JP2002371006A - Prophylactic and/or progress inhibitor against pulmonary fibrosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a prophylactic and/or a progress inhibitor against pulmonary fibrosis, especially drug-induced pulmonary fibrosis. SOLUTION: This prophylactic and/or progress inhibitor against pulmonary fibrosis is characterized by comprising a thrombomodulin-like protein as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an agent for preventing and / or preventing the progression of pulmonary fibrosis, which comprises a thrombomodulin (hereinafter abbreviated as "TM")-like protein.

[0002]

2. Description of the Related Art Pulmonary fibrosis is a disease in which alveolar structures are destroyed due to chronic inflammation in the alveolar wall and an increase in collagen fibers, and eventually leads to respiratory failure. More than 100 factors, such as toxic gases and various drugs, have been identified as triggers that cause early alveolar damage, but the cause is unknown in two-thirds of cases (Japanese Rinsho separate volume, region-specific syndrome series) No. 3, Respiratory Syndrome (Vol. 1), 591-594
P. 1994). In recent years, drug-induced pulmonary fibrosis has been increasing with the increase in drugs used for diagnosis, treatment, prevention, and the like of various diseases. The causative agents of drug-induced pulmonary fibrosis that have been reported to date are over 100 types, and typical agents are shown in Table 1. 1980-
According to a total of 185 cases in Japan in 1989, pulmonary fibrosis due to anticancer drugs and immunosuppressive drugs was the largest at 50.3%,
Next, 23.8 of pulmonary fibrosis caused by antibiotics and chemotherapeutics
%, Pulmonary fibrosis due to gold preparations continues at 15.7%, but the content has changed with the times due to advances in drugs used and countermeasures against side effects, and recently, amiodarone, an antiarrhythmic drug, herbal medicine Shosaikoto, IFN-α and GC
Pulmonary fibrosis due to cytokine therapy such as SF has also been reported (Latest Medicine, 45, 962-972, 1990).
Year).

[Table 1]

[0003] Pulmonary fibrosis caused by anticancer drugs and immunosuppressive drugs causes 1
By 979, 21 were reported as causative agents, including bleomycin, busulfan, cyclophosphamide,
Carmustine, methotrexate, procarbazine and the like were representative. Among them, bleomycin accounts for about half of drug-induced pulmonary fibrosis and is known to develop in a dose-dependent manner. Its frequency of onset is around 10%, and it is often fatal. Needs careful attention. Therefore, recently peplomycin has been used in place of bleomycin,
This time, pulmonary fibrosis due to peplomycin is increasing (Japanese Rinsho Separate Volume, Regional Syndrome Series No. 4, Respiratory Syndrome (2nd volume), pp. 825-827, 1994).

[0004] Pulmonary fibrosis due to antibiotics and chemotherapeutic agents
In the past, the number and type were limited in a small number, but since 1980, the type and incidence have rapidly increased, and it has been reported that more than 30 drugs induce pulmonary fibrosis.
Particularly recently, drug-induced pulmonary fibrosis has been reported for penicillins, macrolides, cephems, tetracyclines, aminoglycosides, antituberculosis drugs and other antibacterial agents (Japan Clinical Clinic Separate Volume, Area-Specific Syndrome Series No. 4, Respiratory Syndrome (2nd volume),
825-827, 1994).

An important point in the diagnosis of drug-induced pulmonary fibrosis is that
A causal relationship between the onset of symptoms such as coughing, dyspnea, and fever is suspected, and interstitial shadows appear diffusely on the chest X-ray at the same time or with a slight delay. This means that eosinophils are increased.
That is, when symptoms such as cough, fever, and dyspnea appear during administration of a drug that may induce pulmonary fibrosis, drug-induced pulmonary fibrosis is suspected, and chest X-ray, CT,
Pulmonary function including arterial blood gas analysis, bronchoalveolar lavage (BA
L) and other tests are performed for diagnosis. In addition, when it is presumed to have developed due to a type IV allergic reaction, it is necessary to prove that a sufficient number of sensitized lymphocytes to the causative drug exist in the peripheral lymphocytes of the patient. In addition, proving macrophage migration inhibitory factor in the lymphocyte culture supernatant and a lymphocyte stimulation test are performed as in vitro test methods.

At present, steroid therapy is used for drug-induced pulmonary fibrosis. However, lesions often remain after steroid therapy. In addition, side effects of steroids and acute exacerbations due to weight loss and discontinuation are often observed, and are not at a level that is satisfactory in clinical practice. Further, when drug-induced pulmonary fibrosis is suspected, it is necessary to first stop the administration of the drug used, so that sufficient treatment of the underlying disease cannot be expected. Therefore, it is desired to prevent or prevent the progression of pulmonary fibrosis by these drugs, to administer even a little ideal drug, and to continue the treatment of the underlying disease to enhance the therapeutic effect. Recent findings have shown pirfenidone to be effective in treating idiopathic pulmonary fibrosis in a Phase II clinical trial in the United States (American Journal of Respiratory and
Critical Care Medicine (American)
journal of respiratory an
d critical care medicin
e) 159, 1061-1069, 1999
Year). However, there are many cases in which treatment with pirfenidone is ineffective, and development of a new therapeutic agent is desired in the future (Internal Medicine, 84, 858-863, 1999).

[0007] TM is a glycoprotein that was discovered by Ezmon et al. In 1981 as an anticoagulant substance present on the surface of vascular endothelial cells, and in 1987 its entire primary structure was revealed by Suzuki et al. TM binds to thrombin and suppresses the procoagulant effect of thrombin, and also exhibits an anticoagulant effect by remarkably promoting the activation of protein C by thrombin. Therefore, TM has been proved or suggested to be effective for the prevention and treatment of systemic hypercoagulable disease (DIC) and various thrombosis. However, it is not known at all that TM or TM-like protein has a preventive or progress-inhibiting effect on pulmonary fibrosis, and it has not been suggested.

It has been suggested that transtracheal administration of activated protein C (hereinafter abbreviated as "APC"), a blood coagulation inhibitor, is effective in a mouse bleomycin-induced lung fibrosis model (Nippon Respiratory System). Academic Journal, 37
Volume extra number, 177 pages, Abstract number 4FB-45, 1999
Year). However, activated protein C is said to be elevated during the inflammatory response to α1-antitrypsin (The International Journal of Biochemistry and Cell Biology (The inter
national journal of bioch
chemistry & cell biology), 29
Vol., Pp. 1501-1511, 1997), which are known to be inactivated (Thrombosis and Hamostasis).
emostasis), 62, 756-762, 1
In 989), there are cases where the effect of APC administered pulmonary administration cannot be expected for some patients. APC is considered to have a short half-life in blood and need to be continuously administered when administered intravenously. Since there are APC neutralizing substances such as protein C inhibitor (hereinafter abbreviated as “PCI”) and α1-antitrypsin in the living body, it is considered that a large amount of APC needs to be administered to achieve the effect. , P
It has been reported that CI has an anticoagulant effect (Thrombos and hemostasis (Thrombos)
is and Haemostasis), volume 84, 5
4-58, 2000).
Causes a decrease in coagulation, which may lead to abnormal coagulation, and furthermore, it is feared that antibody production may be induced by administering a large amount of protein to humans.
Whether APC can be used clinically is completely unknown.

[0009]

In order to prevent and / or prevent the progress of pulmonary fibrosis, in particular, to prevent and / or prevent the progress of drug-induced pulmonary fibrosis, the treatment of the underlying disease with the drug is continued and the therapeutic effect is enhanced. This is a very important issue in that respect. Therefore, an object of the present invention is to provide an agent for preventing and / or preventing pulmonary fibrosis, particularly drug-induced pulmonary fibrosis.

[0010]

Means for Solving the Problems Under the circumstance that the usefulness of the TM-like protein for preventing and preventing the progression of pulmonary fibrosis is unknown, the present inventors have proposed the use of an animal model system to determine the TM-like protein As a result of intensive studies on the effects of preventing and progressing fibrosis, surprisingly, the present inventors have found that TM-like proteins have a strong effect of preventing and progressing pulmonary fibrosis, and completed the present invention.

That is, the present invention provides 1) an agent for preventing and / or preventing progression of pulmonary fibrosis, which comprises a TM-like protein as an active ingredient.

The present invention also provides the agent for preventing and / or progressing pulmonary fibrosis according to 1), wherein 2) the pulmonary fibrosis is drug-induced.

Further, the present invention provides the agent for preventing and / or preventing the progression of pulmonary fibrosis according to 2), wherein the agent is an anticancer agent.

The drug containing the TM-like protein of the present invention comprises:
In a mouse bleomycin-induced lung fibrosis model, it has an effect of suppressing fibrosis of the lung. It has been confirmed that TM-like proteins have a long half-life in vivo, are stable in vivo, and exhibit sustained effects, and exhibit favorable pharmacokinetics as a pharmaceutical (Thrombosis and Haemostasis).
Statis), 73, 805-811, 19
1995). In addition, thrombin is known to promote the proliferation of fibroblasts that produce fibers such as collagen important for fibrosis via its receptor, whereas TM-like protein inhibits the binding of thrombin to its receptor. It is known that cell growth is suppressed by performing (Biokimika et Biophysica actor (Bioc
himica et biophysica act
a), 1451, pp. 173-186, 1999).

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below, but the present invention is not limited thereto.

The active ingredient contained in the agent for preventing and / or progressing pulmonary fibrosis of the present invention is a TM-like protein.
In the present invention, the term "TM-like protein" refers to TM and peptides having an amino acid sequence of a biologically necessary part of TM or analogs thereof. That is,
The TM-like protein used in the present invention includes all proteinaceous substances that bind to thrombin, suppress the procoagulant action of thrombin, and remarkably promote the activation of protein C by thrombin. Therefore, any of those purified from natural products (hereinafter abbreviated as “natural origin”) or those produced by genetic engineering may be used, and modified or chimeric types obtained by genetic engineering techniques may be used. The TM-like protein used in the present invention is not particularly limited as long as it has the above characteristics, and includes human-derived TM or TM-like protein, bovine-derived TM or TM-like protein, and the like.
It may be of natural origin or recombinant. In the case of a pharmaceutical, a human-derived TM-like protein (hereinafter, abbreviated as "human TM-like protein") is desired. Further, a soluble TM-like protein having high solubility is more preferable,
For example, it is a naturally occurring human soluble TM-like protein or a human soluble TM-like protein obtained by genetic recombination.
Particularly preferred is a soluble TM-like protein derived from human urine. In addition, various TM-like proteins having partial structures of the amino acid sequence of TM produced by using genetic recombination technology and the like, which have an action of preventing or preventing the progression of pulmonary fibrosis are also included in the present invention. The TM-like protein is a therapeutic agent for DIC currently under clinical development, has few side effects such as bleeding tendency associated with heparin, and is expected to be highly safe as a pharmaceutical (Thrombosis and hemostasis (Thrombosis and Thrombosis and Hemostasis)). Haem
ostasis) 77, 789-795, 19
1997).

Such TM-like proteins are naturally derived, for example, as human placenta-derived TM and human urine-derived soluble TM-like protein disclosed in JP-A-60-199819. The molecular weight in the non-reduced state disclosed in JP-A-3-86900 is 55,000-
58,000 and 60,000 to 65,000 soluble TM-like proteins and a 72,000 molecular weight in a non-reducing state disclosed in JP-A-3-218399.
± 3,000 and 79,000 ± 3,000 soluble TM-like proteins, Journal of Biochemistry
y), 113, 433-440, 1993, having a molecular weight of 55,000 and 6 in the non-reduced state.
000 soluble TM-like proteins and the European Journal of Biochemistry (Europe
ean Journal of Biochemist
ry), 221 pp. 1079-1087, 1994.
And 63,000 soluble TM-like proteins.

As for the recombinant type, for example, as a recombinant human soluble TM-like protein,
No. 6219, a soluble TM-like protein containing at least the amino acid sequence at positions 345 to 462 from the amino terminal, and WO92 / 00325, which contains at least the amino acid sequence at positions 4-446 from the amino terminal. And soluble TM-like proteins.

Also, Japanese Patent Application Laid-Open Nos. Hei 2-255699, Hei 3-2590084 and Hei 5-213
No. 508150, a TM-like protein disclosed in Japanese Patent Application Laid-Open No. 998/1992, which partially lacks the constituent amino acids of TM.
Publication, WO93 / 15755, WO93 / 25
No. 675 or Japanese Patent Application Laid-Open No. 5-310787 discloses a TM-like protein in which a part of the protein is substituted with another amino acid. For example, those in which oxidation is prevented by substituting methionine with another amino acid (Japanese Patent Application Laid-Open No. 5-508150), or those in which degradation by a protease is prevented by modifying the amino acid sequence (WO93 / 15755) Gazette).

Further, WO 91/04276, WO
No. 91/05803 or JP-A-3-1333
No. 80, a TM-like protein having a sugar chain containing chondroitin and / or chondroitin sulfate, and an O-glycoside sugar chain containing a bovine TM-derived acidic amino acid sequence disclosed in JP-A-6-279497. A binding site is added and a chondroitin sulfate sugar chain is bound to the TM-like protein, or the sugar chain of the O-glycosylation site region disclosed in WO92 / 03149 is modified or the O-glycosylation site region is deleted. A lost TM-like protein; a TM-like protein containing no chondroitin and / or chondroitin sulfate disclosed in JP-A-4-210700. further,
A TM-like protein which may include a part of an amino acid sequence such as human tissue plasminogen activator disclosed in Japanese Patent Publication No. 4-505554 is cited.

The pulmonary fibrosis in which the agent for preventing and / or progressing of the present invention is effective is not particularly limited, and drug-induced pulmonary fibrosis is preferred. The drug-induced pulmonary fibrosis is not particularly limited, for example, anticancer drugs / immunosuppressants, antibiotics / chemotherapeutics, antihypertensive / diuretics, gold preparations, anti-inflammatory / analgesics,
Pulmonary fibrosis induced by antiarrhythmic drugs, herbal medicines, cytokines and the like. More specifically, mention is made of pulmonary fibrosis induced by all the drugs summarized in Table 1.
Among these pulmonary fibrosis, lung fibrosis induced by an anticancer agent / immunosuppressant, antibiotic, or chemotherapeutic agent is preferable, and lung fibrosis induced by an anticancer agent, particularly bleomycin, is more preferable.

The preparation of the present invention may contain a TM-like protein as an active ingredient, and can be produced by any known pharmaceutical production method. The TM-like protein may be a suitable carrier or solvent generally used as a drug, for example, sterile water, physiological saline, vegetable oil, mineral oil, higher alcohol, higher fatty acid, harmless organic solvent, and the like. Engineered human serum albumin, excipients, colorants, emulsifiers, suspending agents, surfactants, dissolution aids, anti-adsorption agents, stabilizers, preservatives,
Injections, oral inhalants, eye drops, nasal absorption suitable for effective ecological administration by appropriately combining with optional ingredients such as humectants, antioxidants, buffers, tonics, analgesics, etc. And oral preparations, preferably oral inhalants or injections, more preferably injections. The preparation form of the injection can be provided, for example, as a lyophilized product or a liquid preparation for injection, etc., and it is particularly preferable to provide the preparation as a lyophilized product. Examples of the formulation of oral inhalants include, for example, aerosol preparations and the like, in which fine particles of TM-like protein or TM-like protein and a pharmaceutical additive are dissolved, or TM-like protein is dissolved or suspended in an appropriate carrier or solvent. It can be provided in the form of a mixture or a lyophilized TM-like protein and a solution for dissolution or suspension in the same package to prepare a solution or suspension at the time of administration.

The method of administration for preventing and / or preventing the progression of pulmonary fibrosis by the agent of the present invention may be, for example, intraarterial injection, intravenous injection, subcutaneous injection, intramuscular injection, pulmonary administration, etc. Pulmonary administration, intravenous injection or continuous intravenous administration is preferred. In addition, subcutaneous administration, intraperitoneal administration, oral administration, and nasal administration can be performed as necessary. The daily dose is not particularly limited because it is determined (increased / decreased) according to the patient's individual body weight, specific symptoms, administration route, type or dose of a drug that induces pulmonary fibrosis, and the findings described below. However, for example, in the case of intravenous administration or pulmonary administration, the amount of TM-like protein is 0.01 to 1 per day.
000 μg / kg, preferably 0.1-500 μg / kg
kg, more preferably 1 to 200 μg / kg, and still more preferably 5 to 100 μg / kg. T
As for the M-like protein, it is desirable to administer the above-mentioned administration solution by a single intravenous injection or intravenously within several hours. Also,
Continuous administration may be performed intravenously over 24 hours. It is also desirable to administer the above dose once or several times a day by transpulmonary administration. When the TM-like protein is in the form of a solid, it is preferable to administer the TM-like protein as a solid fine powder (for example, a powder aerosol) using a metering spray device, a jet milling device, a ball milling device or the like. When the TM-like protein is a solution or a suspension, it is preferable to administer the TM-like protein as water-soluble fine particles (for example, a pressurized aerosol) by using a jet nebulizer or an ultrasonic nebulizer. The administration timing of the TM-like protein
For example, before administration of a drug that is likely to induce pulmonary fibrosis, simultaneous administration (both when the drug that induces pulmonary fibrosis and the TM-like protein are formulated in a single drug form and when they are separately formulated) ), Immediately, continuously or intermittently during administration, or preferably continuously or intermittently after administration, and these administrations can be administered in combination to induce pulmonary fibrosis. More preferably, it is administered simultaneously with the drug. In addition, the agent that induces pulmonary fibrosis may be administered separately or as a mixture.
In the case of anticancer drug-induced pulmonary fibrosis or the like, the TM-like protein or the like may be administered alone or intermittently during the administration period of the anticancer agent or the like. In addition, daily dosage, administration route, administration frequency or administration timing may be based on symptoms such as cough, dyspnea, fever, chest X-ray, CT, pulmonary function including arterial blood gas analysis, bronchoalveolar lavage (BAL) And the like, or a diagnosis of pulmonary fibrosis based on clinical laboratory findings such as an increase in leukocytes and an increase in eosinophils can be determined as appropriate.

[0024]

EXAMPLES The present invention will be described below with reference to experimental examples and examples, but the present invention is not limited to these examples.

Acquisition Example 1 (Acquisition of Soluble TM-like Protein Derived from Human Urine) A protein was prepared according to the method described in Japanese Patent Application Laid-Open No. 3-218399. That is, 100 L of human raw urine was filtered through an acrylic fiber to adsorb and remove urokinase in urine, and the passing urine was desalted and concentrated using an ultrafiltration membrane. Next, DEA
D-cellulose (Whatman) column, DIP-thrombin-agarose chromatography and Sephacryl S-300 (Pharmacia Fine Chemical)
Purification was performed sequentially using a column, and an active fraction was collected (hereinafter abbreviated as “UTM”). This fraction was dialyzed against distilled water overnight and freeze-dried.

Experimental Example 1 (Effect of TM-like protein on mouse bleomycin-induced pulmonary fibrosis model) Male ICR mice (Japan SLC, 12-13 weeks old) were rapidly intravenously administered 150 mg / kg of bleomycin from the tail vein. A mouse bleomycin-induced lung fibrosis model was prepared. In the UTM administration group, immediately before bleomycin administration, 1, 2, 3 and 4 days after bleomycin administration, UTM was rapidly intravenously administered at 3000 TMU / kg or 10000 TMU / kg from the tail vein. The normal group and the control group received a rapid intravenous administration of physiological saline instead of UTM. 21 days after the administration of bleomycin, the lungs were removed, and the amount of hydroxyproline was determined by a method such as Keane MP (The Journal of Immunity).
Munology), Volume 153, 4704-4712
, 1994).

The average increase in the amount of hydroxyproline in each group relative to the normal group (the value obtained by subtracting the average amount of hydroxyproline in the normal group from the average amount of hydroxyproline in each group) is shown in FIG. UTM suppressed the increase in hydroxyproline in the lung due to bleomycin administration.

[0028]

EFFECT OF THE INVENTION TM-like protein is highly safe, has a long half-life in blood, is stable in vivo, and has a long-lasting effect. It has clinical usefulness. Further, the TM-like protein has an effect of preventing and / or preventing progression of pulmonary fibrosis, and particularly can be used as an agent for preventing and / or preventing progression of drug-induced pulmonary fibrosis.

[Brief description of the drawings]

FIG. 1 is a graph showing the increased amount of hydroxyproline in mice in a mouse bleomycin-induced lung fibrosis model used in the present invention.

Claims (3)

[Claims] 1. A method for preventing pulmonary fibrosis and / or comprising thrombomodulin-like protein as an active ingredient.
Or a progress inhibitor. 2. The method of claim 1, wherein said pulmonary fibrosis is drug-induced.
3. The agent for preventing and / or preventing progression of pulmonary fibrosis according to the above. 3. The agent according to claim 2, wherein the agent is an anticancer agent.