WO2001093870A1 - Remedies for diseases caused by fibrin formation and/or endothelial cell damage - Google Patents

Abstract

Fibrinous thrombus formation inhibitors containing, as the active ingredient, one or more biocompatible pigments selected from among sulfonic acid azo pigments, phthalein pigments, triphenylmethane pigments, phenanthridinium pigments, acridine pigments, xanthene pigments, phenothiazine pigments and cyanine pigments; and medicinal compositions containing the inhibitors and drugs acting on fibrin and/or damaged endothelial cells.

Description

 For treating diseases caused by dysfunction and Z or endothelial cell damage

Technical field

 The present invention relates to a medicament for selectively binding to fibrin and damaged endothelial cells and treating a disease caused by fibrin formation and / or endothelial cell damage.

 Akita

Background art

 In the past, thrombus formation in blood vessels was triggered by the adhesion of activated platelets to exposed collagen beneath damaged vascular endothelial cells, and fibrinogen von illlbrand factor linked platelets. It is thought that platelets undergo aggregation, which is followed by fibrin, which in turn attaches blood cells, which leads to thrombus growth.

 From this viewpoint, many antiplatelet agents have been developed for the purpose of preventing and treating various arterial and venous thrombosis.

 However, the evaluation of the effects of these conventional antithrombotic agents is not always constant, and the development of new antithrombotic agents has been desired.

Disclosure of the invention

 Therefore, the present inventors have conducted various studies to develop an antithrombotic agent based on a completely new mechanism of action, and found that certain biocompatible dyes selectively bind to fipurin and / or damaged endothelial cells. The present invention has been found to have the effect of suppressing the formation of fibrin clots, and that these pigments can be carriers of fibrin and / or impaired endothelial cells of other drugs. It was completed.

That is, the present invention relates to a sulfonic acid azo dye, a phthalein dye, Fibrin thrombus containing one or more biocompatible dyes selected from methane dyes, phenanthridinium dyes, acridine dyes, xanthene dyes, phenothiazine dyes, and cyanine dyes as active ingredients It provides a formation inhibitor.

 Further, the present invention provides a drug carrier for fibrin and / or a damaged endothelial cell, comprising the above-mentioned biocompatible dye as an active ingredient.

 The present invention also provides a pharmaceutical composition containing the biocompatible dye, and a drug that acts on fibrin and / or damaged endothelial cells.

 Further, the present invention provides a method for producing a fibrin containing the above-mentioned biocompatible dye, an anticoagulant, a thrombolytic agent, an antiplatelet agent and Z or an endothelial cell repairing agent, and treating diseases caused by Z or endothelial cell damage. It provides a therapeutic agent.

 The present invention also provides use of the above biocompatible pigment for producing a fibrin thrombus formation inhibitor.

 The present invention also provides use of the above biocompatible dye for producing a drug carrier for fibrin and / or damaged endothelial cells.

 The present invention also provides the use of the above biocompatible dye and a drug acting on fibrils and Z or damaged endothelial cells for the production of a pharmaceutical composition.

 The present invention also provides a biocompatible dye, an anticoagulant, a thrombolytic agent, an antiplatelet agent and / or a disorder for producing a therapeutic drug for a disease caused by fibrin formation and Z or endothelial cell damage. It provides use of an endothelial cell repair agent.

 Further, the present invention provides a method for treating a fibrin thrombus, which comprises administering an effective amount of the biocompatible dye.

 Furthermore, the present invention provides a method for delivering a drug to fibrin and / or a damaged endothelial cell, which comprises administering the biocompatible dye and a drug that acts on fibrin and / or a damaged endothelial cell. It is.

Furthermore, the present invention provides the biocompatible dye, an anticoagulant, a thrombolytic agent, It is intended to provide a method for treating a disease caused by fibrin formation and Z or endothelial cell damage, which comprises administering an effective amount of an antiplatelet agent and an agent for repairing damaged or endothelial cells. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a micrograph showing the results of staining of fibrin clots with Evans blue and platelets with eosin. [A: staining with Evansbull-1 (the fibrin portion of arrow a is stained blue), B: after staining with Evansbull-1 Addition of eosin (platelet aggregates in arrow b are stained purple)]. FIG. 2 is a fluorescence micrograph showing the binding of ethidium orbamide to fibrin clots [A: micrograph of thrombus, B: example of ethidium bromide addition, arrow indicates fibrin fluorescent coloring]. FIG. 3 shows that platelets and fibrin adhere to damaged endothelial cells. [A: Platelets (arrows) adhere to damaged endothelial cells. B: Fibrin (arrow) adheres to detached endothelial cells (small arrow) and does not adhere to collagen fibers below (large arrow)]. Fig. 4 shows the results of the canine iliac artery perfusion experiment (A, B, and C melted in order of tPA. When Evans Bull was flown here, the white part (white arrow) became blue and the part was stained blue. Histologically, as in D). Figure

5 shows an endoscopic image of canine iliac artery endothelial cells stained with Evans Bull I.

(A: before staining, B: after staining, arrows indicate damaged endothelial cells, C: micrograph of the same site (arrows)). FIG. 6 is a diagram showing electron microscopic findings of endothelial cells. (A: normal site, B: damaged site stained with Evans blue, emptying of endothelial cells (arrow)). Figure 7 shows the antithrombotic effect of Evans blue (Α: Evans blue administration site, no thrombus observed, Β: site to which Evans blue I was not administered, red thrombus observed). FIG. 8 shows the antithrombotic effect of local administration of Evans blue. (Α: control example, Β: Evans blue administration example). FIG. 9 is a diagram showing the effect of Evans Blue on inhibiting thrombus in the stent. (Α: control case, Β: Evans blue administration case, thrombus not seen Not). FIG. 10 is a view showing a chromatogram of an adduct of Evans blue and batroxobin. BEST MODE FOR CARRYING OUT THE INVENTION

 In the present invention, a biocompatible dye refers to a dye that is used for medical or edible purposes such as pharmaceuticals, dyeing of living bodies or biological materials, clinical tests, etc., and that can be administered to mammals including humans. Specifically, it refers to a dye selected from sulfonic acid azo dyes, phthalein dyes, triphenylmethane dyes, phenanthridinium dyes, acridine dyes, xanthene dyes, phenothiazine dyes, and cyanine dyes. .

Here, as the sulfonic acid-based § zone dye, refers to § zone dye having a Kiichi so ₃ _ in the molecule, for example Evans blue, trypan blue, trypan red, naphthoquinone evening Ren sulfonic acid-based § such as orange B Azo dyes, polar yellow, orange I or orange II, etc., and phthalein-based dyes include, for example, phenolphthalein or phenolsulfophthalein, and trifenylmethane-based dyes, for example, gentian violet And fuchsin (magenta). Examples of phenanthridine-based dyes include ethidium bromide and providedium iodide. Examples of acridine-based dyes include acridine. Orange, acridine yellow, 91 aminoacridine, acriflavine or Xanthene dyes include, for example, rhodamine B, sulforhodamine, fluorescein or eosin, and phenothiazine dyes include, for example, methylene blue, triludin blue or azur (A, B) And the like.

Of the above dyes, sulfonic acid azo dyes (especially naphthalene sulfonic acid azo dyes), phenanthridinium dyes, acridine dyes, phenothiazine dyes, triphenyl methane dyes and saquinten dyes are preferred. , Especially Evansbu Especially preferred are trypan blue, ethidium bromide, acridine orange, methylene blue, triluimble, eosin, gentian violet and fluorescein.

 These dyes selectively bind to fibrin and Z or damaged endothelial cells. And, by such action, the binding between fibrin and fibrin, the binding between fibrin and damaged endothelial cells, the binding between fibrin and platelets, etc. are remarkably suppressed. Therefore, fibrin is useful as a thrombus formation inhibitor that plays a central role in thrombus formation, that is, a fibrin thrombus formation inhibitor. Examples of the disease that can be treated by the action of inhibiting the formation of fibrin thrombus include various thrombosis such as deep vein thrombosis, cerebral thrombosis, cerebral embolism, cerebral infarction, and cardiomyocardial infarction.

 In addition, since the dye has a function of selectively binding to fibrin and Z or damaged endothelial cells, it is useful as a carrier for other drugs to fibrin and / or damaged endothelial cells. When a drug acting on fibrin and Z or a damaged endothelial cell is administered together with the dye or in the form of an adduct with the dye, these drugs are selectively transported to fipurin and Z or the damaged endothelial cell. You.

Here, drugs to be delivered to fibrin and / or damaged endothelial cells include, for example, anticoagulants, thrombolytic agents, antiplatelet agents, repair agents for damaged endothelial cells, therapeutic agents for diseases caused by endothelial cell damage And the like. Is an antiplatelet agent used herein, aspirin, sulfinpyrazone, ticlopidine, Abukishimagu other such Kurobi Dogureru, GPI IbZl I Ia inhibitor, thromboxane A ₂ antagonists, Roitoko Lien antagonists, and the like. Examples of the anticoagulant include an antithrombin agent such as heparin, argatronon, and batroxobin, and a synthetic anticoagulant such as perfaline. Examples of thrombolytic agents include perokinase and plasminogen activator. Examples of the damaged endothelial cell repair agent include vascular endothelial cell growth factor and the like. In addition, examples of the disease caused by the endothelial cell disorder include arteriosclerosis. Among these other drugs, it is preferable to use an anticoagulant, a thrombolytic agent or an antiplatelet agent.

 In other words, the biggest side effect of currently used antithrombotic agents is severe bleeding such as cerebral hemorrhage. To prevent this, it is necessary to reduce the dose or selectively concentrate locally. If it can be collected locally by intravenous administration, the burden on the patient can be reduced and side effects can be prevented. These dyes form adducts with other drugs. Therefore, if a dye such as Evans blue, which selectively binds to the damaged endothelial cells and fibrin, is combined with an antithrombotic agent and administered, it will concentrate at the thrombus localization. In other words, targeted therapy (target therapy or missile therapy) becomes possible.

 Since the biocompatible dye used in the present invention has an action of easily forming an adduct with various drugs in an aqueous solution, it is convenient for the delivery of the drug.

 In the present invention, the mixing ratio of the dye to the other drug is preferably 1: 10000 to 100: 1, more preferably 1: 1100 to 100: 1 by weight.

 When the medicament of the present invention is used, it can be formulated as a composition together with a pharmaceutically acceptable carrier for parenteral administration such as injection, rectal administration, and the like, orally in solid or liquid form.

 Forms of the compositions according to the present invention for injection include sterile aqueous solutions, non-aqueous solutions, suspensions or emulsions. Examples of suitable non-aqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable organic acid esters such as ethyl oleate, and the like. Such compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents.

 The medicament of the present invention is preferably used as an injection among these administration forms, and more preferably, intravenous administration, intraarterial administration and the like.

The dosage of the medicament of the present invention depends on the nature of the components to be administered, the administration route, and the desired treatment period. In general, about 0.1 to 100 mg / kg, particularly about 0.5 to 50 mg / kg, is preferable for the above-mentioned pigment. If desired, the daily dose can be divided and administered in 2 to 4 times. Example

 Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 (Evans blue injection preparation method)

 Evans blue crystals are sterilized, aseptically dissolved in distilled water to a concentration of 2, 5, 10, or 20% by weight, and sealed in 1, 5, or 1 OmL glass or synthetic resin ampules. Stored at room temperature.

Example 2 (Selective binding to fibrin)

 Human arterial or venous blood was collected and allowed to coagulate spontaneously in a test tube to form a thrombus. A part of this thrombus was perfused under a light microscope using physiological saline, and various compounds were administered thereto, and a compound that stains fibrin or platelets was searched. The staining site was confirmed by fibrin staining (TPHA staining) and platelet staining (Gimuza staining).

 As a result, as shown in FIG. 1 and Table 1, fibrin was selectively stained blue at 10% by weight or more of Evans blue.

As a result of a similar examination with a fluorescence microscope, ethidium orbamide, a fluorescent dye, was found to bind to fibrin and platelets as shown in FIG. 2 and Table 1. Connectivity with fibrin

+ Yeo

 Gentian Biolet

 Example 2 (Evidence for selective adherence to fipurin II platelets or damaged endothelial cells)

 Using an anesthetized dog, cause endothelial disorder of the iliac artery to bluish-purple, and perfuse blood to form a thrombus.

After the formation, the site was excised and examined under a microscope and dyed and dyed. As shown in Fig. 3

Both proteins and platelets adhere to the detached endothelial cells or inner elastic plate, but not to the exposed collagen fibers.

Example 4 (Selective binding to fibrin in blood vessels)

 A thrombus was formed in the canine iliac artery and perfused with Evans blue, followed by endoscopy. As shown in FIG. 4, the fibrin was clearly stained. The fibrin was confirmed by staining after formalin fixation. In addition, eosin, fuchsin, trypan blue and methylene blue also selectively stained thrombus in blood vessels.

Example 5

 Fibrinogen and various dyes were mixed in a test tube and dropped into a thrombin solution. This means that the stained fibrin was bound to fibrinogen if possible. As a result, it was found that Evans Bull, Methylene Blue and Trypan Blue were bonded.

Example 6 (Binding property to damaged endothelial cells)

The coronary artery or canine iliac artery removed from the human autopsy heart with the consent of the family was perfused with physiological saline, and the inner surface was rubbed with a cotton ball to create endothelial damage. Then Dye was administered. Figure 5 shows the results of Evansbull single administration. The site stained with Evans blue was endothelial cells detached or vacuolated by electron microscopy as shown in FIG. That is, it was a damaged endothelial cell. Table 1 shows the dyes that bind to damaged endothelial cells.

 Table 2

 Pigment Binding to damaged endothelial cells

 Echijimbu mouth Polo mouth

 Acridine orange W mouth

 Fluorescein TP mouth Example 7 (antithrombotic action)

 Using an anesthetized beagle dog, a balloon catheter was inserted into the iliac artery, and the balloon was inflated to create a vascular disorder model. Next, 0.1 mL of a 10% by weight solution of Evans blue was locally applied under pressure using a porous balloon, and 30 minutes later, the blood vessel was excised, and the wet weight of the thrombus per cm was measured.

 FIG. 7 shows angiography images of the non-administration example (A) and the administration example (B). Figure 8 shows the organizational findings. As a result, thrombus formation due to vascular damage was observed in the non-administered patients, whereas thrombus formation was significantly prevented in the Evans blue-administered patients. The wet weight of the non-administration group was 130 ± 8 mg (mean S.E.R.) on average in 6 cases, whereas the wet weight was significantly lower in the administration group (6 cases), ll ± 2 mg (P <0.05). .

Example 8 (Effect of preventing thrombus in the stent)

 The canine femoral or iliac arteries were dilated with a balloon and then a stent was inserted. Then, using a porous balloon, Evans Blue was administered to the vessel wall at the stent insertion site. As shown in FIG. 9, no thrombus formation was observed at the site where Evans Blue was administered.

Example 9 (Targeted therapy)

Mix Evans blue and tPA (—chain, Mitsui Pharmaceutical), perform intravenous injection, and The effect on canine iliac artery thrombosis at 0 min was examined.

 As a result, as shown in Table 3, a significantly stronger antithrombotic effect was observed than Evans Blue or tPA alone.

 Table 3

 ——— “—one” ¾ ¥ * ”(7kg) —… 丽 通 — (π ¾Γ—

 t PA 1000 units 280

 5000 units 260

 20000 units 98

2mg 265

 10 200

 30 180 t PA + Evans Blue 5000 units

 lOmg 70 *

 * p * 0.05, tPA 5000 units alone or Evans blue lOmg alone

 There is a significant difference compared to intravenous injection.

Example 10 (Proof of Evans Blue and Batroxobin Adduct and Preparation of Injection) Preparation Method 1: Sephadex G-15 (Pharmacia) was packed in a column (3 cm × 65 cm), and distilled water was allowed to flow all day and night. Thereafter, Evans blue (0.1% by weight solution) and batroxobin (20 BU units /] iiL) were mixed in a ratio of 1: 1. The mixture was fractionated into 3 mL portions using a fraction collector. The absorbance at Onm and 62 Onm was measured. Then, the bovine fibrinogen solution was put into the reactor, and the temperature was kept at 37. After that, each fraction was added, and the coagulation time was measured. As a result, as shown in FIG. 10, a substance having an absorbance different from that of Evans blue was found. In addition, a coagulation inhibitory action similar to that of batroxobin, that is, a fibrin production inhibitory action was found. This substance was determined to be an adduct. Therefore, Fraction 2 10-24 Onm was freeze-dried, placed in an lmgZmL (aqueous solution) ampoule, stored in a cold place, and used.

Preparation method 2: Evans blue solution and batroxobin solution (both in ampoules) Is added to an injection pad and mixed immediately as an adduct is formed. Industrial applicability

 ADVANTAGE OF THE INVENTION According to this invention, since formation of a fibrin thrombus can be suppressed selectively, it is useful as an antithrombotic agent. In addition, since this dye selectively binds to fibrin and / or damaged endothelial cells, targeting therapy can be performed in combination with a drug targeting these.

Claims

The scope of the claims 1. One or selected from sulfonic acid azo dyes, phthalein dyes, triphenyl methane dyes, phenanthridinium dyes, acridine dyes, xanthene dyes, phenothiazine dyes, cyanine dyes, or A fibrin thrombus formation inhibitor containing two or more biocompatible pigments as active ingredients.  2. The fibrin thrombus formation inhibitor according to claim 1, which inhibits fibrin-fibrin binding, fibrin-impaired endothelial cell binding and / or fibrin-platelet binding.  3. The biocompatible dye is a naphthylene sulfonic acid-based azo dye, a phenanthridinium-based dye, an acridine-based dye, a phenothiazine-based dye, a triphenylmethane-based dye, or a xanthene-based dye. Fibrin thrombus formation inhibitor.  4. One or more selected from sulfonic acid azo dyes, phthalein dyes, triphenylmethane dyes, phenanthridinium dyes, acridine dyes, xanthene dyes, phenothiazine dyes, cyanine dyes, or A drug carrier containing two or more biocompatible dyes as active ingredients to fipurin and Z or damaged endothelial cells.  5. The drug according to claim 4, wherein the biocompatible dye is a naphthenesulfonic acid-based azo dye, a phenanthridinium-based dye, an acridine-based dye, a phenothiazine-based dye, a triphenylmethane-based dye, or a xanthene-based dye. Carrier. ·  6. One or selected from sulfonic acid azo dyes, phthalein dyes, triphenyl methane dyes, phenanthridinium dyes, acridine dyes, xanthene dyes, phenothiazine dyes, cyanine dyes, or A pharmaceutical composition comprising two or more biocompatible pigments and a drug acting on fipurin and / or damaged endothelial cells. 7. Biocompatible dyes are naphthalenesulfonic acid azo dyes, phenanthridine 7. The pharmaceutical composition according to claim 6, which is a dye, an acridine dye, a phenothiazine dye, a triphenylmethane dye, or a xanthene dye.  8. One or selected from sulfonic acid azo dyes, phthalein dyes, triphenylmethane dyes, phenanthridinium dyes, acridine dyes, xanthene dyes, phenothiazine dyes, cyanine dyes, or A therapeutic agent for a disease caused by fibrin formation and / or endothelial cell damage, comprising two or more biocompatible dyes, and an anticoagulant, a thrombolytic agent, an antiplatelet agent and / or a damaged endothelial cell repair agent.  9. The therapeutic agent according to claim 8, wherein the biocompatible dye is a naphthalenesulfonic acid-based azo dye, a phenanthridinium-based dye, an acridine-based dye, a phenothiazine-based dye, a triphenylmethane-based dye, or a xanthene-based dye.  10. Sulfonate azo dyes, phthalein dyes, triphenylmethane dyes, phenanthridinium dyes, aclidin dyes, xanthene dyes, phenothiazine dyes for the production of fibrin thrombus formation inhibitors Use of one or more biocompatible dyes selected from, cyanine dyes.  11. The use according to claim 10, wherein the inhibition of fibrin thrombus formation suppresses fibrin-fibrin binding, fibrin-damage endothelial cell binding and / or fipurin-platelet binding.  12. The biocompatible dye according to claim 10 or 11, wherein the biocompatible dye is a naphthalenesulfonic acid-based azo dye, a phenanthridinium-based dye, an acridine-based dye, a phenothiazine-based dye, a triphenylmethane-based dye, or a xanthene-based dye. Use of.  13. Sulfonate-based azo dyes, phthalein-based dyes, triphenylmethane-based dyes, phenanthridine-based dyes, acridine-based dyes, xanthene-based dyes, for the production of drug carriers for fibrin and Z or damaged endothelial cells. Use of one or more biocompatible dyes selected from phenothiazine dyes and cyanine dyes. 14. The method according to claim 13, wherein the biocompatible dye is a naphthalenesulfonic acid-based azo dye, a phenanthridine-based dye, an acridine-based dye, or a phenothiazine-based dye. use.  15. Sulfonic acid azo dyes, phthalein dyes, triphenylmethane dyes, phenanthridinium dyes, acridine dyes, xanthen dyes, phenothiazine dyes, cyannine for the production of pharmaceutical compositions Use of one or two or more biocompatible dyes selected from the group consisting of: a dye, and fibrin and a drug acting on endothelial cells.  16. The use according to claim 15, wherein the biocompatible pigment is a naphthalenesulfonic acid-based azo pigment, a phenanthridine-based pigment, an acridine-based pigment, a phenothiazine-based pigment, a triphenylmethane-based pigment, or a xanthene-based pigment.  17. Sulfonate azo dyes, phthalein dyes, triphenylmethane dyes, phenanthridinium dyes, and acridines for the manufacture of therapeutics for diseases caused by fibrin formation and Z or endothelial cell damage One or more biocompatible dyes selected from dyes, xanthene dyes, phenothiazine dyes, cyanine dyes, and anticoagulants, thrombolytic agents, antiplatelet agents and / or impaired endothelial cells Use of repair agents.  18. The use according to claim 17, wherein the biocompatible dye is a naphthalenesulfonic acid-based azo dye, a phenanthridine-based dye, an acridine-based dye, a phenothiazine-based dye, a triphenylmethane-based dye, or a xanthene-based dye.  19. One or more selected from sulfonic acid azo dyes, phthalein dyes, triphenylmethane dyes, phenanthridinium dyes, acridine dyes, xanthene dyes, phenothiazine dyes, and cyanine dyes, or A method for treating fibrin thrombus, comprising administering an effective amount of two or more biocompatible pigments.  20. The method according to claim 19, wherein the fibrin thrombus is caused by fibrin-fibrin binding, fibrin-impaired endothelial cell binding and Z or fipurin-platelet binding. 21. Biocompatible dyes are naphthylene sulfonic acid azo dyes, phenanthridine 21. The treatment method according to claim 19, wherein the treatment method is a dye, an acridine dye, a phenothiazine dye, a triphenylmethane dye, or a xanthene dye.  22. One or selected from sulfonic acid azo dyes, phthalein dyes, triphenyl methane dyes, phenanthridinium dyes, acridine dyes, xanthene dyes, phenothiazine dyes, cyanine dyes, or A method for delivering a drug to fibrin and Z or damaged endothelial cells, which comprises administering at least 2% of a biocompatible dye and a drug that acts on fibrin and Z or damaged endothelial cells.  23. The method according to claim 22, wherein the biocompatible dye is a naphthylene sulfonic acid azo dye, a phenanthridine dye, an acridine dye, a phenothiazine dye, a triphenylmethane dye, or a xanthene dye. Method.  24. One or selected from sulfonic acid azo dyes, phthalein dyes, triphenyl methane dyes, phenanthridinium dyes, acridine dyes, xanthene dyes, phenothiazine dyes, cyanine dyes, or Administration of two or more biocompatible dyes and an effective amount of an anticoagulant, a thrombolytic agent, an antiplatelet agent, and / or an agent for repairing an impaired endothelial cell. For treating a disease caused by  25. The biocompatible dye according to claim 24, wherein the biocompatible dye is a naphthylene sulfonic acid azo dye, a phenanthridine dye, an acridine dye, a phenothiazine dye, a triphenylmethane dye, or a xanthene dye. Treatment method.