JP2006321740A - Post-ischemic angiogenesis promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medicament useful for prevention or treatment of various ischemic diseases by promoting angiogenesis after ischemia and improving blood flow.
A post-ischemic angiogenesis promoter, a post-ischemic blood flow improving agent, and a prophylactic or therapeutic agent for ischemic disease, comprising sphingosine-1-phosphate or a pharmaceutically acceptable salt thereof.
[Selection figure] None

Description

  The present invention relates to a post-ischemic angiogenesis promoter useful for the prevention or treatment of various ischemic diseases.

  For treatment and prevention of ischemic diseases in the heart, brain and periphery represented by myocardial infarction, heart failure, angina pectoris, cerebral infarction, vascular dementia, obstructive arteriosclerosis, Buerger disease, etc. A post-ischemic angiogenesis-promoting agent that promotes neoplasia is effective.

On the other hand, sphingosine-1-phosphate (S1P) is a lipid mediator present in plasma, and is known to have a cell growth promoting action, a cell migration regulating action, an apoptosis inhibiting action and the like in cultured cells. S1P exerts various physiological effects via Edg family G protein-coupled receptors of five subtypes of S1P ₁ to S1P ₅ . Among them, three subtypes of S1P ₁ , S1P ₂ and S1P ₃ are widely distributed throughout the body. The main production enzyme of S1P in vivo is sphingosine kinase 1 (SPHK1).

It has been reported that S1P ₁ receptor knockout mice die due to bleeding caused by angiogenesis in the embryonic period (Non-patent Document 1), and the tendency is further increased in S1P ₁ , S1P ₂ and S1P ₃ triple knockout mice. (Non-patent document 2). Therefore, the sphingosine-1-phosphate (S1P) signal transduction system is considered to play an essential role in embryonic vascular maturation. However, the role of S1P has not been fully elucidated for post-ischemic angiogenesis in adults.

Liu Y, Wada R, Yamashita T, Mi Y, Deng CX, Hobson JP, Rosenfeldt HM, Nava VE, Chae SS, Lee MJ, Liu CH, Hla T, Spiegel S, Proia RL. Edg-1, the G protein- coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation. J. Clin. Invest., 2000 Oct; 106 (8): 951-61 Kono M, Mi Y, Liu Y, Sasaki T, Allende ML, Wu YP, Yamashita T, Proia RL.The Sphingosine-1-phosphate Receptors S1P1, S1P2, and S1P3 Function Coordinately during Embryonic Angiogenesis. J. Biol. Chem., 2004 Jul 9; 279 (28): 29367-73

  An object of the present invention is to provide a medicament useful for preventing or treating various ischemic diseases by promoting angiogenesis after ischemia and improving blood flow.

The present invention includes the following inventions.
(1) A post-ischemic angiogenesis promoter containing sphingosine-1-phosphate or a pharmaceutically acceptable salt thereof.
(2) The post-ischemic angiogenesis promoter according to (1), which is a sustained-release preparation using a biodegradable polymer as a base.
(3) Ischemia according to (2), which is obtained by dissolving sphingosine-1-phosphate or a pharmaceutically acceptable salt thereof and a biodegradable polymer in an organic solvent and then removing the organic solvent. Post-angiogenesis promoter.
(4) The post-ischemic angiogenesis promoter according to (2) or (3), which is a sustained-release microcapsule.
(5) The post-ischemic angiogenesis promoter according to any one of (2) to (4), wherein the biodegradable polymer is a lactic acid-glycolic acid copolymer.
(6) The post-ischemic angiogenesis promoter according to any one of (1) to (5), which is a post-ischemic blood flow improving agent.
(7) The post-ischemic angiogenesis promoter according to any one of (1) to (5), which is a prophylactic or therapeutic agent for ischemic disease.

  ADVANTAGE OF THE INVENTION According to this invention, the pharmaceutical useful for the prevention or treatment of various ischemic diseases can be provided by promoting angiogenesis after ischemia and improving blood flow.

Hereinafter, the present invention will be described in detail.
Sphingosine-1-phosphate used in the present invention may be any of a natural type (D (+) form), a non-natural type (L (-) form), or a mixture thereof. Examples of pharmaceutically acceptable salts of sphingosine-1-phosphate include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, and zinc salts. Examples thereof include salts with inorganic bases and salts with organic bases.

  The sphingosine-1-phosphate used in the present invention is, for example, a general phosphate esterification method using phosphoric acid, diphosphorus pentoxide, polyphosphoric acid, phosphorus oxychloride, etc. (Oil Chemistry 23, 2-8 (1974)). Etc.), a method in which phospholipase D is allowed to act on sphingosylphosphorylcholine (J. Biol. Chem., 269, 3181-3188 (1994)), a method in which sphingosine kinase is allowed to act on sphingosine and the like. Commercial products may also be used.

  The post-ischemic angiogenesis-promoting agent and post-ischemic blood flow-improving agent of the present invention promotes post-ischemic angiogenesis and improves blood flow, thereby improving myocardial infarction, heart failure, angina pectoris, cerebral infarction, It can be used as a therapeutic agent for various ischemic diseases such as vascular dementia, obstructive arteriosclerosis and Buerger's disease. Moreover, if it is administered when an ischemic symptom is observed, the progression of these ischemic diseases and the damage caused by ischemia can be prevented in advance.

Hereinafter, the dosage and formulation of the pharmaceutical preparation of the present invention will be described.
Sphingosine-1-phosphate or a pharmaceutically acceptable salt thereof can be administered to animals and humans as it is or together with a conventional pharmaceutical carrier. The dosage form is not particularly limited, and is appropriately selected and used as necessary. Tablets, capsules, granules, fine granules, powders, sustained-release preparations, suspensions, emulsions, syrups, elixirs And oral preparations such as pills, and parenteral preparations such as injections, suppositories, coatings, and patches.

  The oral preparation is produced according to a conventional method using, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like.

  In this type of preparation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like can be appropriately used in addition to the above-mentioned excipients.

  Examples of the binder include starch, dextrin, gum arabic powder, gelatin, hydroxypropyl starch, methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylpyrrolidone, and macrogol.

  Examples of the disintegrant include starch, hydroxypropyl starch, carboxymethylcellulose sodium, carboxymethylcellulose calcium, carboxymethylcellulose, and low-substituted hydroxypropylcellulose.

  Examples of the surfactant include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, and polysorbate 80.

  Examples of the lubricant include talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, and polyethylene glycol.

  Examples of the fluidity promoter include light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.

  Injections are manufactured according to conventional methods, and generally used as diluents are distilled water for injection, physiological saline, aqueous glucose solution, olive oil, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol and the like. . Furthermore, you may add a bactericidal agent, antiseptic | preservative, and a stabilizer as needed. In addition, from the viewpoint of stability, the injection can be frozen after filling into a vial or the like, the water can be removed by a normal freeze-drying technique, and the liquid can be re-prepared from the freeze-dried product immediately before use. Furthermore, an isotonic agent, stabilizer, preservative, soothing agent and the like may be added as necessary.

  Examples of other parenteral preparations include coating solutions for external use, ointments and the like, patches, suppositories for rectal administration, and the like, which are produced according to a conventional method.

  Moreover, when using the formulation of this invention as a sustained release formulation, it is preferable to use a biodegradable polymer as a base.

  Specific examples of the biodegradable polymer include α-hydroxycarboxylic acids (for example, glycolic acid and lactic acid), hydroxydicarboxylic acids (for example, malic acid and the like), hydroxytricarboxylic acids (for example, citric acid and the like), and the like. Polymers synthesized from one or more by non-catalytic dehydration polycondensation and having a free carboxyl group, or a mixture thereof, poly-α-cyanoacrylic acid ester, polyamino acid (for example, poly-γ-benzyl-L-glutamic acid, etc. ) And maleic anhydride polymers (for example, styrene-maleic acid polymers). The type of polymerization may be random, block or graft. Moreover, when the α-hydroxycarboxylic acids, hydroxydicarboxylic acids, and hydroxytricarboxylic acids have an optically active center in the molecule, any of D-, L-, and DL-forms can be used. Among these, a biodegradable polymer having a free carboxyl group at the terminal, for example, a polymer synthesized from α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, etc.) (eg, lactic acid-glycolic acid copolymer, etc.) ), Poly-α-cyanoacrylic acid ester and the like are preferable. The biodegradable polymer is more preferably a polymer synthesized from α-hydroxycarboxylic acids, and particularly preferably a lactic acid-glycolic acid copolymer.

  When a lactic acid-glycolic acid copolymer or a homopolymer is used as the biodegradable polymer, the composition ratio (mol%) is preferably about 100/0 to about 40/60, and about 85/15 to about 50/50. Further preferred. In this specification, not only a homopolymer such as polylactic acid and polyglycolic acid but also a lactic acid-glycolic acid copolymer may be simply referred to as a lactic acid-glycolic acid polymer. The weight average molecular weight of the lactic acid-glycolic acid polymer is preferably about 3,000 to about 25,000, more preferably about 5,000 to about 20,000.

  The sustained-release preparation is preferably obtained by dissolving the sphingosine-1-phosphate or a pharmaceutically acceptable salt thereof and the biodegradable polymer in an organic solvent, and then removing the organic solvent. it can.

  The organic solvent used for production of the sustained release preparation preferably has a boiling point of 120 ° C. or lower. Examples of the organic solvent include halogenated hydrocarbons (eg, dichloromethane, chloroform, carbon tetrachloride), alcohols (eg, ethanol, methanol, 1,4-butanediol, 1,5-pentanediol), acetic acid, and the like. Examples include ethyl and acetonitrile. These may be mixed and used at an appropriate ratio. When an organic solvent is used alone, for example, dichloromethane, acetonitrile and the like are preferable. When an organic solvent is used as a mixed solvent, for example, a halogenated hydrocarbon (eg, dichloromethane, chloroform, etc.) and an alcohol (eg, ethanol, methanol, 1,4-butanediol, 1,5-pentanediol, etc.) or acetonitrile The combination with is preferable. In particular, a combination of dichloromethane and acetonitrile is widely used. The mixing ratio (volume ratio) of the halogenated hydrocarbon and the alcohol or acetonitrile is about 40: 1 to about 1: 1, preferably about 20: 1 to about 1: 1. In particular, it is desirable to use a halogenated hydrocarbon (such as dichloromethane) alone.

  The sustained-release preparation may be fine particles containing sphingosine-1-phosphate or a pharmaceutically acceptable salt thereof and a microcapsule base (preferably a biodegradable polymer), and specific examples thereof For example, a microcapsule containing one drug core in one particle, a multinuclear microcapsule containing many drug cores in one particle, or a molecular drug dissolved in a microcapsule base Or the microsphere etc. which are disperse | distributing are mentioned.

  Examples of the method for removing the organic solvent in the production of the sustained-release preparation include (a) an underwater drying method (O / W method), (b) a phase separation method (coacervation method), (c) a spray drying method, and A method according to these is used.

  The preparation of the present invention varies depending on the dosage form, administration route and the like, and can be administered from 1 to several times a day to 1 to several times per week to a month.

  In order to exert the desired effect as an oral preparation, the weight of sphingosine-1-phosphate or a pharmaceutically acceptable salt thereof is usually 1 in adults, although it varies depending on the age, body weight and degree of disease of the patient. It is appropriate to take ˜200 mg divided into several times a day.

  In order to exert the desired effect as a parenteral agent, the weight of sphingosine-1-phosphate or a pharmaceutically acceptable salt thereof is usually used in adults, although it varies depending on the age, body weight, and degree of disease of the patient. Intravenous injection, intravenous infusion, subcutaneous injection and intramuscular injection of 1 to 50 mg per day are appropriate.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to these examples.

[Example 1]
Models of post-ischemic angiogenesis in adults include lower limb ischemia models (Couffinhal T, Silver M, Zheng LP, Kearney M, Witzenbichler B, Isner JM. Mouse model of angiogenesis. Am. J. Pathol., 1998 Jun; 152 (6): 1667-79), myocardial infarction models, cerebral ischemia models, and the like have been established. In the following examples, an angiogenic action of sphingosine-1-phosphate (S1P) on acute skeletal muscle ischemia using S1P intramuscular administration and physiologically enhanced S1P synthesis using a mouse lower limb ischemia model SPHK1 transgenic mice were examined.

(1) Method
Mice 8-12 weeks old, male C57BL / 6J and SPHK1 transgenic mice were used. For transgenic mice (C57BL / 6J background), wild-type C57BL / 6J of the same litter was used as a control.

On the day before the creation of the unilateral lower limb ischemia model, hair removal from the abdomen to the lower leg was performed with a hair removal cream. The left lower limb muscle was exposed by pentobarbital (70 mg / kg) anesthesia and lower left abdominal left midline incision. The femoral artery and vein were exfoliated, ligated with 8-0 silk directly under the ligament ligament and under the popliteal artery branch, and excised together with the side branch. The wound was washed with physiological saline, and after hemostasis was confirmed, the wound was sutured with 4-0 silk thread.

Preparation of S1P Sustained Release Preparation Lactic Acid-Glycolic Acid Copolymer (PLGA-7510 manufactured by Wako Pure Chemical Industries, Ltd .; DL-lactic acid / glycolic acid composition ratio (mol%) 75:25, weight average molecular weight 10,000) 60 mg was dissolved in 1 ml of dichloromethane (Wako Pure Chemical Industries), 1 mg of sphingosine-1-phosphate (manufactured by Biomol) was dispersed in 1 ml of dichloromethane (Wako Pure Chemical Industries) using a sonicator, and both were combined and 0.4 ml Was washed with dichloromethane to make a 2.4 ml solution. To this oil layer, 12 ml of 2.5% polyvinyl alcohol (manufactured by Sigma) was added, and an O / W emulsion was prepared with a probe sonicator. Polyvinyl alcohol was added as an emulsifier. The emulsion was stirred overnight at room temperature using a magnetic stirrer, and dichloromethane was evaporated to solidify the PLGA microcapsules. The process of collecting PLGA microcapsules solidified by ultracentrifugation (44000 RPM, 5 minutes) and dispersing in purified water was repeated three times to remove polyvinyl alcohol. Finally, it was dissolved in 2 ml of purified water, lyophilized in vacuum and stored in a desiccator under negative pressure. A microcapsule containing no sphingosine-1-phosphate was used for the control group. 1 mg of microcapsules purified at the time of use was dissolved in 500 μL of sterile physiological saline to prepare an injection solution.

1/2000 (volume ratio) of a dimethyl sulfoxide (DMSO) solution of sphingosine-1-phosphate was added to a physiological saline containing 0.1% BSA (fatty acid free) administered with a drug to prepare an injection solution. In the control group, physiological saline containing 0.05% DMSO and 0.1% BSA was used. 10 μl each was administered daily from immediately after the surgery to a total of 6 sites, 4 sites inside the thigh and 1 site each inside and outside the gastrocnemius muscle.

  In an experiment using a sustained-release preparation, 1 mg of microcapsules was dissolved in 0.5 ml of physiological saline to prepare an injection solution. Immediately after the operation, 4 sites were placed inside the thigh and 1 site each on the medial and external gastrocnemius muscles for a total of 6 sites. 10 μl each was administered using a 27G needle.

Blood flow measurement using a laser Doppler blood flow meter Anesthetize a mouse with pentobarbital, warm it for about 10 minutes in a cage placed on a constant temperature bath at 38 ° C, and then laser doppler blood flow meter on a 37 ° C hot plate. Blood flow was measured using (Moor Instruments (UK)). The blood flow from the lower leg to the fingertip was quantified on both sides and expressed in the ratio of affected side / healthy side.

On the 10th day after the quantification of the capillary density, the mouse was sacrificed by deep anesthesia, and the triceps surae muscle was fixed and used as a frozen specimen. The cross section was subjected to immunohistochemical staining using rat anti-mouse CD31 antibody (Pharmingen) and ABC kit (Vectastain). The number of capillaries per unit area per 10 visual fields was counted and averaged with a 400 × optical microscope.

One-way analysis of variance was used for statistical multi-group comparisons, and Student's t-test was used for two-group comparisons.

(2) Results / Discussion FIG. 1 shows changes in the affected / healthy ratio when a non-sustained release formulation of sphingosine-1-phosphate (S1P) was administered every day. On the seventh day after the operation, blood flow increased 2.5 times in the sphingosine-1-phosphate 10 ⁻⁸ M administration group as compared to the control group.

  FIG. 2 shows changes in the affected / healthy ratio when a single sustained release preparation of sphingosine-1-phosphate (S1P) is administered once. In the case of a sustained-release preparation, an increase in blood flow was observed with a significant difference (p <0.05) on the 5th and 7th days even after single administration.

  FIG. 3 shows the capillary density on day 10 when a non-sustained release formulation of sphingosine-1-phosphate (S1P) was administered every day.

  Furthermore, when a transgenic mouse of sphingosine kinase 1 (SPHK1), which is the main production enzyme of sphingosine-1-phosphate (S1P), was produced and examined in the same manner, on the 9th day after the operation, it was found that A blood flow increase of about 1.5 times was observed (FIG. 4).

  From the above, it was revealed that sphingosine-1-phosphate (S1P) exhibits angiogenesis promoting action and blood flow increasing action in adult ischemic tissue.

It is a figure which shows the change of the blood flow ratio of the affected side / healthy side when the non-sustained release formulation of sphingosine-1-phosphate (S1P) is administered every day. It is a figure which shows the change of the affected-side / health-side blood flow ratio when a sustained-release preparation of sphingosine-1-phosphate (S1P) is administered once. It is a figure which shows the capillary density of the 10th day when the non-sustained release formulation of sphingosine-1-phosphate (S1P) is administered every day. It is a figure which shows the change of the blood flow ratio after the operation in a SPHK1 transgenic mouse (SK) and a littermate wild type individual (WT) after an operation.

Claims (7)

  A post-ischemic angiogenesis promoter containing sphingosine-1-phosphate or a pharmaceutically acceptable salt thereof.   The post-ischemic angiogenesis promoter according to claim 1, which is a sustained-release preparation using a biodegradable polymer as a base.   The post-ischemic angiogenesis promoter according to claim 2, obtained by dissolving sphingosine-1-phosphate or a pharmaceutically acceptable salt thereof and a biodegradable polymer in an organic solvent and then removing the organic solvent. .   The agent for promoting angiogenesis after ischemia according to claim 2 or 3, which is a sustained-release microcapsule.   The post-ischemic angiogenesis promoter according to any one of claims 2 to 4, wherein the biodegradable polymer is a lactic acid-glycolic acid copolymer.   The agent for promoting post-ischemic angiogenesis according to any one of claims 1 to 5, which is a post-ischemic blood flow improving agent.   The agent for promoting post-ischemic angiogenesis according to any one of claims 1 to 5, which is a prophylactic or therapeutic agent for ischemic diseases.

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