JP5103618B2 - Inhibitor of abnormal vasoconstriction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a water-soluble drug composition specifically inhibiting calcium-independent abnormal vasoconstriction and can be used as an injection. <P>SOLUTION: The inhibitor of abnormal vasoconstriction comprises N-ä2-[4-(2,2-dimethylpropyonyloxy)phenylsulfonylamino]benzoyl}aminoacetate or a pharmacologically acceptable derivative thereof as an active ingredient. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

  The present invention relates to a therapeutic agent for a disease caused by abnormal blood vessel contraction, and more particularly, to an inhibitor of epivascular contraction comprising a compound capable of suppressing calcium-independent abnormal blood vessel contraction as an active ingredient.

In this specification, the following terms are defined as follows. Abnormal contraction of blood vessels refers to abnormal contraction (also referred to as spasm) of vascular smooth muscle, in particular, contraction independent of an increase in cytoplasmic calcium ion (Ca ²⁺ ) concentration, that is, Ca ²⁺ independent. Membrane raft refers to the structure on the cell membrane that is rich in cholesterol and sphingolipids formed on the surface of blood vessels, and is important as a signal transduction field for vascular disease, and disappears by removing cholesterol. Yes.

  Abnormal contraction of blood vessels and accompanying sudden blood circulation disorders cause cerebral infarction, angina pectoris, myocardial infarction, and so on. It has become a serious problem. In particular, “cerebral vascular spasm” that occurs frequently after subarachnoid hemorrhage is a disease in which most of the blood vessels in the brain contract and widen the infarcted state of the brain, despite the success of subarachnoid hemorrhage surgery. It is causing a serious situation where the patient's life cannot be saved.

As for the contraction of blood vessels, “normal contraction” depending on Ca ²⁺ and “abnormal contraction” shown in the above definition are known, and a considerable part of the normal contraction including its mechanism has been clarified. However, little is known about the mechanism and countermeasures for abnormal contraction. Therefore, in the case of cerebral infarction, angina pectoris, myocardial infarction, etc. caused by abnormal contraction of blood vessels, conventionally, as a means for suppressing the normal contraction of the mechanism, that is, treatment by inhibiting Ca ²⁺ channel In addition, calcium antagonists and the like are used in actual medical settings (Patent Documents 1 and 2).
However, the true cause of the above diseases is abnormal blood vessel contraction, and as a result, a countermeasure that suppresses normal contraction can be expected to have an immediate effect in terms of reducing the amount of contraction in terms of the overall blood vessel contraction phenomenon. However, the current situation is not an essential treatment. Therefore, a treatment method capable of specifically suppressing abnormal blood vessel contraction itself has been eagerly desired.
Patent application title: Calcium channel inhibitor JP-A-9-176005 L-carnitine or alkanoyl L-carnitine-containing pharmaceutical composition in combination with omega-3 polyunsaturated fatty acid Suppressor of abnormal smooth muscle contraction Monthly Bioindustry November 2003 issue “Eicosapentaenoic acid (EPA) prevents vasospasm” Nakao F, Kobayashi S .; et al. 2002. Circ. Res. 91: 953-960. Shirao S, Kobayashi S. et al. et al. 2002. Circ. Res. 91: 112-119. Somlyo A. V. 2002. Circ. Res. 91: 83-84 (Editorials).

  In view of the above-mentioned present situation, the present invention is a water-soluble drug that can specifically inhibit abnormal contraction of blood vessels and can be used as an injection as a means for dealing with symptoms that require immediate effect. An object is to provide a composition.

In order to solve the above problems, the present inventors proceeded with analysis using a vasoconstriction model of mammals, a vasospasm model of mice, etc., and for vasospasm, sphingosylphosphorylcholine (sphingosylphosphorylcholine), which is a kind of sphingolipid, (Hereinafter abbreviated as SPC) plays an important role, SPC contracts vascular smooth muscles independently of Ca ²⁺ through activation of Rho kinase, and another protein called Fyn is activated by SPC. Thus, it has been clarified that binding to a structure called membrane raft on the vascular smooth muscle cell membrane wall plays an important role in the activation of Rho kinase. These facts suggest the possibility that vasospasm can be treated without inhibiting the normal contraction of blood vessels if a series of Rho kinase activation processes by SPC stimulation can be inhibited somewhere (non-patent literature). 1-4). Based on this prediction, various compounds have been studied, and by the oral administration of eicosapentaenoic acid (EPA), one of the highly unsaturated fatty acids contained in fish, etc., abnormal blood vessel contraction can actually be prevented. I have found out (Patent Document 3). However, since EPA is a fatty acid and is unsuitable as an injection, it searches for a compound having a higher immediate effect and available as an injection, and a compound represented by the following chemical formula (I), that is, N- {2 It was found that [4- (2,2-dimethylpropyonyxy) phenylsulfonylamino] benzoyl} aminoacetate (drug name: cyberestat) has a high inhibitory effect on abnormal vascular contraction, and the present invention was completed.

  Cibelestat itself is a known compound and has already been used as a therapeutic agent for emphysema and rheumatoid arthritis. Its effect is to specifically inhibit elastase (neutrophil elastase), which acts on the inflammatory response in the body, thereby causing diseases caused by excessive action of elastase, such as emphysema, rheumatoid arthritis, atherosclerosis Is to treat. The present inventors examined this substance as a therapeutic agent for abnormal contraction of blood vessels from a viewpoint completely different from the effect known so far of elastase inhibition, and sodium of sivelestat that has already been confirmed to be safe. The present inventors have found the fact that salts and other soluble salts have a fundamentally different effect from the conventionally known effect of elastase-specific inhibitory effect and can be used as an injection.

A first aspect of the invention is a compound represented by the chemical formula (1), or, as an active ingredient a derivative selected from at least one of its alkali metal salts, alkaline earth metal salts, ammonium salts, An inhibitor of abnormal blood vessel contraction is provided.

According to a second aspect of the present invention, there is provided the abnormal blood vessel contraction inhibitor according to the first aspect, wherein the derivative is a sodium salt.

According to a third aspect of the present invention, there is provided the vascular abnormal contraction inhibitor according to the first or second aspect, wherein the vascular abnormal contraction is calcium-independent abnormal contraction.

According to a fourth aspect of the present invention, there is provided the vasoconstriction inhibitor according to any one of the first to third aspects, wherein the administration form is an injection.

  By using an inhibitor of abnormal vascular contraction provided by the present invention, an injectable therapeutic drug that is immediately effective for calcium-independent vascular abnormal contraction for which no therapeutic method has been established at all has been provided. It becomes possible to do. The compound (cyberestat) provided by the present invention has been confirmed to be safe as a therapeutic agent for pneumonia, and is expected to be usable as a new therapeutic agent very quickly.

  The best mode for carrying out the present invention will be described below. The first aspect of the present invention provides an inhibitor of abnormal vascular contraction comprising as an active ingredient a compound represented by the above-mentioned chemical formula (I), that is, civerestat or a pharmacologically acceptable derivative thereof. The pharmacologically acceptable derivative is preferably at least one derivative selected from alkali metal salts, alkaline earth metal salts, and ammonium salts, and more preferably a sodium salt. Hydrates may also be used. In the present specification, the compounds represented by the formula (I) and pharmacologically acceptable derivatives thereof are collectively referred to as cyberestat. The present inventors have pioneered the world that eicosapentaenoic acid (EPA), a kind of unsaturated fatty acid contained in fish, has a specific effect on abnormal blood vessel contraction. However, in the process of its analysis, it has been found that a specific three-dimensional structure of EPA is necessary for abnormal vasoconstriction. We searched with attention and arrived at Cibelestat in the process. As shown in FIG. 1, EPA and cibelestat have completely different structural formulas, but when the present inventors performed molecular mechanical optimization on the structure, as shown in FIG. A rich U-shaped structure is expected, and under this expectation, as shown in the examples below, when Cibelestat was applied to an abnormal vasoconstriction model, the calcium-independent contraction of vascular smooth muscle was specifically detected. It became clear that it inhibited.

  Therefore, the inhibitor of abnormal vasoconstriction provided by the present invention is preferably a so-called “calcium-independent vasoconstriction” which is not a vasoconstriction known as normal vasoconstriction, that is, a G protein-binding agonist in the body, for example, It is applicable to abnormal vasoconstriction caused by thromboxane or SPC stimulation, more preferably vasoconstriction caused by activation of Rho kinase-Fyn protein in a membrane raft on a vascular cell membrane by SPC stimulation. The suppression of abnormal vasoconstriction by EPA, which has been clarified so far by the present inventors, is that EPA suppresses the activation of the Rho kinase-Fyn protein signaling system present in the membrane raft. As with EPA, Siberestat has a three-dimensional structure rich in hydrocarbons as shown in FIG. 2, and thus probably enters membrane rafts like EPA and suppresses abnormal contraction.

  The inhibitor of abnormal vascular contraction provided by the present invention can be used as a therapeutic agent for various diseases caused by abnormal contraction of blood vessels. The dosage form may be appropriately changed depending on the patient's condition and is not intended to limit the present invention. For example, it may be administered in the form of an injection or oral medicine, particularly when severe symptoms are exhibited. Is difficult to administer orally, so it is preferable to administer it as an immediate injection. The concentration of sivelestat and other auxiliary components contained in the inhibitor may be properly used according to the purpose of drug discovery depending on the state and type of the disease, and the present invention is not limited. As the concentration, a concentration within the range of 0.1 mM to 1 mM, more preferably within the range of 0.2 to 0.3 mM is effective as shown in the following examples. Examples of the present invention are shown below, but the present invention is not limited to the examples.

(Adjustment of porcine coronary artery specimens) All animal experiments in this example were conducted in accordance with relevant laws and regulations. The coronary arteries were removed from the pig heart treated at the slaughterhouse by the following method. A 2-3 cm left anterior descending branch was removed from the origin of the coronary artery, and immediately cooled Krebs solution (123 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl ₂ , 1.2 mM MgCl ₂ , 12 mM KH ₂ PO ₄ , 15.5 mM NaHCO ₃ , 11.5 mM glucose, bubbled with 95% O ₂ + 5% CO ₂ ) and stored. After carefully removing the connective tissue and vascular outer membrane around the blood vessel, a 1 × 4 mm strip was prepared. In the experiment in which vascular endothelial cells were removed, the endothelial surface of the strip was gently wiped with a cotton swab, and it was confirmed that there was no vascular relaxation reaction with 1 μM bradykinin.

(Isometric tension measurement) The prepared coronary artery smooth muscle strip is suspended in a 5 ml organ chamber (filled with 37 ° C. Krebs solution), one end is fixed, and the other end is a transducer (TB-612T, Nihon Kohden). And isometric tension was measured. The strip is loaded with a static tension of approximately 300 mg, the generated tension is 0% of the static tension in Krebs solution, a G protein-coupled agonist, and U46619, an analog of thromboxane that causes contraction of the blood vessel wall ( The tension at the time of administration of 0.1 μM, Cayman chemical) was expressed as 100%, and this was used as a vasoconstriction model. Cibelestat sodium (Ono Pharmaceutical Co., Ltd.) has low solubility in Krebs solution, and after dissolving in DMSO, the upper limit of the administration concentration was 0.3 mM.
Using the above vasoconstriction model, the effect of sivelestat on vascular smooth muscle contraction was examined. As shown in FIG. 3, sivelestat sodium relaxed coronary artery smooth muscle pre-contracted with U46619 in a concentration-dependent manner. In FIGS. 3A, B, and C, the horizontal axis of the graph represents time, and the lower white box represents the period in which U46619 (A, B) and potassium solution (C) are reacted. The vertical axis of the graph represents the relative value of the vascular smooth muscle tension. The arrow shown on the graph in the figure represents the administration time point of cibelestat, and the accompanying number represents the concentration (M) of cibelestat. FIG. 3A shows the results for the vascular smooth muscle from which the endothelial tissue was removed, and a clear relaxation reaction was observed when 0.1-0.3 mM of sivelestat was applied. FIG. 3B is a result of providing a smooth muscle strip with an endothelial tissue attached thereto, and a clear relaxation effect of cyberestat was observed as in FIG. 3A. From the results of FIGS. 3A and 3B, it was shown that Siberestat has an effect of suppressing the contraction of vascular smooth muscle regardless of the presence or absence of endothelial tissue. On the other hand, in the precontraction due to high-concentration potassium ion (K ⁺ ) depolarization stimulation (model of normal contraction), the relaxation effect due to Siberestat was not observed as compared with the precontraction due to U46619 (FIG. 3C). From the comparison of FIGS. 3A, B and C, it has been clarified that cibelestat has a relaxing effect only on abnormal contraction and does not act on normal contraction.

(Smooth muscle cell membrane potential measurement) In order to confirm whether the relaxation effect of sivelestat treatment was observed due to suppression of contraction, or caused by relaxation of the muscle itself via potassium channels, sivelestat The resting membrane potential at the time of treatment was measured. The smooth muscle strip from which the endothelial cells were removed was fixed in a 3 ml organ chamber with the endothelial side facing up. The chamber was perfused with Krebs solution at 37 ° C. at a flow rate of 3 ml / min, and a very small glass electrode (electric resistance at the tip = 50-80 MΩ) filled with 3 M KCl was inserted from the endothelium side, and the membrane potential was measured. The electric signal was amplified with an amplifier (Intra767, World Precision Instruments), and the potential change was observed over time with an oscilloscope (SS7802, IWATSU).
FIG. 4 shows the result. FIG. 4A shows the time-dependent change in potential due to cybelestat and Levchromalim (10 μM) treatment that activates potassium channels and induces hyperpolarization. In the graph, the potential changes in the range of the box showing Levcrokalim treatment. On the other hand, the potential does not fluctuate in the range of the box indicating the cibelestat treatment, and the cibelestat treatment does not affect the resting membrane potential, that is, the cibelestat activates the potassium channel present in the vascular smooth muscle. It became clear that it did not cause muscle relaxation. FIG. 4B shows the equilibrium value of the potential in each treatment, while Levcrokalim is around −70 mV, while Siberestat is around −50 mV as in the case of no treatment, confirming the result of FIG. 4A.

(Simultaneous measurement of tension and intracellular calcium concentration) Smooth muscle strips from which endothelial cells have been removed were loaded with Fura-2, a Ca ²⁺ sensitive fluorescent dye, and isometric tension measurement and intracellular calcium ion concentration [Ca ²⁺ ]. i was measured simultaneously. Strips were placed in Krebs solution containing 12.5 μM AM Fura-2AM (Dojindo) with a fat-soluble acetoxymethyl ester (AM) group and incubated at 37 ° C. for 4 hours (95% O ₂ +5 Bubbling with% CO ₂ ). While performing isometric tension measurement in a quartz organ chamber, two-wavelength excitation at 340 nm and 380 nm was performed using a fluorometer (CAM-230, JASCO). Fluorescence at 500 nm of 340 nm excitation light and 380 nm excitation light was measured, and the ratio (F340 / F380) was measured as [Ca ²⁺ ] i. The change in tension and [Ca ²⁺ ] i was expressed as 0% in Krebs solution and 100% in 118 mM K + solution (reversed K + concentration and Na + concentration of Krebs solution).
FIG. 5 shows these results. The graph in FIG. 5A shows the change with time of [Ca ²⁺ ] i, and the time axis coincides with the graph in FIG. 5A (result of tension measurement). When Cibelestat is administered to a smooth muscle strip contracted by increasing [Ca ²⁺ ] i at U46619, smooth muscle is relaxed without decreasing [Ca ²⁺ ] i (no change in the upper graph). Became clear. Moreover, as shown in FIG. 5B, when Ca2 ⁺ is cumulatively administered in the presence of U46619 and high concentration K + and a [Ca2 ⁺ ]-tensile curve is drawn, the treatment with U46619 is compared to the case of high concentration K + alone (graph ◇). This graph shifts to the left (graph ○), which indicates that “high sensitivity” occurs in which contraction occurs at a low concentration of calcium. On the other hand, when the cibelestat treatment is performed after the U46619 treatment, this curve is changed. Shifted to the right and downward (graph ●), it was shown that sivelestat suppressed the increase in calcium sensitivity by U46619. As has been clarified in Non-Patent Document 3 by the present inventors, the increase in calcium sensitivity is a phenomenon that is characteristic of abnormal blood vessel contraction caused by SPC stimulation. It shows effectiveness as an inhibitor of abnormal contraction.

(Skinned method) Smooth muscle strips from which endothelial cells had been removed were treated with β-escin (Katayama Chemical) to make small holes in the cell membrane while preserving the information transmission mechanism of the cell membrane. 150-200 mm × 2 mm strip of intracellular fluid containing 30 mM β-escin (74.1 mM Potassium methanolate sulfate, 2 mM Magnesium methanolate sulfate, 4.5 mM MgATP, 1 mM EGTA, 10 mM creatine phosphate, 30 mM PIPES, 1 μM FCCP After incubating with leupeptin, 1 μM calmodulin), a contractile response to pCa6.3 solution (intracellular fluid containing low concentration Ca2 + buffered with 10 mM EGTA) was observed. For skinned specimens, maximum contraction was obtained at pCa4.5. Using this skinned specimen, the effect of Siberestat was confirmed.
FIG. 6 shows these results. In FIG. 6A, the contraction reaction (upward swell) seen in the range indicated by the box of U46619 + GTP was completely suppressed by the Shibelestat treatment (black box). FIG. 6B shows this as a bar graph, and shows that the contraction (left side) induced by the stimulation of U46619 + GTP was suppressed to almost 0 by the Shibelestat treatment (right side). FIG. 6C shows the result of processing Cibelestat without U46619 + GTP in the above experimental system, and since no contraction suppressing effect is seen here, the influence of Cibelestat suppresses the contraction of the blood vessel itself. Instead, it was shown to suppress contractions caused by certain stimuli such as U46619. That is, the contraction (calcium-independent contraction) obtained by administering U46619 (30 nM) and GTP (10 μM) in a pCa6.3 solution was completely suppressed by 0.3 mM cyberestat, It was shown that sivelestat has no effect on the dependent contraction (in pCa6.3 solution).

  As clarified in the above examples, the drug mainly composed of cibelestat provided by the present invention is expected to specifically suppress abnormal contraction of blood vessels without affecting normal contraction, It can be used in the medical industry as a therapeutic agent for various diseases caused by abnormal blood vessel contraction.

The chemical structures of cibelestat and eicosapentaenoic acid are compared and shown. The chemical structures of civerestat and eicosapentaenoic acid are shown in comparison with a focus on their steric structures. Figure 7 shows the effect of sivelestat on vasoconstriction caused by U46619 (thromboxane analog) administration (A, B) and high concentration potassium solution treatment (C). (A) The measurement result in the vascular smooth muscle which removed the endothelium is shown. (B) shows the measurement results with vascular smooth muscle with the endothelium attached. (C) The measurement result in the high concentration potassium solution pretreatment is shown. The influence on the resting membrane potential of Siberestat treatment is shown. (A) shows time-dependent changes in intracellular calcium concentration and tension in a specimen that has been treated with sivelestat in the presence of U46619. (B) shows the effect of sivelestat treatment on the increase in calcium sensitivity caused by U46619 treatment. The influence of the cibelestat process on the vascular smooth muscle contraction in the presence of U46619 using the skinned method is shown.

Claims (4)

The compounds of formula (1), or its alkali metal salts, alkaline earth metal salts, as an active ingredient a derivative selected from at least one of an ammonium salt, an inhibitor of vascular abnormalities contraction.

The abnormal vasoconstriction inhibitor according to claim 1, wherein the derivative is a sodium salt . The abnormal blood vessel contraction inhibitor according to claim 1 or 2, wherein the abnormal blood vessel contraction is calcium-independent abnormal contraction. The vascular abnormal contraction inhibitor according to any one of claims 1 to 3, wherein the administration form is an injection .

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