JP5158715B2 - Smooth muscle contraction inhibitor - Google Patents

Description

  The present invention relates to a smooth muscle contraction inhibitor. More specifically, the present invention relates to an inhibitor of smooth muscle calcium-independent abnormal contraction, a therapeutic and preventive agent for cardiovascular diseases, and an Fyn / Rho kinase signal transduction system inhibitor.

  It is known that smooth muscle contraction includes contraction depending on an increase in cytoplasmic calcium ion concentration and contraction independent of this.

  That is, in the case of cell membrane depolarization such as nerve stimulation, the calcium ion concentration in the cytoplasm is increased to form a calcium ion-calmodulin complex, and the myosin light chain kinase is activated by this complex and the myosin light chain kinase is activated. Is considered to be phosphorylated and cause contraction. Such calcium ion-dependent smooth muscle contraction is considered normal contraction.

  On the other hand, some vasoconstrictors, such as sphingosylphosphorylcholine (SPC), have enhanced calcium ion sensitivity via cytoplasmic Rho kinase and cause contraction exceeding the calcium ion concentration increase or contraction independent of the calcium ion concentration. It has been known. These are calcium ion-independent contractions and are considered abnormal contractions. For example, SPC activates Fyn, one of the Src family tyrosine kinases, further activates Rho kinase, phosphorylates the myosin light chain without increasing the cytoplasmic calcium concentration, and is independent of calcium ions. It is thought to cause the smooth muscles to contract (Anesthesia, Vol. 47, No. 5, 530-540, 1998).

  As such a calcium ion-independent smooth muscle contraction inhibitor, Rho kinase inhibitor fasdylic acid or Y-27632 is known (Nature, 389, October 30, No. 990-994). 1997). However, since these drugs also have an action of suppressing calcium-dependent contraction, there are limits to their therapeutic effects as well as side effects. Therefore, the present condition is that a drug having an action of selectively suppressing only calcium-independent contraction is desired.

  JP 2001-261556 also discloses that eicosapentaenoic acid (EPA) is independent of calcium ion by inhibiting Fyn activation without affecting calcium ion-dependent normal smooth muscle contraction. It has been reported that only abnormal contraction is selectively suppressed.

  However, in Japanese Patent Application Laid-Open No. 2001-261556, eicosapentaenoic acid sodium salt has a smooth muscle contraction inhibitory action, whereas eicosapentaenoic acid ethyl ester and eicosapentaenoic acid triglyceride have a smooth muscle contraction inhibitory action. It is stated that there is no.

  In addition, eicosapentaenoic acid can only be taken internally and may not be able to accurately respond to acute vascular disease.

  An object of the present invention is to provide a suppressor and a preventive agent for smooth muscle contraction independent of calcium ion without affecting normal smooth muscle contraction dependent on calcium ion.

  The present inventors have found that lipids having a specific acyl group, and octadecapentaenoic acid and octadecatetraenoic acid solve the above problems, and have completed the present invention.

  That is, the present invention comprises a smooth muscle contraction inhibitor characterized by containing at least one selected from the group consisting of octadecapentaenoic acid and octadecatetraenoic acid, salts thereof, and esters thereof. It is.

  The present invention is also a smooth muscle contraction inhibitor containing lipid, wherein at least one acyl chain of the lipid is selected from the group consisting of octadecapentaenoic acid, octadecatetraenoic acid and eicosapentaenoic acid. It is a smooth muscle contraction inhibitor characterized by being at least 1 type.

  Moreover, this invention is said smooth muscle contraction inhibitor whose lipid is a simple lipid or complex lipid.

  Moreover, this invention is said smooth muscle contraction inhibitor whose simple lipid is ceramide or a glyceride.

  Moreover, this invention is said smooth muscle contraction inhibitor whose glyceride is a triglyceride, a diglyceride, or a monoglyceride.

  Moreover, this invention is said smooth muscle contraction inhibitor whose complex lipid is phospholipid.

  In the present invention, the phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, and lysates thereof. It is said smooth muscle contraction inhibitor characterized by these.

  Moreover, the present invention contains at least one selected from the group consisting of octadecapentaenoic acid and octadecatetraenoic acid, salts thereof, and esters thereof, and treats cardiovascular disease It is an agent.

  Further, the present invention is a therapeutic agent for cardiovascular diseases containing lipid, wherein at least one acyl chain of the lipid is selected from the group consisting of octadecapentaenoic acid, octadecatetraenoic acid and eicosapentaenoic acid It is a therapeutic agent for cardiovascular disease, characterized in that it is at least one kind.

  Moreover, this invention is said circulatory system disease therapeutic agent whose lipid is a simple lipid or complex lipid.

  Moreover, this invention is said circulatory system disease therapeutic agent whose simple lipid is a ceramide or a glyceride.

  Moreover, this invention is said circulatory system disease therapeutic agent whose glyceride is a triglyceride, a diglyceride, or a monoglyceride.

  Moreover, this invention is said circulatory system disease therapeutic agent whose complex lipid is phospholipid.

  In the present invention, the phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, and lysates thereof. The therapeutic agent for circulatory system diseases described above.

  Moreover, this invention is said therapeutic agent whose circulatory system disease is vasospasm.

  The present invention is also the above therapeutic agent, wherein the vasospasm is selected from the group consisting of cerebral vasospasm, coronary artery vasospasm, pulmonary vasospasm, mesenteric vasospasm, and finger vasospasm.

  Moreover, the present invention contains at least one selected from the group consisting of octadecapentaenoic acid and octadecatetraenoic acid, salts thereof, and esters thereof, and prevents cardiovascular disease It is an agent.

  The present invention is also a lipid-containing cardiovascular disease preventive agent, wherein at least one acyl chain of the lipid is selected from the group consisting of octadecapentaenoic acid, octadecatetraenoic acid and eicosapentaenoic acid. It is a circulatory system disease preventive agent characterized by being at least one kind.

  Moreover, this invention is said circulatory system disease preventive agent whose lipid is a simple lipid or complex lipid.

  Moreover, this invention is said circulatory system disease preventive agent whose simple lipid is ceramide or a glyceride.

  Moreover, this invention is said circulatory system disease preventive agent whose glyceride is a triglyceride, a diglyceride, or a monoglyceride.

  Moreover, this invention is said circulatory system disease preventive agent whose complex lipid is phospholipid.

  In the present invention, the phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, and lysates thereof. It is a circulatory system disease preventive agent characterized by these.

  Moreover, this invention is said prophylactic agent whose circulatory system disease is vasospasm.

  The present invention is the above preventive agent, wherein the vasospasm is selected from the group consisting of cerebral vasospasm, coronary artery vasospasm, pulmonary vasospasm, mesenteric vasospasm, and finger vasospasm.

  The present invention also includes an Fyn / Rho kinase signal comprising at least one selected from the group consisting of octadecapentaenoic acid and octadecatetraenoic acid, salts thereof, and esters thereof. It is a transmission system inhibitor.

  The present invention is also a lipid-containing Fyn / Rho kinase signaling system inhibitor, wherein at least one acyl chain of the lipid is composed of octadecapentaenoic acid, octadecatetraenoic acid, and eicosapentaenoic acid. It is an Fyn / Rho kinase signal transduction system inhibitor, characterized by being at least one selected from the group.

  The present invention is also the above Fyn / Rho kinase signal transduction system inhibitor, wherein the lipid is a simple lipid or a complex lipid.

  Moreover, this invention is said Fyn / Rho kinase signal transduction system inhibitor whose simple lipid is a ceramide or a glyceride.

  Moreover, this invention is said Fyn / Rho kinase signal transmission system inhibitor whose glyceride is a triglyceride, a diglyceride, or a monoglyceride.

  Moreover, this invention is said Fyn / Rho kinase signal transduction system inhibitor whose complex lipid is phospholipid.

  In the present invention, the phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, and lysates thereof. The Fyn / Rho kinase signal transduction system inhibitor described above.

  ADVANTAGE OF THE INVENTION According to this invention, the inhibitor and preventive agent of smooth muscle contraction independent of calcium ion can be provided, without affecting normal smooth muscle contraction dependent on calcium ion.

  The smooth muscle targeted by the present invention is not particularly limited with respect to organs and parts, and examples thereof include vascular smooth muscle, digestive organ smooth muscle, respiratory smooth muscle, uterine smooth muscle, etc., and particularly blood vessels. Smooth muscle can be mentioned as a preferred example.

  Although there is no restriction | limiting in particular as octadecapentaenoic acid used for this invention, For example, all-cis-3,6,9,12,15-octadecapentaenoic acid can be mentioned as a preferable example.

  Moreover, there is no restriction | limiting in particular as octadecatetraenoic acid used for this invention, For example, all-cis-6,9,12,15-octadecatetraenoic acid can be mentioned as a preferable example.

  Moreover, there is no restriction | limiting in particular as eicosapentaenoic acid used for this invention, For example, all-cis-5,8,11,14,17-eicosapentaenoic acid can be mentioned as a preferable example.

  Octadecapentanoic acid and octadecatetraenoic acid may be in the form of an acid as well as in the form of a salt such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, and sodium salt is particularly preferred. It can be mentioned as a salt. In addition, octadecapentaenoic acid and octadecatetraenoic acid may be in the form of esters such as methyl ester, ethyl ester, butyl ester and glyceryl ester, and may be in the form of phospholipids such as phosphatidylcholine and other complex lipids. May be. Among these, ethyl ester can be mentioned as a particularly preferable ester.

  The method for producing octadecapentaenoic acid, octadecatetraenoic acid, and eicosapentaenoic acid used in the present invention is not particularly limited, and it is extracted from natural products such as seaweed, as well as by chemical synthesis and production by microorganisms. Can also be manufactured.

  The lipid having a specific acyl group used in the present invention is not particularly limited as long as at least one acyl chain is octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid, and examples thereof include simple lipids and complex lipids. be able to.

  When the simple lipid is a glyceride, the position of glycerol to which octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid binds may be any of α-position, β-position and γ-position, at least One acyl chain may be octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid. The acyl chain other than octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid is not particularly limited, and examples thereof include saturated or unsaturated fatty acids having 1 to 30 carbon atoms. Further, if at least one acyl chain is octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid, the number of acyl chains bound to glycerol may be three, two or one. Hereinafter, the monoacylglyceride whose acyl chain is eicosapentaenoic acid and the diacylglyceride whose at least one acyl chain is eicosapentaenoic acid may be referred to as “EPA-monoglyceride” and “EPA-diglyceride”, respectively.

  The method for producing a simple lipid in which at least one acyl chain used in the present invention is octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid is not particularly limited. For example, in the case of glyceride, Glycerol and octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid are bound by an enzyme such as lipase and fractionated by chromatography or the like. Furthermore, a method of chemically synthesizing glycerol with octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid, or a method of separating from natural products can be used.

  When the simple lipid is ceramide, it can be produced by combining sphingosine and octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid with an enzyme such as SCDase and fractionating by chromatography or the like. it can. Furthermore, a method of chemically synthesizing sphingosine and octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid can also be used.

  The complex lipid used in the present invention is not particularly limited as long as it contains a carbon atom, oxygen atom, hydrogen atom, phosphorus atom and / or nitrogen atom and / or sulfur atom in its molecule. Lipids can be mentioned as a preferred example.

  The phospholipid used in the present invention is not particularly limited as long as it is a lipid containing phosphoric acid. For example, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin and their A lyso form can be mentioned. Among these, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol can be mentioned as preferable phospholipids. These phospholipids can be used alone or in combination of two or more.

  When the complex lipid is a glycerophospholipid, the position of glycerol to which octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid binds may be either α-position or β-position. When the glycerophospholipid has two acyl chains, the other acyl chain is not particularly limited, and examples thereof include saturated or unsaturated fatty acids having 1 to 30 carbon atoms. You may have a branch chain. The other acyl chain may be octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid. Among these, phosphatidylcholine in which at least one acyl chain is octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid can be mentioned as a preferred example. Hereinafter, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol whose at least one acyl chain is eicosapentaenoic acid may be referred to as “EPA-PC”, “EPA-PE”, and “EPA-PG”, respectively.

  The method for producing the complex lipid used in the present invention is not particularly limited. For example, in the case of a phospholipid, lysophospholipid and octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid are mixed with an enzyme such as phospholipase. It can be produced by binding and fractionating by chromatography or the like. Furthermore, a method of chemically synthesizing from lysophospholipid and octadecapentaenoic acid, octadecatetraenoic acid or eicosapentaenoic acid, or a method of separating from natural products can be used.

  Cardiovascular diseases targeted by the present invention are not particularly limited, and examples thereof include vasospasm such as cerebral vasospasm, coronary artery vasospasm, pulmonary vasospasm, mesenteric vasospasm, and finger vasospasm. Can do.

  In the present invention, the “Fyn / Rho kinase signal transduction system inhibitor” means a series of proteins that are activated by Fyn tyrosine kinase by stimulation such as SPC and further activated by Rho kinase, thereby enhancing protein phosphorylation. Those that inhibit all or part of the pathway.

  The dosage form of the smooth muscle inhibitor, the therapeutic or preventive agent for circulatory system diseases, and the Fyn / Rho kinase signal transduction system inhibitor of the present invention is not particularly limited and is appropriately determined depending on the purpose. Oral preparations such as tablets, powders and capsules, injections, eye drops, nasal drops, patches and the like.

  In addition, the smooth muscle inhibitor, the therapeutic or preventive agent for cardiovascular diseases, and the Fyn / Rho kinase signal transduction system inhibitor of the present invention can contain a pharmaceutically acceptable carrier and other components.

  There are no particular limitations on the administration method of the smooth muscle inhibitor, the therapeutic or preventive agent for cardiovascular disease, and the Fyn / Rho kinase signal transduction system inhibitor of the present invention, and it is appropriately determined according to each purpose. Examples of the administration method include oral, intraarterial, intravenous, intraperitoneal, subcutaneous, intradermal, transdermal, and transmucosal. In particular, in the case of a therapeutic agent for cardiovascular disease, administration by injection into an artery or vein can be mentioned as a preferred example.

  The dosage of the smooth muscle inhibitor, the therapeutic or preventive agent for cardiovascular disease, and the Fyn / Rho kinase signal transduction system inhibitor of the present invention is appropriately determined according to each purpose, and its dosage form, administration method, It will be adjusted as appropriate according to the degree of symptoms, sex, age, weight, and the like. For example, when it is administered into an artery or vein as a therapeutic agent for cardiovascular disease, it is usually 1 to 200 mg, preferably 5 to 100 mg, more preferably 10 to 50 mg of octadecapentaenoic acid or octadecatetra. The enoic acid or lipid is administered once or divided into several times a day. In addition, when orally administered as a therapeutic agent for cardiovascular disease, usually 0.1 to 9 g, preferably 0.5 to 6 g, more preferably 1 to 3 g of octadecapentaenoic acid, octadecatetraenoic acid or Lipids are administered once a day or divided into several times. Furthermore, when administered orally as a prophylactic agent for cardiovascular disease, the dose is generally smaller than when administered as a therapeutic agent.

<< Example 1: Preparation of n-3 octadecapentaenoic acid and n-3 octadecatetraenoic acid >>
An Antarctic Ocean haptophyte B strain was cultured as follows. 400 mL of the medium was placed in a 500 mL flask, inoculated with haptoalgae B strain, and statically cultured at 4 ° C. for 6-9 weeks under a white fluorescent lamp for 24 hours under light conditions. As the medium, 1 mL of Daigo 1MK medium for marine microalgae (Nippon Pharmaceutical Co., Ltd.) was used per 100 mL of artificial seawater (Marine Art High, Senju Pharmaceutical Co., Ltd.).

  After culturing, the cells were collected by centrifugal sedimentation to obtain algal bodies having a wet weight of 11 g. Lipid extraction was performed from algal cells according to a conventional method (Blight, EG & Dyer WJ (1959) Can. J. Biochem. Physiol. 37: 911-7) to obtain 428 mg of lipid. The total lipid obtained was reacted in methanol containing 10% acetyl chloride at 100 ° C. for 3 hours to obtain 167 mg of hexane extract. The fatty acid composition of this hexane extract was 11% myristic acid, 5% palmitic acid, 4% n-3 octadecatetraenoic acid, 14% n-3 octadecapentaenoic acid, and 8% n-3 docosahexaenoic acid. It was.

  Next, thin layer chromatography was performed to increase the composition of highly unsaturated fatty acids. The hexane extract is applied to silica gel (silica gel 60, Merck), and a developing solvent hexane: diethyl ether = 19: 1 is used to obtain a highly unsaturated fatty acid methyl ester fraction containing 3 or more double bonds in the molecule. 37 mg was recovered. The fatty acid composition of this methyl ester mixture is n-3 octadecatetraenoic acid 14%, n-3 octadecapentaenoic acid 36%, n-3 docosahexaenoic acid 34%, and myristic acid and palmitic acid are almost detected. There wasn't.

  N-3 octadecapentaenoic acid methyl ester and n-3 octadecatetraenoic acid methyl ester in this highly unsaturated fatty acid methyl ester mixture were purified by liquid chromatography. The column used was an ODS reverse phase column (TSK-GEL ODS-120T, inner diameter 4.6 mm, length 250 mm, Tosoh Corporation), and the solvent was detected with an ultraviolet absorptiometer of acetonitrile: water = 85: 15, 205 nm wavelength. As a result, n-3 octadecapentaenoic acid methyl ester and n-3 octadecatetraenoic acid methyl ester were recovered. The recovered n-3 octadecapentaenoic acid methyl ester and n-3 octadecatetraenoic acid methyl ester were 12 mg and 6 mg, respectively, and their purities were 99.7% and 91.0% by gas chromatography analysis, respectively. Met.

  The obtained n-3 octadecapentaenoic acid methyl ester and n-3 octadecatetraenoic acid methyl ester were reacted in acetonitrile: water = 9: 1 solution containing 0.5N HCl at 100 ° C. for 3 hours, Free n-3 octadecapentaenoic acid and free n-3 octadecatetraenoic acid were used.

<< Example 2: Preparation of n-3 octadecatetraenoic acid-bonded ceramide >>
n-3 octadecatetraenoic acid (Cayman Chemical Company) 12.8 μmol, bovine brain cerebroside-derived D-sphingosine (SIGMA) 5.12 μmol, SCDase (Takara Bio) 80 mU, 25 mM phosphorus containing 0.1% Triton X-100 The resultant was dispersed in 3.2 ml of sodium acid buffer (pH 7.0), and a synthesis reaction was performed at 37 ° C. for 40 hours. After a total of 14 enzyme synthesis reactions, lipid extraction was performed according to a conventional method (Blight, EG & Dyer WJ (1959) Can. J. Biochem. Physiol. 37: 911-7).

  The obtained lipid fraction was subjected to a silica gel-TLC plate for preparation (silica gel 60, 0.5 mm thickness, Merck) and developed with diethyl ether: acetic acid (10: 0.1, v / v). The synthesized spot of n-3 octadecatetraenoic acid-binding ceramide was fractionated and extracted from silica gel to obtain 3.2 mg of n-3 octadecatetraenoic acid-binding ceramide.

<< Example 3: Preparation of EPA-PC >>
Eicosapentaenoic acid (EPA) (180 mg) and egg yolk-derived lysophosphatidylcholine (110 mg) were dispersed in glycerol (5500 mg), and porcine pancreas-derived phospholipase A2 (23 mg), formamide (0.5 ml) and CaCl2 ( ₃ μmol) were added. The synthesis reaction was carried out for 48 hours while stirring at 50-200 rpm. After completion of the reaction, the lipid fraction was collected by the Folch method (J. Biol. Chem. 146, 35-44 (1942)).

  The obtained lipid fraction was applied to a silica gel-TLC plate for preparation (silica gel 60, 0.5 mm thickness, Merck) and developed with chloroform: methanol: water (65: 25: 4, v / v / v). . The synthesized EPA-bound phosphatidylcholine (EPA-PC) spot was fractionated and extracted from silica gel to obtain 90 mg of EPA-PC. The prepared EPA-PC contained 46.6% EPA.

<< Example 4: Preparation of EPA-PE and EPA-PG >>
200 mL of the medium was put into a 1000 mL flask, and the EPA-producing bacterium Shewanella benthica ATCC 43991 strain was inoculated and cultured at 28 ° C. for 1 week for a total of 28 shakes. The medium used was LB + Na medium (1.0% tryptone, 0.5% yeast extract, 3.0% sodium chloride).

  After culturing, the cells were collected by a centrifugal sedimentation method to obtain bacterial cells having a wet weight of 36 g. Total lipids were extracted from the cells according to a conventional method (Blight, EG & Dyer WJ (1959) Can. J. Biochem. Physiol. 37: 911-7) to obtain 468 mg of lipid. The obtained total lipid was applied to a thin layer chromatography (TLC) plate (silica gel 60, Merck) and developed using a developing solvent chloroform: methanol: water = 65: 25: 4. PE and PG spots rich in EPA were scraped and extracted from silica gel to obtain 49 mg and 27 mg of lipid, respectively. These PE and PG containing a large amount of EPA were designated as EPA-PE and EPA-PG, respectively. EPA-PE and EPA-PG contained 38% and 39% EPA, respectively.

<< Example 5: Preparation of EPA-PS >>
After 30 mg of EPA-PC synthesized in Example 2 was dissolved in 2.5 ml of ethyl acetate, 1 ml of 0.2 M acetate buffer (pH 5.6) containing 3.4 M L-serine was added. Furthermore, 1 unit of phospholipase D (derived from Streptomyces sp.) Was added to start the reaction, and then phosphatidyl group transfer reaction was performed for 24 hours while stirring at 300 to 400 rpm in a dark place at 37 ° C. After completion of the reaction, the lipid fraction was collected by the Folch method (J. Biol. Chem. 146, 35-44 (1942)).

  The obtained lipid fraction was applied to a silica gel-TLC plate for preparation (silica gel 60, 0.5 mm thickness, Merck) and developed with chloroform: methanol: water (65: 25: 4, v / v / v). . The synthesized EPA-bound phosphatidylserine (EPA-PS) spot was fractionated and extracted from silica gel to obtain 12 mg of EPA-PS. The prepared EPA-PS contained 47% EPA.

<< Example 6: Preparation of enzyme-synthesized EPA-PG >>
After 50 mg of EPA-PC synthesized in Example 2 was dissolved in 2.2 ml of ethyl acetate, 1.2 ml of 0.2 M acetate buffer (pH 5.6) containing 14 mg of bovine serum albumin was added. Further, 400 μl of glycerol and 2.4 units of phospholipase D (derived from Streptomyces sp.) Were added to start the reaction, and then a phosphatidyl group transfer reaction was performed for 4 hours while stirring at 300-400 rpm in a dark place at 37 ° C. . After completion of the reaction, the lipid fraction was collected by the Folch method (J. Biol. Chem. 146, 35-44 (1942)).

  The obtained lipid fraction was applied to a silica gel-TLC plate for preparation (silica gel 60, 0.5 mm thickness, Merck) and developed with chloroform: methanol: water (65: 25: 4, v / v / v). . The synthesized EPA-bound glycerol (EPA-PG) spot was fractionated and extracted from silica gel to obtain 20 mg of enzyme-synthesized EPA-PG. The prepared enzyme-synthesized EPA-PG contained 50% EPA.

<< Example 7: Preparation of EPA-monoglyceride and EPA-diglyceride >>
300 mg of EPA and 5 g of glycerol were sufficiently dispersed in the presence of 9 mg of α-tocopherol, and then 30 mg of Lipozyme RM IM (Novozymes A / S) was added thereto and stirred at 300-400 rpm in the dark at 37 ° C. A time synthesis reaction was performed. After completion of the reaction, partitioning was performed using diethyl ether: water (1: 1, v / v), and a lipid fraction was collected from the diethyl ether layer.

  The obtained lipid fraction was subjected to a preparative silica gel-TLC plate (silica gel 60, 0.5 mm thickness, Merck) and developed with n-hexane: ether (60:40, v / v). After fractionating the spots of synthesized EPA-monoglyceride, EPA-diglyceride (collected as a mixture of 1,2-EPA-diglyceride and 1,3-EPA-diglyceride), extraction from silica gel and 80 mg of EPA-monoglyceride, 90 mg of EPA-diglyceride was obtained.

<< Example 8: Tension test >>
<Sample>
A porcine coronary artery (left anterior descending branch) was cut about 3 cm 1 cm distal from the main trunk bifurcation, and a Krebs solution (123 mM NaCl, 4.7 mM) previously aerated with a mixed gas (95% O ₂ , 5% CO ₂ ) and ice cooled. KCl, 15.5 mM NaHCO ₃ , 1.2 mM KH ₂ PO ₄ , 1.2 mM MgCl ₂ , 1.25 mM CaCl ₂ , 11.5 mM D-glucose). The following sample preparation was performed while exchanging the Krebs solution every 15 minutes. After removing the fat around the blood vessel, the outer membrane was removed, the endothelium was removed with a cotton swab, and a smooth muscle strip 1 mm × 4 mm was prepared using a razor.

<Sample>
The octadecapentaenoic acid obtained according to Example 1 was used after diluting with Krebs solution to a final concentration after replacing the solvent with ethanol. The EPA-PC obtained according to Example 3 was used after adjusting the concentration. As EPA-PE and EPA-PG, those obtained in accordance with Example 4 were used by adjusting to the final concentration of EPA based on the content of EPA. Octadecatetraenoic acid and octadecapentaenoic acid, which were obtained according to Example 1, were used after diluting to a final concentration with Krebs solution after replacing the solvent with ethanol. EPA-monoglyceride and EPA-diglyceride were used according to Example 7 after the solvent was replaced with ethanol and diluted with Krebs solution to a final concentration. EPA-PS was obtained according to Example 5, after volatilizing the solvent with nitrogen gas, adding a small amount of Krebs solution and suspending by sonication, and further diluting this with Krebs solution to the final concentration. Used. Enzyme-synthesized EPA-PG and octadecatetraenoic acid ceramide were used according to Examples 6 and 2, after the solvent was replaced with ethanol, diluted to a final concentration with Krebs solution. .

<Device>
The wire on which the sample was suspended was connected to a transducer (FD pickup: Nihon Kohden), and the tension was detected with a recorder (desktop pen recorder U-228: Pantos) through an amplifier (distortion pressure amplifier: Nihon Kohden). The specimen was placed in a 5 mL Magnus tube maintained at 37 ° C., and the test solution was maintained at 37 ° C. and loaded with a mixed gas.

<Measurement method>
As a reference (100%) contraction, a calcium-dependent contraction of depolarization with 118 mM high potassium solution is caused for 15 minutes, and then the operation of relaxing for 15 minutes with Krebs solution is repeated, and the waveform of tension due to high potassium depolarization becomes stable. When 40 mM potassium solution was added and the contraction reached a plateau, 1 μM bradykinin was added to confirm the presence or absence of endothelium. It was judged that the endothelium was removed because the smooth muscle was not relaxed by bradykinin. Thereafter, the cells were relaxed with Krebs solution for 20 minutes and stimulated with SPC 30 μM. Further, when the tension due to SPC stimulation reached the maximum and plateau, octadecapentaenoic acid, EPA-PC, and EPA (reference example) were added so that the final concentration was 60 μM. EPA-PE and EPA-PG were added based on the content of EPA so that the final concentration of EPA was 60 μM. Octadecatetraenoic acid was added to a final concentration of 60 μM. EPA-monoglyceride and EPA-diglyceride were added based on the content of EPA so that the final concentration of EPA was 60 μM. EPA-PS and enzyme synthesized EPA-PG were added based on the content of EPA so that the final concentration of EPA was 60 μM. Octadecatetraenoic acid ceramide was added based on the content of octadecatetraenoic acid so that the final concentration of octadecatetraenoic acid was 60 μM.

<Result>
FIG. 1 shows an example using EPA, FIG. 2 shows an example using octadecapentaenoic acid, FIG. 3 shows an example using EPA-PC, FIG. 4 shows an example using EPA-PE, and FIG. 5 shows an example using EPA-PG. FIG. 6 shows an example with octadecatetraenoic acid, FIG. 7 shows an example with EPA-monoglyceride, FIG. 8 shows an example with EPA-diglyceride, and FIG. 9 shows an example with EPA-PS. FIG. 10 shows an example using enzyme synthesis EPA-PG, and FIG. 11 shows an example using octadecatetraenoic acid ceramide.

  Neither endothelium-dependent relaxation reaction by bradykinin was observed, indicating that the endothelial cells are removed blood vessels.

  1 to 11, octadecapentaenoic acid and EPA-PC, EPA-PE, EPA-PG, octadecatetraenoic acid, EPA-monoglyceride, EPA-diglyceride, EPA-PS, enzymatic synthesis EPA-PG, octadeca It can be seen that ceramide tetraenoic acid suppresses abnormal contraction due to SPC as in EPA.

  ADVANTAGE OF THE INVENTION According to this invention, the inhibitor and preventive agent of smooth muscle contraction independent of calcium ion can be provided, without affecting normal smooth muscle contraction dependent on calcium ion.

FIG. 1 is a diagram showing contraction suppression by EPA against SPC stimulation in smooth muscle tissue. FIG. 2 is a diagram showing contraction suppression by octadecapentaenoic acid for SPC stimulation in smooth muscle tissue. FIG. 3 is a diagram showing the suppression of contraction by EPA-PC for SPC stimulation in smooth muscle tissue. FIG. 4 is a diagram showing the suppression of contraction by EPA-PE for SPC stimulation in smooth muscle tissue. FIG. 5 is a diagram showing the suppression of contraction by EPA-PG for SPC stimulation in smooth muscle tissue. FIG. 6 is a diagram showing the suppression of contraction by octadecatetraenoic acid for SPC stimulation in smooth muscle tissue. FIG. 7 is a diagram showing the suppression of contraction by EPA-monoglyceride for SPC stimulation in smooth muscle tissue. FIG. 8 is a diagram showing the suppression of contraction by EPA-diglyceride against SPC stimulation in smooth muscle tissue. FIG. 9 is a diagram showing contraction suppression by EPA-PS for SPC stimulation in smooth muscle tissue. FIG. 10 is a diagram showing contraction suppression by enzyme-synthesized EPA-PG in response to SPC stimulation in smooth muscle tissue. FIG. 11 is a diagram showing contraction suppression by octadecatetraenoic acid ceramide in response to SPC stimulation in smooth muscle tissue.

Claims (32)

A smooth muscle contraction inhibitor comprising at least one selected from the group consisting of octadecapentaenoic acid and salts thereof, and esters thereof ,
A smooth muscle contraction inhibitor, wherein the ester is at least one selected from the group consisting of methyl ester, ethyl ester, butyl ester, and glyceryl ester. A smooth muscle contraction inhibitor containing lipid,
At least one of the acyl chains, characterized in that it is a octadecadienoic penta-enoic acid, smooth muscle contraction inhibitor of the lipid. The smooth muscle contraction inhibitor according to claim 2, wherein the lipid is a simple lipid or a complex lipid. The smooth muscle contraction inhibitor according to claim 3, wherein the simple lipid is ceramide or glyceride. The smooth muscle contraction inhibitor of Claim 4 whose glyceride is a triglyceride, a diglyceride, or a monoglyceride. The smooth muscle contraction inhibitor according to claim 3, wherein the complex lipid is a phospholipid. The phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin , and their lyso forms, and sphingomyelin , The smooth muscle contraction inhibitor according to claim 6. A therapeutic agent for cardiovascular diseases, comprising at least one selected from the group consisting of octadecapentaenoic acid and salts thereof, and esters thereof ,
A therapeutic agent for cardiovascular disease, wherein the ester is at least one selected from the group consisting of methyl ester, ethyl ester, butyl ester and glyceryl ester. A therapeutic agent for cardiovascular disease containing lipids,
At least one of the acyl chains, characterized in that it is a octadecadienoic penta-enoic acid, therapeutic agents for diseases of the circulatory system of the lipid. The therapeutic agent for cardiovascular disease according to claim 9, wherein the lipid is a simple lipid or a complex lipid. The therapeutic agent for cardiovascular disease according to claim 10, wherein the simple lipid is ceramide or glyceride. The therapeutic agent for cardiovascular disease according to claim 11, wherein the glyceride is triglyceride, diglyceride, or monoglyceride. The therapeutic agent for cardiovascular disease according to claim 10, wherein the complex lipid is a phospholipid. The phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin , and their lyso forms, and sphingomyelin , The therapeutic agent for cardiovascular disease according to claim 13. The therapeutic agent according to any one of claims 8 to 14, wherein the cardiovascular disease is vasospasm. The therapeutic agent according to claim 15, wherein the vasospasm is selected from the group consisting of cerebral vasospasm, coronary artery vasospasm, pulmonary vasospasm, mesenteric vasospasm, and finger vasospasm. An agent for preventing cardiovascular disease, comprising at least one selected from the group consisting of octadecapentaenoic acid and salts thereof, and esters thereof ,
A cardiovascular disease preventive agent, wherein the ester is at least one selected from the group consisting of methyl ester, ethyl ester, butyl ester and glyceryl ester. A preventive agent for cardiovascular disease containing lipids,
At least one of the acyl chains, characterized in that it is a octadecadienoic penta-enoic acid, cardiovascular disease prevention agents of the lipid. The cardiovascular disease preventive agent according to claim 18, wherein the lipid is a simple lipid or a complex lipid. The cardiovascular disease preventive agent according to claim 19, wherein the simple lipid is ceramide or glyceride. The circulatory disease preventive agent according to claim 20, wherein the glyceride is triglyceride, diglyceride, or monoglyceride. The cardiovascular disease preventive agent according to claim 19, wherein the complex lipid is a phospholipid. The phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin , and their lyso forms, and sphingomyelin , The preventive agent for cardiovascular disease according to claim 22. The preventive agent according to any one of claims 17 to 23, wherein the cardiovascular disease is vasospasm. The prophylactic agent according to claim 24, wherein the vasospasm is selected from the group consisting of cerebral vasospasm, coronary artery vasospasm, pulmonary vasospasm, mesenteric vasospasm, and finger vasospasm. An Fyn / Rho kinase signal transduction system inhibitor comprising at least one selected from the group consisting of octadecapentaenoic acid and salts thereof, and esters thereof ,
The Fyn / Rho kinase signal transduction system inhibitor, wherein the ester is at least one selected from the group consisting of methyl ester, ethyl ester, butyl ester, and glyceryl ester. A lipid-containing Fyn / Rho kinase signaling system inhibitor comprising:
At least one of the acyl chains, characterized in that it is a octadecadienoic penta-enoic acid, Fyn / Rho kinase signaling pathway inhibitor of lipid. 28. The Fyn / Rho kinase signaling pathway inhibitor according to claim 27, wherein the lipid is a simple lipid or a complex lipid. 29. The Fyn / Rho kinase signal transduction system inhibitor according to claim 28, wherein the simple lipid is ceramide or glyceride. 30. The Fyn / Rho kinase signal transduction system inhibitor according to claim 29, wherein the glyceride is triglyceride, diglyceride, or monoglyceride. The Fyn / Rho kinase signal transduction system inhibitor according to claim 28, wherein the complex lipid is a phospholipid. The phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin , and their lyso forms, and sphingomyelin , The Fyn / Rho kinase signal transduction system inhibitor according to claim 31.

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