New vascular disorder treatment

Abstract

Provided is a medical agent for effectively treating a vascular disorder caused by the excessive synthesis and secretion of type IV collagen protein from vascular endothelial cells.　Provided are [1] a therapeutic agent for a vascular disorder associated with the excessive synthesis and secretion of type IV collagen protein, [2] a therapeutic agent for diseases caused by the excessive synthesis and secretion of type IV collagen protein, [3] a pharmaceutical composition for treating a vascular disorder associated with the excessive synthesis and secretion of type IV collagen protein, [4] a pharmaceutical composition for treating diseases caused by the excessive synthesis and secretion of type IV collagen protein from vascular endothelial cells, and [5] an agent for inhibiting the excessive synthesis and secretion of type IV collagen protein from vascular endothelial cells, each of which contains sirolimus as an active ingredient.

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for vascular disorders containing sirolimus as an active ingredient. [Background technology]

[0002] Vascular disorders can cause a variety of serious symptoms. Peripheral vascular disorders, in particular, can cause Raynaud's phenomenon and necrosis, significantly reducing the patient's quality of life. In cases where severe symptoms occur in various tissues, not just peripheral ones, they can even lead to death. Therefore, treatment of vascular disorders that affect the entire body is extremely important.

[0003] Traditionally, many peripheral vascular disorders or blood flow disorders, including Raynaud's phenomenon, have been thought to be caused by the abnormal accumulation of extracellular matrix, resulting in capillary loss and functional decline due to hypoxia. There are various hypotheses as to the causes of this, but the direct cause remains unknown. It is believed that some reason, such as environmental factors, external stimuli, or internal immune abnormalities, triggers abnormalities in vascular endothelial cells and vascular smooth muscle cells, and that the accumulation of collagen protein, among the excess extracellular matrix, is one of the causes of vascular wall thickening (e.g., Non-Patent Document 1).

[0004] To date, vascular disorders have been treated with symptomatic therapy, including the administration of drugs with vasodilatory effects, but no treatments that fundamentally remove the cause of the disease (radical treatments) have been performed. At the very least, no drugs that directly act on the accumulation of extracellular matrix in blood vessels have been found.

[0005] Peripheral vascular disorders, including Raynaud's phenomenon, can occur in association with scleroderma. The Scleroderma Treatment Guidelines (Japanese Dermatological Association Systemic Scleroderma Clinical Practice Guidelines) state that digitalis preparations, including digoxin, are the basic treatment for pulmonary hypertension associated with scleroderma. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Armando Gabrielli,et al.,N Engl J Med. 2009;360(19):1989-2003 Summary of the Invention [Problem to be solved by the invention]

[0007] In the treatment of vascular disorders, it is desirable to perform treatment that fundamentally removes the cause of the disease, but to date, no drugs capable of such treatment have been disclosed. Furthermore, no drugs have been disclosed for peripheral vascular disorders that occur in association with scleroderma. In light of these circumstances, the present invention aims to provide a drug that effectively treats peripheral vascular disorders caused in part by abnormal accumulation of extracellular matrix, particularly excessive synthesis and secretion of type IV collagen protein. [Means for solving the problem]

[0008] As a result of investigations, the present inventors have found for the first time that type IV collagen gene is mainly highly expressed in vascular endothelial cells derived from iPS cells of patients with vascular disorders. Type IV collagen protein is abundantly present mainly in the basement membrane adjacent to vascular endothelial cells and is synthesized and secreted by vascular endothelial cells. They have also found that the synthesis and secretion of type IV collagen protein in vascular endothelial cells can be effectively inhibited by applying sirolimus to vascular endothelial cells, thereby completing the present invention.

[0009] That is, the present invention includes the following [1] to [5]. [1] A therapeutic agent containing sirolimus as an active ingredient for vascular disorders associated with excessive synthesis and secretion of type IV collagen protein. [2] A therapeutic agent containing sirolimus as an active ingredient for diseases caused by excessive synthesis and secretion of type IV collagen protein from vascular endothelial cells. [3] A pharmaceutical composition for treating vascular disorders associated with excessive synthesis and secretion of type IV collagen protein, comprising sirolimus as an active ingredient and a pharmaceutically acceptable carrier. [4] A pharmaceutical composition for treating a disease caused by excessive synthesis and secretion of type IV collagen protein from vascular endothelial cells, comprising sirolimus as an active ingredient and a pharmaceutically acceptable carrier. [5] An agent containing sirolimus as an active ingredient that inhibits excessive synthesis and secretion of type IV collagen protein from vascular endothelial cells. [Effects of the Invention]

[0010] According to the present invention, it is possible to inhibit the synthesis and secretion of type IV collagen protein in vascular endothelial cells, thereby effectively treating vascular disorders such as peripheral vascular disorders that occur due to excessive accumulation of extracellular matrix containing collagen. [Brief explanation of the drawings]

[0011] [Figure 1] This figure shows the results of confirming the expression status of type IV collagen genes in vascular endothelial cells derived from healthy adults and scleroderma patients (one case, two cell lines). COL4A1 represents the type IV collagen α1 gene, COL4A2 represents the type IV collagen α2 gene, COL4A5 represents the type IV collagen α5 gene, and COL4A6 represents the type IV collagen α6 gene. [Figure 2] FIG. 1 shows the difference in type IV collagen protein amount depending on the presence or absence of sirolimus in the medium. [Figure 3] FIG. 1 shows the difference in type IV collagen protein amount depending on the presence or absence of digoxin in the medium. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention relates to a method for treating a rheumatoid arthritis, comprising administering sirolimus as an active ingredient to a patient. [1] A therapeutic agent for vascular disorders associated with excessive synthesis and secretion of type IV collagen protein. [2] A therapeutic agent for diseases caused by excessive synthesis and secretion of type IV collagen protein. [3] A pharmaceutical composition for treating vascular disorders associated with excessive synthesis and secretion of type IV collagen protein. [4] A pharmaceutical composition for treating diseases caused by excessive synthesis and secretion of type IV collagen protein from vascular endothelial cells, and [5] Inhibitor of excessive synthesis and secretion of type IV collagen protein from vascular endothelial cells Regarding.

[0013] Sirolimus (also known as rapamycin) is a metabolite of the actinomycete Streptomyces hygroscopicus isolated from soil on Easter Island, and was discovered as a macrolide antibiotic in the 1970s. Sirolimus was subsequently found to have immunosuppressive effects, and is approved in 55 countries worldwide as a drug to prevent rejection after kidney transplants.

[0014] The vascular disorders that are the target of treatment by the therapeutic agent for vascular disorders associated with excessive synthesis and secretion of type IV collagen protein according to the present invention are typically peripheral vascular disorders, but are not limited thereto. For example, vascular disorders caused by the overproduction of extracellular matrix containing collagen, such as those caused by scleroderma, can be treated by the therapeutic agent according to the present invention. Specific examples include Raynaud's syndrome, which is a typical symptom of scleroderma, and the associated pulmonary hypertension, interstitial lung disease, digestive fibrosis, renal fibrosis, cardiac fibrosis, and other vascular disorders.

[0015] In the therapeutic agent according to the present invention for a disease caused by excessive synthesis and secretion of type IV collagen protein from vascular endothelial cells, which comprises sirolimus as an active ingredient, the disease is selected from the following (1) to (3): (1) Peripheral vascular disease caused by scleroderma (2) Raynaud's syndrome (3) The following diseases associated with Raynaud's syndrome: pulmonary hypertension, interstitial lung disease, fibrosis of the digestive system, fibrosis of the kidneys, fibrosis of the heart, or other vascular lesions

[0016] The pharmaceutical composition of the present invention contains sirolimus as an active ingredient and a pharmaceutically acceptable carrier, and can be used in the form of an oral preparation or an external preparation that is applied directly to the affected area. In particular, it is preferable to use it in the form of an oral preparation for patients who have developed vascular disorders due to systemic diseases. Such pharmaceutical compositions can be produced by conventionally known methods.

[0017] The oral preparation of the present invention can be, for example, a tablet, an orally disintegrating tablet, a capsule, or a granule. The oral preparation contains pharmaceutically acceptable carriers such as commonly used excipients, additives, and / or vehicles, to the extent that the effects of the active ingredient, sirolimus, are not impaired. Examples of such pharmaceutically acceptable carriers that can be used include carriers selected from binders, disintegrants, lubricants, plasticizers, surfactants, stabilizers, antioxidants, coating agents, sweeteners, colorants, and flavoring agents.

[0018] Specific examples of carriers that can be used include binders such as anhydrous lactose, lactose hydrate, crystalline cellulose, mannitol, macrogol 400, polyethylene glycol, polyoxyethylene (160) polyoxypropylene (30) glycol, glycerin monooleate, and calcium sulfate; binders such as dextran, low-substituted hydroxypropyl cellulose, corn starch, partially pregelatinized starch, carmellose sodium, and hypromellose; disintegrants such as sodium carboxymethyl starch; and lubricants such as magnesium stearate, talc, sodium stearyl fumarate, stearic acid, and light anhydrous silica. Examples of suitable coating agents include eudragit, carnauba wax, hypromellose, and derivatives thereof. Examples of suitable coating agents include ethanol, methyl methacrylate, methyl acrylate, methyl methacrylate, methyl acrylate copolymer, methyl meth ...

[0019] The topical preparation of the present invention can be in the form of a commonly used ointment, cream, lotion, gel, etc. Such topical preparations can be prepared by dissolving or dispersing the active ingredient sirolimus in a suitable solvent, followed by blending with pharmaceutically acceptable carriers such as commonly used excipients and additives, including gelling agents, thickeners, pH adjusters, and stabilizers.

[0020] Usage / Dosage The oral preparation, which is one embodiment of the therapeutic agent according to the present invention, can be administered in the same manner as a normal oral preparation, for example, by taking it with water. The dosage is determined appropriately depending on the type of target disease, the severity of the disease, and the condition of the patient to be treated. Usually, 1 to 4 mg of sirolimus is orally administered to an adult per day.

[0021] The topical preparation, which is one embodiment of the therapeutic agent according to the present invention, can be used in the same manner as a typical topical preparation used for skin diseases, such as by directly applying it to the affected area. The dose is determined appropriately depending on the type of target disease, the severity of the disease, and the condition of the subject (patient). Typically, an amount equivalent to 0.1 to 2 mg of sirolimus per day for an adult can be directly applied to the skin around the affected area. [Example]

[0022] The present invention will be described below with reference to specific embodiments, but it will be understood that the present invention is not limited to these embodiments, and that various changes and modifications therein can be made by those skilled in the art without departing from the scope or spirit of the present invention as defined in the appended claims.

[0023] (Example) (1) Cultivation of vascular endothelial cells derived from patient iPS cells The cells used were iPS cells derived from healthy adults and scleroderma patients established at Kumamoto University (Zhongzhi Wang, et al., JDS 2016 Nov;84(2):186-196.) Because scleroderma is known to cause Raynaud's syndrome, a type of peripheral vascular disorder, these cells were used as a vascular disorder model.

[0024] iPS cells derived from healthy adults and scleroderma patients were cultured under the conditions listed in Table 1 to induce differentiation into vascular endothelial cells.

[0025] [Table 1]

[0026] The obtained cells were sorted using flow cytometry to identify only cells positive for VE-cadherin (CD144) and PECAM-1 (CD31), which are vascular endothelial cell markers. The morphology and expression of the marker genes confirmed that the sorted cells were vascular endothelial cells.

[0027] The isolated vascular endothelial cells derived from iPS cells from healthy adults and scleroderma patients were cultured in Humedia EB-2 + Growth Factor medium (hereinafter referred to as serum-containing medium) containing 10% FBS as shown in the bottom row of Table 2, and expanded for three passages. The cultured cells were expanded to approximately 1 × 10 5 Each well was seeded onto a 6-well plate (coated with type I collagen) and cultured for one day in an incubator at 37°C and 5% CO2.

[0028] [Table 2]

[0029] After confirming that the cells had settled to the bottom of the plate, the supernatant medium was removed and the plate medium was washed with PBS. The entire plate was divided into two. For the cells used for RNA extraction, serum-containing medium was used, containing 0.25% DMSO as a solvent control, or test substance 1, containing 1 μmol / L sirolimus and 0.25% DMSO. For the cells used for protein extraction from the cell supernatant, Humedia EB-2 + Growth Factor medium (see the top row of Table 2, but without FBS) (hereafter referred to as serum-free medium) was used, and medium was prepared under the same conditions as above. The prepared medium was added to the plate containing the cells and cultured for 3 days in an incubator at 37°C and 5% CO2.

[0030] (2) Confirmation of type IV collagen gene expression in vascular endothelial cells Among the cultured cells obtained in (1) above, cells cultured in serum-containing medium were used to confirm the expression level of type IV collagen gene in vascular endothelial cells derived from iPS cells from healthy adults and scleroderma patients using the following procedure.

[0031] After culturing for three days, the supernatant (medium) was removed from the plate, and the inside of the plate was washed with PBS. An RNA extraction reagent (product name: Sepasol®-RNA I Super G, manufactured by Nacalai Tesque) was added to recover the cells, and the cell suspension was then transferred to a container. Chloroform was added, the mixture was stirred, and the mixture was centrifuged at 4°C and 12,000 rpm for 15 minutes. The separated chloroform layer was then placed in a separate container, and RNA was purified using ethanol precipitation. Reverse transcriptase (product name: SuperScript TM III Reverse Transcriptase, Invitrogen TM cDNA was synthesized using a fluorescent reagent (product name: THUNDERBIRD® SYBR® qPCR Mix, manufactured by Toyobo Co., Ltd.), and the expression level of the type IV collagen gene was confirmed by qPCR.

[0032] The results are shown in Figure 1. Compared to vascular endothelial cells from healthy adults, it was confirmed that type IV collagen genes (particularly type IV collagen α5 and α6 genes) were expressed at higher levels in vascular endothelial cells derived from scleroderma patients. As mentioned above, scleroderma patients are known to develop Raynaud's syndrome, a peripheral vascular disorder, as one of its typical symptoms. Therefore, these results strongly suggest that high expression of type IV collagen genes is involved in vascular wall thickening.

[0033] (3) Confirmation of the effect of sirolimus on the synthesis and secretion of type IV collagen protein in vascular endothelial cells In the above (1), the amount of type IV collagen protein released into the medium was quantified using the supernatant of the cells cultured in serum-free medium by the following method. First, the proteins released into the medium were concentrated. This procedure requires serum-free cultivation to avoid the effects of proteins contained in serum. After culturing cells in serum-free medium for three days, the cell supernatant was collected. Four volumes of chilled acetone (4 times the volume of the collected medium) were added and the cells were left to stand overnight at -20°C. The next day, the cells were centrifuged at 1000xG for 10 minutes at 4°C. The supernatant was removed, and the precipitate was mixed with 75% ethanol and centrifuged again under the same conditions. After removing the ethanol, the precipitate was dried and dissolved in RIPA buffer. Each sample was adjusted to a protein concentration of 1 mg / ml using a protein quantification reagent (Bradford Protein Assay, Bio-Rad). After adjustment, the protein was denatured in a 95°C heat block and subjected to Western blotting. Each sample was then loaded onto a gel (SuperSep TM The gel was applied to a 5-12% gel of ACE (Fujifilm Wako Pure Chemical Industries, Ltd.), electrophoresed, and then transferred to a membrane (Immobilon-P membrane, Merck). The primary antibody was Goat Anti-Type IV Collagen antibody (Southern Biotech), and the secondary antibody was an HRP-conjugated antibody (Bio-Rad). After antibody reaction, type IV collagen protein was detected using an HRP chemiluminescence reagent (Immobilon® Western Blotting Detection Reagent, Merck) on a luminometer image analyzer (ImageQuant LAS 4000mini, GE Healthcare Japan).

[0034] The results are shown in Figure 2. As shown in this figure, the amount of type IV collagen protein was significantly lower in the medium containing test substance 1 (sirolimus) than in the solvent control medium. This result indicates that the inclusion of sirolimus inhibited the synthesis and secretion of type IV collagen protein in vascular endothelial cells. As mentioned above, scleroderma causes thickening of the vascular wall for some reason. Combined with our experimental results shown in (2) above, this suggests that high expression of the type IV collagen gene in vascular endothelial cells is a contributing factor to this disorder. Furthermore, because sirolimus has the effect of reducing type IV collagen protein, this drug has been shown to be effective as a therapeutic agent for peripheral vascular disorders, such as Raynaud's syndrome, caused by the overproduction of extracellular matrix containing collagen.

[0035] (Comparative Example) The same procedure as in the example above (1) was carried out using a medium containing 10 nmol / L of test substance 2 (digoxin) instead of the medium containing test substance 1, and the effect of the presence of digoxin on the expression of type IV collagen was confirmed.

[0036] The results are shown in Figure 3. As shown in this figure, the amount of type IV collagen protein in the medium containing digoxin was barely reduced, suggesting that digoxin has little therapeutic effect on peripheral vascular disease. Digitalis preparations containing digoxin are listed in the Scleroderma Treatment Guidelines (Japanese Dermatological Association Systemic Scleroderma Clinical Practice Guidelines) as the basic treatment for pulmonary hypertension associated with scleroderma. Furthermore, the efficacy of cardiac glycoside analogs, including digoxin, on fibrotic diseases has been reported in several publications. Our results contradict this conventional wisdom, demonstrating that digoxin is not effective, at least for peripheral vascular disease. On the other hand, sirolimus, whose therapeutic effect is not mentioned or suggested in the guidelines, is effective in treating peripheral vascular disease caused by the overproduction of extracellular matrix, including type IV collagen.

Claims

1. A therapeutic agent for vascular disorders associated with excessive synthesis and secretion of type IV collagen protein due to high expression of type IV collagen α5 and α6 genes from vascular endothelial cells, comprising sirolimus as an active ingredient.

2. The therapeutic agent according to claim 1, wherein the vascular disorder is caused by scleroderma.

3. The therapeutic agent according to claim 1 or 2, wherein the vascular disorder is a peripheral vascular disorder.

4. 4. The therapeutic agent according to claim 3, wherein the peripheral vascular disorder is selected from Raynaud's syndrome, pulmonary hypertension associated with Raynaud's syndrome, interstitial lung disease, digestive fibrosis, renal fibrosis, cardiac fibrosis, and other vascular lesions.

5. A pharmaceutical composition for treating vascular disorders associated with excessive synthesis and secretion of type IV collagen protein due to high expression of type IV collagen α5 and α6 genes from vascular endothelial cells, comprising sirolimus as an active ingredient and further comprising a pharmaceutically acceptable carrier.

6. The pharmaceutical composition described in claim 5, which is an oral agent or an external agent.

7. An inhibitor of excessive synthesis and secretion of type IV collagen protein caused by high expression of type IV collagen α5 and α6 genes from vascular endothelial cells, comprising sirolimus as an active ingredient.

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