JP2003238441A - Neovascularization inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a neovascularization inhibitor useful as an excellent prophylactic and remedy for neovascular diseases including malignant tumor, proliferative diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, and psoriasis, useful as a diagnostic agent for the neovascular diseases, and useful for a drug delivery system to newborn blood vessels so as to prevent and treat the neovascular diseases. <P>SOLUTION: This neovascularization inhibitor contains a substance which inhibits an action of an endothelium-derived hyperpolarizing factor (EDHF) and controls neovascularization as an active ingredient. A potassium ion channel blocker which blocks a potassium ion channel in a endothelial cell or the like, a potassium ion-liberating substance which increases a potassium ion concentration in a local area, and a gap-junctional inhibitor which controls and inhibits gap junction are each used for the neovascularization inhibitor. Further, an EDHF inhibitor, such as an epoxyeicosatrienoic acid inhibitor for controlling and inhibiting action of epoxyeicosatrienoic acid which is a product of a cytochrome P-450 epoxygenase and is estimated to have vasodilative action similar to that of the EDHF, is used for the neovascularization inhibitor. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an endothelium-derived hyperpolarizing factor (E).
DHF) to suppress angiogenesis by causing vascular contraction by inhibiting the action of DHF), and to malignant tumors, proliferative diabetic retinopathy, and age-related diseases using the angiogenesis inhibitor The present invention relates to prophylactic / therapeutic agents and diagnostic agents for angiogenic diseases such as macular degeneration, rheumatoid arthritis and psoriasis.

[0002]

BACKGROUND OF THE INVENTION Angiogenesis is understood to be the process by which new blood vessels are created from existing blood vessels. The first step is the production of protease by vascular endothelial cells, migration, proliferation and differentiation of endothelial cells. Is required to digest the basement membrane and interstitial extracellular matrix due to enhanced protease production, which is followed by endothelial cell migration and proliferation. Such angiogenesis is physiologically observed in limited areas such as follicles, corpus luteum, and endometrium,
Pathologically, tumor angiogenesis, diabetic retinopathy, and angiogenesis of arteriosclerotic lesions are well known. In addition, angiogenesis is regulated on the balance of many inducers and suppressors, and the inducer is dominant in pathological conditions such as tumor blood vessels, diabetic retinopathy, and rheumatoid arthritis. It is said.

For example, tumor cells are vascular endothelial growth factor (VEGF),
It is known to produce various angiogenesis-inducing factors such as basic fibroblast growth factor (bFGF), IL-8, thymidine phosphorylase, etc. Among them, a receptor expressed on the surface of vascular endothelial cells VEGF, which binds to and transmits a cell proliferation signal, has received attention. An anti-tumor effect is expected by suppressing VEGF that is deeply involved in tumor angiogenesis, and an anti-VEGF antibody that directly acts on VEGF and specifically inhibits its activity as a substance that suppresses VEGF, There are soluble VEGF receptor, oligo RNA aptamer (RNA-type protease inhibitor), etc., anti-VEGF receptor antibody that acts on VEGF receptor to inhibit VEGF activity, VEGF-derived peptide, tyrosine kinase, etc.
As a method of suppressing F, a method of introducing an antisense VEGF gene into VEGF-producing cells has been attracting attention. In addition, even if VEGF, which is one of the many known angiogenesis-inducing factors, is taken as an example, there are five isoforms in human due to differences in RNA splicing, and PLGF (placental) having a structure similar to VEGF
growth factor), VEGF-B, VEGF-C, VE
GF-D and the like are also known, and VEGF-C, VEGF-
It is said that D is involved in the proliferation of lymphatic endothelium and VEGF-B is involved in the migration of endothelium, and each of them has an isoform like VEGF. As described above, it can be seen that there are a large number of reported so-called angiogenesis-inducing factors and angiogenesis-suppressing factors.

In malignant tumors, new blood vessels are created,
By forming a tumor vascular network, the tumor proliferates significantly and tumor cell metastasis is increased through the tumor blood vessels. Therefore, focusing on the causal relationship between tumor growth and angiogenesis,
Various angiogenesis inhibitors have been reported. As such an angiogenesis inhibitor, in addition to the above VEGF inhibitor, marimasat having a protease activity-inhibiting action, TNP-470 that inhibits the proliferation of vascular endothelial cells, and other mechanisms have not been clarified, but angiogenesis inhibition Although effective thalidomide, endostatin, etc. have been reported, some of these angiogenesis inhibitors have serious side effects and, in general, long-term administration is required. Can not be determined in a short time,
There are some whose effects are doubtful.

Apart from angiogenesis, restructuring of blood vessels, which is understood as an adaptive phenomenon that changes its structure in response to changes in the environment surrounding blood vessels, is also known. Blood vessels sense physiological and physical stimuli in the blood and synthesize and secrete various physiologically active substances to change the tonicity of the blood vessels in the short term and the structure of the blood vessels in the long term. Known to change. For example, for vascular endothelial cells, endothelium-derived relaxing facto (EDRF)
r) is present and its identity is nitric oxide (NO), and that prostacyclin, which has both vasodilatory action and platelet aggregation inhibitory action, is produced in vascular endothelial cells and the natriuretic peptide Family (AN
(P, BNP, CNP) have a vasorelaxant effect, and endothelin made by vascular endothelial cells has a vasoconstrictor effect, etc., and substances with a vasoconstrictor effect are involved in vascular remodeling. It is beginning to be understood that it has.

It has also been known that acetylcholine activates muscarinic receptors, releases EDRF from vascular endothelium, hyperpolarizes the smooth muscle cell membrane potential of blood vessels, and causes a vasorelaxation reaction. And EDRF
If the action of is suppressed by oxyhemoglobin (which binds to and inactivates NO), etc., the vasorelaxation reaction is diminished,
The hyperpolarization reaction of the vascular smooth muscle cell membrane potential is not affected, and the hyperpolarization reaction of the vascular smooth muscle cell membrane due to acetylcholine disappears when the endovascular membrane is removed. The polarization reaction was shown to have a factor different from EDRF released from the vascular endothelium, and was named endothelium-derived hyperpolarizing factor (EDHF). EDRF and EDHF released from vascular endothelium by acetylcholine
Have been shown to relax vascular smooth muscle by different mechanisms, and in particular, the production or release of EDHF in vascular endothelium requires an increase in intracellular Ca ²⁺ concentration, which is necessary. It is considered that there is a release of Ca ^{2+ from} the intracellular storage site and an inflow from the outside of the cell.

[0007] In addition, hyperpolarization reaction of smooth muscle by EDHF
Is the extracellular fluid K⁺The extracellular fluid increases when the concentration decreases.
K⁺If the concentration is increased, it is attenuated, and EDHF
K of vascular smooth muscle cell membrane⁺Increase the conductance of
In fact, hyperpolarization of vascular smooth muscle is ATP sensitive K⁺Channel
Is inhibited by glibengramid, a selective inhibitor of
No, but nonselective K⁺Channel blocker tetrabutyrate
Since it is suppressed by lumonium (TBA), E
DHF is K of vascular smooth muscle cell membrane⁺Activated channels
Therefore, it is considered that the cell membrane potential of vascular smooth muscle is hyperpolarized.
But which K⁺It ’s known to affect the channel.
Not in. Then, by the action of EDHF, the vascular smooth muscle is thinned.
Hyperpolarization of the plasma membrane potential leads to voltage dependence of the vascular smooth muscle cell membrane
Sex Ca²⁺The opening probability of the channel decreases and Ca²⁺Cells of
Inflow into the cell is reduced and intracellular Ca ²⁺Blood with decreasing concentration
It is caused by relaxation of the duct and EDHF agonists.
The endothelium-derived vasorelaxant that has been subjected to
 synthase) and the proteins involved in the synthesis of prostacyclin.
Inhibitor of cyclooxygenase
It won't block, but K⁺Channel blocker
It is known to be inhibited by

Furthermore, in the arteriole of the third branch of the rat mesenteric artery, the vasorelaxation response to acetylcholine is ED.
Although it was only slightly attenuated by some RF inhibitors, when the extracellular fluid K ⁺ concentration was increased, hyperpolarization of vascular smooth muscle was abolished and vasorelaxation was also abolished, whereas rat aorta was abolished. In the common iliac artery, acetylcholine-induced vasorelaxation is almost eliminated by the NO synthase inhibitor. Therefore, EDRF (NO) is more important than EDHF in acetylcholine-mediated vasorelaxation in thick blood vessels, and coronary vasorelaxation It is considered that EDHF is important for the regulation of peripheral vascular resistance and the like.

Using human umbilical vein cells, Ca ²⁺
It has been reported that the proliferation of bFGF-induced human endothelial cells is suppressed by using an activated K ⁺ channel blocker, iberiotoxin (Wiecha J, Munz B, et al.
al.:Blockade of Ca2 + -activated K + channels inhibit
s proliferation of human endothelial cells induced
by basic fibroblast growth factor.J Vasc Res 1998
Sep-Oct; 35 (5) 363-371), but according to this report, the proliferation of human endothelial cells by bFGF was Ca ^{2 +} active K
^Although Iberiotoxin, which is a ⁺ channel blocker, suppresses activation of Ca ²⁺ -activated K ⁺ channels, it suggests that activation of Ca 2 ⁺ -activated K ⁺ channels may be important for angiogenesis and revascularization. There is no mention of whether it can affect contraction. Moreover, there is no mention of whether or not tumor tissue can be suppressed by suppressing it. Also, at the cellular level, K ⁺ channel blockers suppress the growth of tumor cells, but this K ⁺ channel is not different from normal cells,
It has been suggested that the antitumor effect does not occur only by having the property of blocking the K ⁺ channel, and only those having an affinity for the tumor cell membrane cause the antitumor effect (Dubois JM, et al .: Role. of potassium channels in
mitogenesis. Prog Biophys Mol Biol 1993; 59: 1-2
1.). Furthermore, although iberiotoxin is a BK _Ca channel inhibitor, it is believed that it does not substitute for caribdotoxin in terms of EDHF inhibition, and that the K ⁺ channel for EDHF would not be a BK _Ca channel. This suggests that although iberiotoxin is a K ⁺ channel inhibitor, it has little effect on vascular contraction as an EDHF inhibitor (Paul M. Vanhoutte “Endoth.
elium-Dependent Hyperpolarizations ”109-116, 200
0). In addition, 18β-glycyrrhetinic acid, a gap junction inhibitor, was mainly used in EDH among the endothelial cell-dependent relaxation in guinea pig aorta.
It has also been reported to suppress relaxation by F (JJPVo
l. 49, No. 3, 267-274, 1999).

On the other hand, bisphosphonates, which are therapeutic agents for hypercalcemia, are involved in calcium metabolism, and are considered to be the causative agent of elevated blood calcium in cancer, PTHRP (parathyroi).
It is said to be effective against hypercalcemia by suppressing the effect of d hormone-related proteins or peptides). It is also known that this bisphosphonate suppresses metastasis of cancer to bones and internal organs, and suppresses pain caused by cancer. Furthermore, it has been reported (WO00 / 71104) that intravascular administration of bisphosphonate suppresses angiogenesis and causes an anti-cancer effect. However, intravascular administration of bisphosphonate causes angiogenesis. The suppression mechanism has not yet been elucidated.

[0011]

The object of the present invention is to provide an excellent prophylactic / therapeutic agent for angiogenic diseases such as malignant tumor, proliferative diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, psoriasis, and the like. It is intended to provide a diagnostic agent for angiogenic diseases and an angiogenesis inhibitor useful for a drug delivery system to new blood vessels in the prevention / treatment of angiogenic diseases.

[0012]

[Means for Solving the Problems] The lumen of blood vessels is covered with one layer of cells called vascular endothelium, and these cells are not a mere septum according to recent research, and produce various vascular smooth muscle agonist substances. It has become clear that it will be released. One of them is that the lumen of blood vessels is covered with a layer of cells called vascular endothelium, and these cells produce and release various vascular smooth muscle agonists, not just septates, according to recent studies. I understand. EDHF is one of them, and when EDHF acts on vascular smooth muscle cells, the vascular smooth muscle cells hyperpolarize (the inside of the cell is normally kept at -50 mV with respect to extracellular fluid, When EDHF acts, the membrane potential becomes hyperpolarized to about -70 mV), and it has been recognized in recent years that EDHF thus named is physiologically and pathophysiologically important. It is said that there are many unclear points about the target molecule.

[0013] It is not easy to determine the diagnosis of cancer clinically, and until the 1980s before the spread of CT, determination of tumor blood vessels by angiography was used for the diagnosis of cancer. However, in the case of a tissue such as a kidney, which has a large blood flow, the contrast enhancement effect of the normal tissue overlaps the tumor and the tumor cannot be visualized, so that it is necessary to reduce the blood flow of the normal tissue. When noradrenaline or adrenaline, which contracts blood vessels in normal tissues, is administered first, and then a contrast test is performed, normal blood vessels contract, but tumor blood vessels do not contract, and contrast is often observed in a relatively embossed manner. Known to some specialists, it was called pharmacological angiography. However, due to the spread of CT, tumor blood vessels can be visualized without performing pharmacological angiography.
It was a technology that was rarely used in the 0's. It was also known that there is a difference in blood flow change between tumor blood vessels and normal blood vessels when blood pressure changes occur. However, it was believed that tumor blood vessels lacked a system to control their blood flow. On the other hand, as described above, it has been recently reported that administration of bisphosphonate from an artery has an effect of embolizing a tumor and an effect of suppressing angiogenesis. Based on these findings, the present inventors suspect that the angiogenesis-suppressing action of bisphosphonate may be caused not by the mere embolization effect of arteries but by contracting new blood vessels, which are tumor blood vessels of cancer. Thought. The reason is that PTHRP, a causative agent of hypercalcemia that bisphosphonate inhibits, has a vasodilatory effect, and bisphosphonate may inhibit not only hypercalcemia but also vasodilatory effect. Because there is a nature.

Bisphosphonate is a therapeutic drug for hypercalcemia and is known to be particularly effective for cancer hypercalcemia. PTH discovered as a peptide derived from cancer as a causative agent of hypercalcemia of this cancer
RP is known. Bisphosphonate can suppress hypercalcemia by inhibiting the action of this peptide PTHRP. Other than that, it is known that bisphosphonate can suppress the metastasis of cancer to the internal organs and can treat bone metastasis, but its mechanism of action is not known.
PTHRP is a peptide related to parathyroid hormone and is known to have a vasodilatory action in addition to causing an increase in blood calcium. This vasodilatory action has a vasodilatory action that is not due to NO, and is inhibited by tetrabutylammonium chloride, and is therefore attributed to the action of EDHF.

The above-mentioned bisphosphonate, PTHRP,
And from the relationship of EDHF, bisphosphonate
We suspected that it might be blocking not only the hypercalcemia of THRP but also the action of EDHF. If bisphosphonates are only involved in calcium metabolism, the reason for inhibiting visceral metastases of cancer is unclear. Also, if bisphosphonate only causes embolism, embolism due to bisphosphonate administered intravenously or orally will occur in the lungs, liver, etc.,
Effectiveness is poor unless it is injected from the artery,
Embolization of the lungs and liver becomes more problematic, and it is difficult to explain the cancer visceral metastasis-suppressing effect of intravenous injection or oral administration.
If it has a vasoconstrictor effect, it is effective even when passing through veins, lungs, and heart. Moreover, EDRF (NO
It is said that EDHF has a strong effect on peripheral small blood vessels.
Is similar to the relationship between normal blood vessels that contract with noradrenaline and tumor vessels that do not.
The former relationship is a vasodilatory effect and the latter relationship is a contractile effect. However, due to the similarity of their distribution, inhibiting EDHF results in a strong contraction on the neovascular side of the tumor, and the flow of new blood vessels is increased. I thought it might be hindered.

EDHF relaxes blood vessels by a mechanism different from EDRF. EDHF activates the K ⁺ channel of vascular smooth muscle cell membrane from vascular endothelial cell membrane, resulting in voltage-dependent Ca of vascular smooth muscle cell membrane. open probability of ²⁺ channel is reduced, reducing the influx into the cells Ca ^2+, vessels are said to be caused to relax by reducing the intracellular Ca ²⁺ concentration. This is consistent with the involvement of PTHRP in calcium metabolism and EDHF action. Then, the following hypothesis was devised based on the report that bisphosphonate as an inhibitor thereof has not only inhibition of hypercalcemia but also neovascular inhibition and anticancer action by embolization effect of blood vessels. The action of endothelium-dependent hyperpolarization reaction of EDHF by paraclines such as PTHRP produced by cancer has the action of retaining and strengthening the relaxation expansion of new blood vessels, and the action of bisphosphonate which inhibits it is a mere embolic effect. Instead, I suspected that the tumor might have decreased blood flow due to the strong contraction on the new blood vessel side. In other words, it has a vascular endothelium-dependent hyperpolarization effect,
A substance that inhibits the action of EDHF that relaxes tumor new blood vessels that are microvessels, that is, an EDHF inhibitor, contracts tumor new blood vessels and blood vessels that are continuous with tumor blood vessels, causes tumor infarction, and has an excellent anticancer effect. I thought I might have it.

Therefore, EDHF was added to the tumor-transplanted mice.
It was confirmed that the EDHF inhibitor exerts an excellent anti-cancer effect by administering tetrabutylammonium chloride, which is an inhibitor of E. coli, and examining the tumor growth inhibitory effect. This tumor growth inhibitory effect was considered to be delay of progression of lesions, reduction, necrosis of lesions due to interruption of nutrient supply to tumor tissues by anti-neovascular action, and healing as a result thereof, and the present invention was completed.

That is, according to the present invention, an angiogenesis inhibitor (claim 1) comprising a substance which inhibits the action of endothelium-derived hyperpolarizing factor (EDHF) and suppresses angiogenesis as an active ingredient, and The angiogenesis inhibitor according to claim 1 (claim 2), which has a vasoconstricting action and suppresses angiogenesis as an active ingredient, and the angiogenesis inhibitor is a K ⁺ channel. A blocker, wherein the angiogenesis inhibitor according to claim 1 or 2 (claim 3) or K ⁺ channel blocker is tetrabutylammonium salt, tetraethylammonium salt, caribdotoxin, apamin, oleyloxyethylphosphorylcholine. Or an agent for inhibiting angiogenesis (claim 4) according to claim 3, which is one or more substances selected from ketoconazole. , Substances that suppress angiogenesis
3. An angiogenesis inhibitor (claim 5) according to claim 1 or 2, which is a K ⁺ free substance, or a substance which suppresses angiogenesis, is a gap junction inhibitor. The angiogenesis inhibitor according to 1 or 2 (claim 6) or the gap junction inhibitor is heptanol, Gap26, Gap27 or 18-β glycyl retinoic acid, wherein the angiogenesis inhibitor according to claim 6 ( Claim 7) or the substance which suppresses angiogenesis is an epoxyeicosatrienoic acid inhibitor, wherein the angiogenesis inhibitor according to claim 1 or 2 (claim 8).
9. Angiogenesis according to any one of claims 1 to 8, which specifically acts on new blood vessels such as malignant tumor, proliferative diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, psoriasis and the like. It relates to an inhibitor (claim 9).

The present invention also provides a prophylactic / therapeutic agent for angiogenic diseases (claim 10), which comprises the angiogenesis inhibitor according to any one of claims 1 to 9, and an angiogenic disease,
The preventive / therapeutic agent for an angiogenic disease according to claim 10, which is malignant tumor, proliferative diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis or psoriasis (claim 11).
Or the angiogenesis inhibitor according to any one of claims 1 to 9,
A diagnostic agent for an angiogenic disease characterized by containing a marker substance (claim 12);
The proliferative diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis or psoriasis, and the diagnostic agent for an angiogenic disease (claim 13) according to claim 12, or any one of claims 1 to 9. Or the described angiogenesis inhibitor and prevention of angiogenic diseases
The present invention relates to a drug delivery system for new blood vessels in the prevention / treatment of angiogenic diseases, which comprises a therapeutic drug (claim 14).

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION As an angiogenesis inhibitor of the present invention, the action of endothelium-derived hyperpolarizing factor (EDHF), that is, endothelium-dependent hyperpolarizing reaction (Endthelium dependent hyp
It is not particularly limited as long as it has a substance that inhibits erpolarization) and suppresses angiogenesis as an active ingredient, but a substance having a microvasoconstrictor action is preferable. Here, the endothelium-dependent hyperpolarization reaction is , A hyperpolarization reaction that does not occur in vascular smooth muscle without endothelium but occurs in vascular smooth muscle with endothelium. Such an angiogenesis inhibitor of the present invention,
And K ⁺ channel blocker that blocks the K ⁺ channel in endothelial cells and the like, and K ⁺ free substance which can increase the K ⁺ concentration in the local cell and at the site of contact of the cells, by current or molecular weight of 1000 or less of about electrolyte ions Gap junction inhibitors that suppress and inhibit gap junctions (gap junctions), which is a type of cell control mechanism that directly transfers and exchanges substances through the pores of channels and is also called cell-cell communication from a functional perspective, and cytochrome p450 epoxygenase Product, EDHF
An epoxy eicosatotrienoic acid inhibitor that suppresses / inhibits the action of epoxy eicosatrienoic acid, which is said to have a vasodilatory effect similar to, can be mentioned. In applying these, for example, K ⁺ channel blocker and K ⁺ A free substance or the like can be used in combination.

As the above K ⁺ channel blocker,
Tetrabutyl-ammonium
Salt, tetraethyl-ammoniu
m) salt, caribdotoxin, apamin, oleyloxyethyl phosphorylcholine, ketoconazole, 4-aminopyr
idine), dendrotoxins, phencyclidine, phalloidin
din), 9-aminoacridine, margatoxin, imperator toxin, LY97241, Gaboon viper venom, (+)-tubocurarine, noxiustoxin (Noxiustoxi
n), Penitrem-A, Penetrem-Cet
iedil), Trifluoroperazin
e), Haloperidol, Leiurotoxin 1 and Glibenclamid
mide), tolbutamide (Tolbutamide), phentolamine (Phentolamine), cyclazindol (Ciclaz
indol), lidocaine (Lidocaine), quinidine (Quinid)
ine), mast cell degranulating peptide (M
ast cell degranulating peptide), aditoxin 2
(Agitoxin-2), Hanatoxin, Tytyus toxin K-alpha, Licorice (Lico)
rice) or Glycyrrhizic aci
d), Carbenoxolone (Glycyrrhet)
inic acid), toluene (Toluene), ethanol (Etha
nol), Cottonseed oil with low dietary
K ⁺ ), Sr ⁺⁺ , Ba ⁺⁺ , Cs ⁺⁺ , Mg ^{++ and the} like can be mentioned, and one or more selected from these can be used.

The above-mentioned tetrabutylammonium salt is one in which a hydrogen atom of an ammonium ion is replaced with a butyl group, and the tetraethylammonium salt is one in which a hydrogen atom of an ammonium ion is replaced with an ethyl group. Examples of the salts include halogens such as fluorine, chlorine, bromine and iodine, and pharmaceutically acceptable salts such as phosphorus oxides and carbonates. The above-mentioned caribdotoxin is a scorpion (Reylus quinketratos) toxin having a molecular weight of 4300 and has an effect of blocking the K ⁺ channel of the cell membrane. Caribdotoxins act on and inhibit K ⁺ channels that open when intracellular Ca ²⁺ increases. Apamin, a honey bee toxin that has an action similar to that of caribdotoxin, is a basic peptide composed of 18 amino acids and having a molecular weight of about 2000. Apamin is a central nervous system excitatory action and excitement and desuppression in various cells. It has an action, and it is said that the action is due to the selective inhibition of the calcium-dependent potassium channel of the cell membrane activated by ATP, which is an enzyme that acts as a catalyst for the reaction of D-glucose. The combined use of caribdotoxin and apamin is suitable as a K ⁺ channel blocker.

The above-mentioned oleyloxyethylphosphorylcholine is a compound having a substituent in which oleyl, which is a monovalent acid group derived from oleic acid, and oxyethylene are bonded, and is triethylamine dissolved in an ether solvent or the like under a nitrogen stream. It can be obtained by dropwise addition of a halogenated oleyloxyethylphosphorane solution such as phosphorane chloride.
The above ketoconazole has an action of inhibiting cortisol synthesis, has low toxicity, and has 1-acetyl-4- [4- [2- (2,4-dichlorophenyl) -2.
-Imidazol-1-ylmethyl-1,3-dioxolan-4-ylmethoxy] phenyl] piperazine, including the free base form of ketoconazole, or a pharmaceutically acceptable salt thereof, its stereochemical isomers and its compatible isomers. You may stay. Preferred ketoconazole compounds are the free base (±)-(cis) form. Cotoconazole can be produced by a known method.

[0024] As the K ⁺ release material may be any one so long as it is a substance which can increase the K ⁺ concentration in the local releases K ^+, be mentioned potassium chloride and other potassium salts specifically By injecting these K ⁺ free substances directly from an artery, it is possible to suppress the action of EDHF in angiogenesis and suppress new blood vessels. Further, as a gap junction inhibitor that suppresses / inhibits gap junction (gap junction), connexin (connexin), which is a generic term for protein subunits that constitute the gap junction protein particle connexon, particularly connexins 37, 40, 43, etc. Heptanol, Gap2, which is an inhibitor / inhibitor
6 (connexin mimetic peptide; eg ^37,40 Gap2
6, ⁴³ Gap26 etc.), Gap27 (connexin mimetic
peptide; eg ^37,43 Gap27, ⁴⁰ Gap27
Etc.), 18β-glycyrrhetinic (18β-glycyrrhetinic
acid) and the like. And again EDHF
Examples of the epoxyeicosatrienoic acid inhibitor having a vasodilatory action similar to that of cytochrome p450 epoxygenase include proadiphane, miconazole, and 17-octadecino. Icic acid (17-octadecynoic acid) etc. can be mentioned.

Further, as the angiogenesis inhibitor of the present invention, those which specifically act on new blood vessels such as malignant tumor, proliferative diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, psoriasis and the like are preferable.

The prophylactic / therapeutic agent for angiogenic diseases of the present invention may be any as long as it contains the angiogenesis inhibitor of the present invention.
Malignant tumors, proliferative diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, psoriasis and the like can be preferably mentioned.
Among them, the preventive / therapeutic agent for angiogenic diseases of the present invention,
It is effective because it acts effectively on malignant tumors and can suppress the growth and metastasis of tumor cells. In addition, the angiogenesis inhibitor of the present invention does not cause a significant increase or decrease in the number of red blood cells, the number of white blood cells or the weight of hemoglobin, does not cause immunosuppression, has little side effect, and is effective in treating or preventing angiogenic diseases. Excellent ones are preferred. The prophylactic / therapeutic agent for angiogenic diseases of the present invention containing the angiogenesis inhibitor of the present invention suppresses angiogenesis by contracting blood vessels formed by the angiogenic disease or by suppressing angiogenesis in the angiogenic disease. By doing so, expansion of the lesion can be prevented.

The above-mentioned prophylactic and therapeutic agents containing the angiogenesis inhibitor of the present invention can be administered orally or parenterally, and commonly used dosage forms such as powder, granules, capsules, syrups, It can be administered orally in the form of a suspension or the like, or in the form of a solution, emulsion, suspension or the like, it can be parenterally administered to a vein or artery in the form of an injection. Alternatively, it may be administered intranasally in the form of a spray. In addition, the above-mentioned prophylactic and therapeutic agents include pharmaceutically acceptable conventional carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrating agents, solubilizing agents, solubilizing agents, isotonic agents. Various formulation ingredients such as agents can be appropriately added. The amount of the prophylactic / therapeutic drug used is not particularly limited and can be appropriately adjusted and used depending on the type of disease, the site of the lesion, the progress of the medical condition, and the physical strength of the patient.

The diagnostic agent for angiogenic diseases of the present invention may be any agent as long as it contains the angiogenesis inhibitor of the present invention and a marker substance and is capable of detecting the site of neovascularization. , Those in which an angiogenesis inhibitor and a marker substance are bound, or those in which a part of the angiogenesis inhibitor is replaced with a marker substance such as a radioisotope, can be advantageously used as a diagnostic agent for angiogenic diseases. You can The angiogenic diseases to be diagnosed include malignant tumors, proliferative diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis,
Psoriasis and the like can be preferably mentioned. Moreover, the marker substance is not particularly limited as long as it is a conventionally known marker substance for diagnosis, and specific examples thereof include a fluorescent substance, a radioisotope, and a contrast agent. In particular, when the angiogenesis inhibitor is a protein or peptide, a marker protein or peptide tag can be preferably used as a marker substance. The marker protein is not particularly limited as long as it is a conventionally known marker protein, and specific examples thereof include alkaline phosphatase, Fc region of antibody, HRP and GFP, and a peptide tag. As H
A tag, Myc tag, His tag, FLAG tag, GS
Specific examples of conventionally known peptide tags such as T tags and Xpess tags can be given. When the diagnostic agent for angiogenic diseases of the present invention is used, the angiogenesis inhibitor of the present invention causes blood vessels to contract and occlude new blood vessels, resulting in slow or stagnant blood flow at that site. Can be detected with a marker substance.

As a drug delivery system to new blood vessels in the prevention / treatment of angiogenic diseases of the present invention, a system using the angiogenesis inhibitor of the present invention, an angiogenesis inhibitor of the present invention and prevention of angiogenic diseases There is no particular limitation as long as it is a system containing a therapeutic agent, and by utilizing the drug delivery system for neovascularization of the present invention, the angiogenesis inhibitor of the present invention causes the blood vessels to contract and form new blood vessels. As a result of the occlusion, a drug such as a carcinostatic agent can be retained at the occluded site, and the same effect as that obtained by locally administering the drug such as a carcinostatic agent to the lesion site can be produced.

[0030]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples. Example 1: Anti-neovascular effect by tetrabutylammonium chloride The anti-neovascular effect was investigated using tetrabutylammonium chloride (TBA) which is an EDHF inhibitor. First,
The applied dose of TBA was examined in normal DDY mice (male, 9 weeks old). 2 mM TBA from mouse tail vein
When 0.5 ml was administered, it resulted in cardiac arrest, so 2
When 0.3 ml of mM TBA was administered and administration was performed, the movement of mice became poor. Next, when 0.2 ml of 2 mM TBA was administered, there was no particular problem in mice,
The following test was conducted by administering 0.2 ml of M TBA.

It was investigated how tetrabutylammonium chloride acts on tumors. Tumor is S-1
A sarcoma of 80 was grown in the ascites of DDY mice, and the ascites was prepared and used. 20 μl of the tumor cells
(4-5 × 10 ⁶ cells) were injected intramuscularly in the left thigh of male 9-week-old DDY mice. After 3 days, the tumor was palpated in the thigh, and 24 tumors confirmed to be transplanted were randomly divided into 3 groups (A, B, C). For group A, no intravenous injection was used as a control, and for group B, TBA (2 mM,
0.2 ml) was administered once through the tail vein, and once every two days for group C, TBA (2 mM, 0.2 ml) was administered through the tail vein a total of 5 times. After 9 days from the start of administration, the drug was killed under ether anesthesia, the skin was peeled off, both thighs were cut at the hip joint, the weights of both thighs were measured, and the weight of the thigh on the tumor side to the thigh on the normal side was measured. The value obtained by subtracting the weight was taken as the tumor weight, and the weight was tabulated and tested. The results are shown in Table 1 and FIG. In Fig. 1, the tumor weights obtained as the difference between the weight of the tumor leg and the weight of the normal leg in each of the groups A, B, and C are shown in the filled range, and the average value is represented by a straight line in the filled range. Indicated.

[0032]

[Table 1]

In t-test, p = 0.03 between groups A and C
4, p = 0.509 between groups A and B, p between groups B and C
Was 0.167. From this, it was confirmed that there were significant differences among the A, B, and C groups. Therefore, T
The antitumor effect of BA was confirmed, and it was shown to have an anti-neovascular effect.

Example 2: Test on the effect of administration of tetrabutylammonium chloride on blood cell count For 9-week-old DDY mice to which tetrabutylammonium chloride was administered in the above-mentioned Example 1, red blood cell count, white blood cell count, and hemoglobin weight were automatically measured for blood cells. It measured with the counter. The results are shown in Table 2. In the table, the number of red blood cells is RBC (unit: 10,000 cells / mm ³ ) and the number of white blood cells is WBC (unit: cells / m ³ ).
m ³ ) and the hemoglobin weight are shown in Hg (unit: g / dl). As is clear from Table 2, in the group C to which tetrabutylammonium chloride was administered multiple times, the number of white blood cells was slightly low, but it was not enough to cause immunosuppression. The effect on blood cells was small.

[0035]

[Table 2]

[0036]

EFFECTS OF THE INVENTION By using the angiogenesis inhibitor of the present invention capable of suppressing neovascularization, malignant tumors that are angiogenic diseases, proliferative static diabetic retinopathy, age-related macular degeneration,
It enables treatment and diagnosis of rheumatoid arthritis and psoriasis.

[Brief description of drawings]

FIG. 1 is a diagram showing the tumor weight reduction state of DDY mice administered with the angiogenesis inhibitor of the present invention.

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Claims (14)

[Claims] 1. An angiogenesis inhibitor comprising a substance that inhibits the action of endothelium-derived hyperpolarizing factor (EDHF) and suppresses angiogenesis as an active ingredient. 2. A substance having a microvascular contracting action and suppressing angiogenesis as an active ingredient.
The angiogenesis inhibitor as described. 3. The substance which suppresses angiogenesis is a K ⁺ channel blocker, wherein the substance is K ⁺ channel blocker.
The angiogenesis inhibitor as described. 4. The K ⁺ channel blocker is one or more substances selected from tetrabutylammonium salt, tetraethylammonium salt, caribdotoxin, apamin, oleyloxyethylphosphorylcholine or ketoconazole. The angiogenesis inhibitor according to claim 3. 5. The angiogenesis inhibitor according to claim 1 or 2, wherein the substance that suppresses angiogenesis is a K ⁺ free substance. 6. The angiogenesis inhibitor according to claim 1, wherein the substance that suppresses angiogenesis is a gap junction inhibitor. 7. The angiogenesis inhibitor according to claim 6, wherein the gap junction inhibitor is heptanol, Gap26, Gap27 or 18-β glycyl retinoic acid. 8. The angiogenesis inhibitor according to claim 1, wherein the substance that suppresses angiogenesis is an epoxyeicosatrienoic acid inhibitor. 9. The method according to any one of claims 1 to 8, which specifically acts on new blood vessels such as malignant tumor, proliferative diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, psoriasis and the like. Angiogenesis inhibitor. 10. A prophylactic / therapeutic agent for an angiogenic disease, which comprises the angiogenesis inhibitor according to any one of claims 1 to 9. 11. The prevention / treatment of angiogenic disease according to claim 10, wherein the angiogenic disease is malignant tumor, proliferative diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis or psoriasis. medicine. 12. A diagnostic agent for angiogenic diseases, which comprises the angiogenesis inhibitor according to any one of claims 1 to 9 and a marker substance. 13. The diagnostic agent for angiogenic disease according to claim 12, wherein the angiogenic disease is malignant tumor, proliferative diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis or psoriasis. 14. A new blood vessel in the prevention / treatment of an angiogenic disease, which comprises the angiogenesis inhibitor according to any one of claims 1 to 9 and a preventive / therapeutic agent for an angiogenic disease. Drug delivery system.