Therapeutic agent for diseases associated with blood flow disorder

Abstract

Provided is a therapeutic agent for diseases associated with blood flow disorders. A therapeutic agent according to the present disclosure is for diseases associated with blood flow disorders and contains autotaxin or lysophosphatidic acid.

Description

Therapeutic agent for diseases associated with blood flow disorders

[0001] The present invention relates to a therapeutic agent for diseases associated with blood flow disorders.

[0002] For example, systemic sclerosis (SSc) is an autoimmune disease that causes fibrosis of the skin and internal organs, and additionally presents with blood flow disorders and the like. Progression of blood flow disorders may lead to the development of refractory skin ulcers.

[0003] For example, as disclosed in Non-Patent Document 1, Tracleer (registered trademark in Japan) tablets are said to be effective in suppressing the onset of digital ulcers in systemic sclerosis.

[0004] Incidentally, autotaxin is known to be required for angiogenesis during embryonic development (Non-Patent Document 2).

[0005] Janssen Pharma Co., Ltd., Tracleer (registered trademark in Japan) tablets 62.5mg package insert, launched in June 2005, revised in February 2024 (1st edition)van Meeteren, L. A. et al., Mol. Cell. Biol., Vol. 26, p. 5015-5022, 2006

[0006] However, for diseases associated with blood flow disorders, for example in the case of systemic sclerosis, although prophylactic agents such as the aforementioned Tracleer (registered trademark in Japan) tablets are known, no effective therapeutic agent has been known. Furthermore, although autotaxin has been suggested to be involved in angiogenesis, its association with blood flow disorders has not been known.

[0007] An object of one aspect of the present invention is to provide a therapeutic agent for diseases associated with blood flow disorders.

[0008] In order to solve the above problem, the therapeutic agent according to one aspect of the present invention is a therapeutic agent for a disease associated with blood flow disorders, containing autotaxin or lysophosphatidic acid.

[0009] According to one aspect of the present invention, a therapeutic agent for diseases associated with blood flow disorders can be provided.

[0010] These figures show microscopic images of vascular endothelial cells obtained from iPS cells derived from healthy individuals and vascular endothelial cells obtained from iPS cells derived from SSc patients. These figures show the length of lumen formation in vascular endothelial cells obtained from iPS cells derived from healthy individuals and vascular endothelial cells obtained from iPS cells derived from SSc patients. These figures show microscopic images of vascular endothelial cells obtained from iPS cells derived from SSc patients, with and without autotaxin administration. These figures show the change in absorbance over time in vascular endothelial cells obtained from iPS cells derived from healthy individuals and vascular endothelial cells obtained from iPS cells derived from SSc patients.

[0011] One embodiment of the present invention will be described below, but the present invention is not limited thereto. Unless otherwise specified in this specification, "A to B" representing a numerical range means "A or greater, B or less".

[0012] [1. Therapeutic Agent] The therapeutic agent according to one embodiment of the present invention is a therapeutic agent for diseases involving blood flow disorders, containing autotaxin or lysophosphatidic acid. In this specification, "treatment of diseases involving blood flow disorders" means improving at least the blood flow disorder, and / or the resulting pain and / or skin ulcers, in a subject (patient) suffering from a disease involving blood flow disorders.

[0013] The aforementioned disease may be a disease accompanied by Raynaud's phenomenon, or it may not be a disease accompanied by Raynaud's phenomenon. Raynaud's phenomenon is a phenomenon in which the small arteries in the fingers and toes constrict strongly in response to cold, causing the fingers and toes to turn pale.

[0014] One example of the aforementioned disease is systemic sclerosis (SSc). Systemic sclerosis is a designated intractable chronic disease that causes fibrosis of the skin and internal organs, and is characterized by Raynaud's phenomenon, skin sclerosis, pulmonary fibrosis, and reflux esophagitis. In particular, vascular lesions, including Raynaud's phenomenon which is seen in more than 80% of systemic sclerosis cases, give rise to conditions such as intractable peripheral skin ulcers, pulmonary hypertension, and scleroderma renal crisis, which have a significant impact on the patient's daily life and prognosis. In systemic sclerosis, symptoms are caused by a complex interplay of three events: fibrosis, impaired blood flow, and immune abnormalities, but the relationship between these is still not well understood.

[0015] There is no known cure for systemic sclerosis; only symptomatic treatments are available. In particular, regarding circulatory impairment and the resulting skin ulcers in systemic sclerosis, while preventative agents are known, no effective treatments have been found.

[0016] Treatment for skin symptoms caused by peripheral circulatory disorders in SSc patients involves vasodilators or antiplatelet agents, but currently, these have not shown sufficient efficacy. Previous reports (Kuwana et al., Lancet, Vol. 364, No. 9434, p. 603-610, 2004 and Shirai et al., Arthritis Rheumatol., Vol. 67, Issue 2, p. 498-507, 2015) have shown that vascular endothelial progenitor cells supplied from the bone marrow of SSc patients are reduced compared to healthy individuals, and impaired angiogenesis has been pointed out as one of the causes of peripheral circulatory disorders in SSc.

[0017] In systemic sclerosis, while the pathogenesis of tissue fibrosis, including its relationship to Transforming Growth Factor (TGF)-β1 signaling, is becoming clearer, vascular lesions remain largely unanalyzed. This is because monolayer plate cultures of vascular endothelial cells alone cannot adequately evaluate the structure specific to blood vessels. To analyze vascular structure, it is necessary to reconstruct blood vessels, where simple structures become hierarchical and complex, using collected vascular endothelial cells. For this to work, vascular endothelial cells must retain their diversification capacity (Nishikawa, Proceedings of the 120th Japan Medical Association Symposium, 2001), but selectively purifying such vascular endothelial cells in large quantities from patient tissue is difficult. To solve this problem, the inventors generated iPS cells from healthy individuals and SSc patients, as described below, and then induced vascular endothelial cells from these iPS cells via hematopoietic endothelial cells. Comprehensive microarray analysis using RNA from these vascular endothelial cells revealed that vascular endothelial cells derived from SSc patients showed reduced autotaxin expression compared to vascular endothelial cells derived from healthy individuals. Furthermore, it was confirmed that administering autotaxin to vascular endothelial cells derived from SSc patients restored lumen formation ability. Restoration of lumen formation ability in vascular endothelial cells means that blood vessels are formed normally, which is expected to improve peripheral circulatory disorders, Raynaud's phenomenon, impaired blood flow, and / or associated pain and / or skin ulcers.

[0018] In addition to systemic sclerosis, other diseases mentioned above include systemic lupus erythematosus and mixed connective tissue disease. These diseases can also cause impaired blood flow and / or resulting pain and / or skin ulcers. It is believed that administering autotaxin to patients can improve the lumen-forming ability of vascular endothelial cells, thereby improving impaired blood flow and / or resulting pain and / or skin ulcers, not only in systemic sclerosis but in other diseases as well. Preferably, the disease mentioned above is systemic sclerosis.

[0019] Autotaxin (ATX) is an enzyme that breaks down lysophosphatidylcholine (LPC) to produce lysophosphatidic acid (LPA). Lysophosphatidic acid is known to cause organ fibrosis. For this reason, autotaxin has traditionally been known as a marker for liver fibrosis, among other things.

[0020] As mentioned above, systemic sclerosis is accompanied by tissue fibrosis in addition to Raynaud's phenomenon. Autotaxin was thought to promote tissue fibrosis by producing lysophosphatidic acid, but surprisingly, this experiment revealed that it also has a beneficial effect on vascular endothelial damage. As mentioned above, the inventors have confirmed that lumen formation ability is restored by administering autotaxin, and it is speculated that a similar effect may be obtained by lysophosphatidic acid produced by autotaxin.

[0021] Autotaxins may be those obtained by known genetic recombination techniques, protein synthesis methods, and purification methods, or commercially available recombinant proteins may be used.

[0022] The autotaxin contained in the therapeutic agent does not necessarily have to be a full-length protein. For example, a partial autotaxin protein that exhibits activity to improve the lumen formation ability of vascular endothelial cells may be used. Alternatively, a mutant protein in which some amino acids of autotaxin are substituted, deleted, added, or inserted may be used that exhibits activity to improve the lumen formation ability of vascular endothelial cells. Whether or not it has activity to improve the lumen formation ability of vascular endothelial cells can be confirmed by the method described in the examples.

[0023] In the therapeutic agent, components other than autotaxin or lysophosphatidic acid are not particularly limited and may include, for example, buffering agents, pH adjusters, isotonic agents, preservatives, antioxidants, high molecular weight polymers, excipients, solvents, antibacterial agents, etc.

[0024] Examples of the buffering agent include phosphoric acid or phosphate, boric acid or borate, citric acid or citrate, acetic acid or acetate, carbonate or carbonate, tartaric acid or tartrate, ε-aminocaproic acid, and trometamol. Examples of the phosphate include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. Examples of the borate include borax, sodium borate, and potassium borate. Examples of the citrate include sodium citrate, disodium citrate, and trisodium citrate. Examples of the acetate include sodium acetate and potassium acetate. Examples of the carbonate include sodium carbonate and sodium bicarbonate. Examples of the tartrate include sodium tartrate and potassium tartrate.

[0025] Examples of the pH adjusting agents include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and potassium hydroxide.

[0026] Examples of the isotonic agents include ionic isotonic agents (e.g., sodium chloride, potassium chloride, calcium chloride, magnesium chloride) and nonionic isotonic agents (e.g., glycerin, propylene glycol, sorbitol, mannitol).

[0027] Examples of the aforementioned preservatives include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

[0028] Examples of the aforementioned antioxidants include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

[0029] Examples of the high molecular weight polymers include methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose phthalate, carboxymethylethylcellulose, cellulose phthalate acetate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, and atelocollagen.

[0030] Examples of the excipients include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.

[0031] Examples of the aforementioned solvents include water, physiological saline solution, and alcohol.

[0032] Examples of the aforementioned antibacterial agents include β-lactam, aminoglycoside, tetracycline, lincomycin, chloramphenicol, macrolide, ketolide, polypeptide, and glycopeptide antibiotics; and synthetic antibacterial agents such as pyridonecarboxylic acid (quinolone), fluoroquinolone, oxazolidinone, and sulfonamide drugs.

[0033] The amount of autotaxin or lysophosphatidic acid contained in the therapeutic agent is not particularly limited. The amount of autotaxin may be, for example, 0.001% to 100% by weight, 0.01% to 100% by weight, 0.1% to 100% by weight, 0.1% to 95% by weight, 0.1% to 90% by weight, 0.1% to 80% by weight, 0.1% to 70% by weight, 0.1% to 60% by weight, 0.1% to 50% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, or 0.1% to 10% by weight, based on the total weight of the therapeutic agent.

[0034] The amount of components other than autotaxin or lysophosphatidic acid contained in the therapeutic agent is not particularly limited. The amount of components other than autotaxin or lysophosphatidic acid may be, for example, 0% to 99.999% by weight, 0% to 99.99% by weight, 0% to 99.9% by weight, 5% to 99.9% by weight, 10% to 99.9% by weight, 20% to 99.9% by weight, 30% to 99.9% by weight, 40% to 99.9% by weight, 50% to 99.9% by weight, 60% to 99.9% by weight, 70% to 99.9% by weight, 80% to 99.9% by weight, or 90% to 99.9% by weight, relative to the total weight of the therapeutic agent.

[0035] The form of administration of the aforementioned therapeutic agent is not limited; for example, it may be a topical preparation or an oral preparation. For example, if it is a topical preparation, it can be administered locally to the site where blood flow disorders and / or associated pain and / or skin ulcers are occurring. Specific dosage forms of topical preparations include, for example, topical solid preparations, topical liquid preparations (liniments, lotions), spray preparations (topical aerosols, pump sprays), ointments, creams, gels, and patches (tapes, poultices). Specific dosage forms of oral preparations include tablets, capsules, granules, powders, oral liquid preparations, syrups, oral jellies, and oral films.

[0036] [2. Treatment Method] One embodiment of the present invention also includes a method for treating a disease involving blood flow impairment, comprising the step of administering the above-described therapeutic agent to a subject. The subject may be a subject who requires treatment for a disease involving blood flow impairment. For example, the subject may be a subject suffering from a disease involving blood flow impairment. The subject may be a subject exhibiting blood flow impairment, and / or associated pain and / or skin ulcers.

[0037] A subject to be administered may be a human or a non-human mammal (for example, livestock, companion animals and experimental animals). Non-human mammals are not particularly limited, and examples thereof include monkeys, chimpanzees, cattle, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.

[0038] For example, when the therapeutic agent is administered as an external preparation, the administration method is not particularly limited as long as the administration method contacts the administration site, and examples thereof include application, sticking, spraying, and immersion. The administration site is, for example, skin, and specific examples include fingers of limbs and the like.

[0039] The present invention is not limited to the respective embodiments described above, and various modifications can be made within the scope recited in the claims. Embodiments obtained by appropriately combining technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0040] One embodiment of the present invention may include the following configurations. <1> A therapeutic agent for a disease associated with blood flow disorder, comprising autotaxin or lysophosphatidic acid. <2> The therapeutic agent according to <1>, wherein the disease is systemic scleroderma.

[0041] One example of the present invention will be described below.

[0042] [1. Induction of vascular endothelial cells from iPS cells] Human iPS cells derived from healthy subjects and SSc patients were each cultured in a culture dish. As said human iPS cells, according to the method described in PLoS One. 2018 Jul 25;13(7): e0200790, iPS cells established from human skin fibroblasts and blood cells of 2 healthy subjects (HC-1, HC-2) and 2 SSc patients (SSc-1, SSc-2), respectively, were used.

[0043] When the cultured human iPS cells became subconfluent, they were detached from the culture dish and seeded onto a Matrigel (registered trademark in Japan)-coated dish. Next, StemPro ＴＭ -34 SFM (Gibco TM ) supplemented with 2 mM L-Glutamine (GibcoTM The culture medium was prepared by adding the following: 50 ng / mL of BMP4 (R & D Systems, Minneapolis, MN) and bFGF (WAKO, Japan). The culture medium was added to a dish coated with Matrigel (registered trademark of Japan), and human iPS cells were cultured for 24 hours.

[0044] Next, the cells were cultured for 2 days with 40 ng / mL VEGF (Invitrogen, Waltham, MA) and 50 ng / mL bFGF added to the culture medium, and then for 3 days with an additional 40 ng / mL VEGF, 50 ng / mL bFGF, and 20 μmol / L SB431542 (Miltenyi Biotec, Teterow, Germany) added. On day 6, the cells were stained with CD31 antibody (WM59; BioLegend, San Diego, CA) and CD144 antibody (16B1, eBioscience), and only the cells in the compartments positive for both antibodies were collected using FACS Aria II (BD Biosciences, San Jose, CA).

[0045] The harvested cells were cultured in Humedia-EB2 (KURABO) containing 10% FBS, 20 ng / mL VEGF, 25 ng / mL bFGF, and penicillin / streptomycin solution (ScienCell, final concentration penicillin 100 units / mL, streptomycin 100 μg / mL). Using this method, vascular endothelial cells (iPSECs) were induced from iPS cells.

[0046] [2. Evaluation of lumen-forming ability] IPSECs cultured in the above-mentioned Humedia-EB2 until reaching subconfluence were cultured overnight in a dish containing growth factor-free medium supplemented with 0.5% serum. Next, IPSECs were detached from the dish and seeded at 10000 cells / well in a 96-well plate coated with Matrigel (registered trademark in Japan). Eight hours later, the state of formed lumens was photographed using a fluorescence microscope (BZ-9000, KEYENCE), and the total length of lumens was evaluated from the obtained images using Image J (National Institutes of Health, Bethesda, MD).

[0047] [3. Microarray analysis] RNA was recovered from the IPSECs used in [2. Evaluation of lumen-forming ability], and gene expression levels were converted into Z-scores using Agilent Feature Extraction Software (9.5.1.1). In the comparison between the healthy subject group and the SSc patient group, the top 20 genes with a Z-score of 2.0 or higher or the bottom 20 genes with a Z-score of -2.0 or lower were extracted. Among these, 18 genes common to four comparisons between 2 healthy subjects and 2 SSc patients were listed as candidates. Next, actual gene expression was confirmed by qPCR. Among genes with a significant difference observed between the healthy subject group and the SSc patient group, the mechanism of action of candidate genes was considered based on previous reports, and genes thought to be involved in angiogenesis were investigated. As a result, only two relevant genes were obtained, including the ATX gene. Among these, improvement in lumen formation was observed only when ATX was added.

[0048] [4. ATX addition experiment] Subconfluent IPSECs were cultured overnight in growth factor-free medium supplemented with 0.5% serum. The next day, recombinant ATX-GST fusion protein (Sigma-Aldrich) or GST protein (Sigma-Aldrich) diluted in Humedia-EB2 containing 10% FBS to a final concentration of 20 nM was added to a 96-well plate coated with Matrigel (registered trademark in Japan). The IPSECs were suspended in Humedia-EB2 containing 10% FBS. The obtained suspension was adjusted to a cell concentration of 1 × 10 ４The cells were seeded in the aforementioned dish, with each cell individually contained. Separately, a 96-well dish coated with Matrigel (a registered trademark in Japan) was prepared by adding only the solvent and seeding the cells using the above suspension. After 8 hours of incubation, the state in which the lumen had formed was photographed using a fluorescence microscope (BZ-9000, KEYENCE), and the length of the lumen was evaluated from the obtained images. Note that GST protein stands for glutathione S-transferase and is commonly used as a tag in genetic engineering.

[0049] [5. Evaluation Results] Figure 1 shows microscopic images of iPSECs from healthy individuals and iPSECs from SSc patients, obtained in [2. Evaluation of Lumen Formation Ability]. The reticular structure in Figure 1 is the lumen. From Figure 1, it can be seen that iPSECs from SSc patients have reduced lumen formation ability compared to iPSECs from healthy individuals, as the lumen formation is more sparse.

[0050] Figure 2 shows the length of lumen formation in iPSECs derived from healthy individuals and iPSECs derived from SSc patients, respectively. A Welch's t-test using GraphPad Prism:Version 10.2.3 (403) revealed a significant difference in lumen formation length between iPSECs from healthy individuals and iPSECs from SSc patients (P < 0.01). Therefore, quantitatively, it can be seen that iPSECs derived from SSc patients have reduced lumen formation ability compared to iPSECs derived from healthy individuals.

[0051] Figure 3 shows microscopic images of iPSECs derived from SSc patients, comparing those administered ATX (ATX-treated group) and those not administered ATX (untreated group). Endothelial cells cultured in dishes containing the recombinant ATX protein described above belong to the ATX-treated group, while endothelial cells cultured in dishes containing only the solvent belong to the untreated group. From Figure 3, it can be seen that the lumens are more densely formed in the ATX-treated group compared to the untreated group, resulting in longer lumens and improved lumen formation ability. From these findings, it is suggested that ATX is effective in treating diseases involving blood flow disorders, including SSc.

[0052] [6. Reference Experiment] The proliferative capacity of iPSECs derived from healthy individuals and iPSECs derived from SSc patients obtained in [1. Induction of Vascular Endothelial Cells from iPS Cells] was evaluated using Cell Counting Kit-8 (Registered Trademark in Japan) (Dojin Chemical Laboratories Co., Ltd.).

[0053] Figure 4 shows the time-dependent changes in absorbance of iPSECs from healthy individuals and iPSECs from SSc patients, measured using Cell Counting Kit-8 (registered trademark in Japan). The horizontal axis represents the elapsed time from immediately after cell adhesion (0 hours), and the vertical axis represents the difference between the absorbance measured at each time point and the absorbance measured immediately after cell adhesion (0 hours). A larger value on the vertical axis indicates a higher cell count.

[0054] As shown in Figure 4, there is no significant difference in proliferative capacity between iPSECs derived from healthy individuals and iPSECs derived from SSc patients. This suggests that the reduced lumen formation ability in SSc patients is not due to angiogenesis impairment as mentioned in Non-Patent Literature 2, but rather to impairment of vascular endothelial cell function. The effect of autotaxin administration on improving such impairment of vascular endothelial cell function is previously unknown and represents a new finding. Furthermore, Figures 1C and 1D, and Figure 2 show that morphological abnormalities of vascular endothelial cells (cell shrinkage and aggregation) occur in SSc patients, and Figure 3 shows that these morphological abnormalities of vascular endothelial cells are improved in the ATX-administered group. Thus, the improvement of vascular endothelial cell morphological abnormalities by autotaxin administration is also a surprising finding.

[0055] One aspect of the present invention can be used in the treatment of diseases involving impaired blood flow.

Claims

1. A therapeutic agent containing autotaxin or lysophosphatidic acid for the treatment of diseases involving impaired blood flow.

2. The therapeutic agent according to claim 1, wherein the disease is systemic scleroderma.

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