonary disease according to the above.