Composition for regulating cell proliferation and regulation method of cell proliferation

Abstract

The present invention provides a composition for regulating cell proliferation, said composition containing a regulator of D-amino acid content, or a composition that activates an mTOR-related pathway. The present invention also provides a regulation method of cell proliferation in a subject, said method comprising administering a regulator of D-amino acid content in vivo to a subject requiring the same. The present invention also provides a regulation method of cell proliferation in vitro or ex vivo, said method comprising a step for applying a regulator of D-amino acid content to a cell, a biological tissue or an organ and culturing. The present invention also provides a use of a regulator of D-amino acid content to produce a pharmaceutical composition for regulating cell proliferation.

Description

Composition for modulating cell proliferation and method for modulating cell proliferation

[0001] The present invention relates to compositions and methods for modulating cell proliferation.

[0002] Cells are generally recognized as the most basic building blocks of living organisms. They possess metabolic pathways, such as glycolysis and the citric acid cycle, for constant vital activity, and are endowed with genetic information for self-renewal and replication, along with the ability to express that information. Humans are composed of over 30 trillion somatic and germ cells. The majority of somatic cells are of approximately 200 types, including proliferative dividing cells that continually divide and proliferate (e.g., myeloblasts, basal cells, etc.), differentiated dividing cells that divide and proliferate while differentiating (e.g., bone marrow cells, neuroblasts, myoblasts, etc.), reversible postmitotic cells that do not normally proliferate (e.g., stem cells, smooth muscle cells, lymphocytes, etc.), and fixed postmitotic cells that have lost the ability to divide (e.g., neurons, cardiac myocytes, red blood cells, etc.).

[0003] In physiological regenerative tissues, there are cell types that undergo a cycle of regeneration, function, and death under normal conditions (e.g., epithelial cells, blood cells, etc.); in conditional regenerative tissues, there are cells that normally do not proliferate but proliferate and regenerate under special circumstances such as injury (e.g., organ parenchymal cells, vascular endothelial cells, connective tissue, etc.); and in non-regenerative tissues, there are cell types that are highly organized and differentiated but do not have the ability to spontaneously generate or proliferate (e.g., nerve cells, skeletal muscle, cardiac muscle cells, etc.).

[0004] When cells can no longer proliferate due to their mitotic and differentiation lifespans, which are determined by telomeres, they undergo cell death, and the sum of these events is thought to represent the aging of an individual. Regenerative cells experience a decrease in overall cell number due to apoptosis, and the increased burden on each cell promotes further apoptosis, resulting in a decrease in the number of parenchymal cells (atrophy) in tissues and organs, resulting in decreased function. Tumors are cells that grow autonomously without normal control due to some abnormality in the cells' genes, while malignant tumors (cancers) are those that undergo invasive cell proliferation and metastasis. Cell fusion, gene transfer into cells, and cell culture are techniques that artificially manipulate cellular mechanisms and abnormalities to control and modify genetic and physiological functions. These techniques are used in the research and development of cell and tissue functions, regenerative medicine, pharmaceuticals, etc. In cell and tissue culture, differentiation and proliferation are controlled by setting conditions such as temperature, atmosphere, and medium components (glucose content, growth factors (e.g., epidermal growth factor, insulin-like growth factor, etc.), nutrients, etc.). Non-Patent Document 1 describes how amino acid metabolism and regulation regulate the proliferation capacity of human pluripotent stem cells.

[0005] In recent years, advances in the technology for identifying and analyzing chiral amino acids have enabled quantitative research into trace amounts of D- and L-amino acids in mammals and other living organisms. This has shed light on the existence and functions of some D-amino acids, which, due to previous technological limitations, were treated as total amino acids (D-amino acids + L-amino acids) or simply as L-amino acids. It has been reported that the amounts of D-amino acids in living organisms, tissues, cells, and body fluids vary depending on their ingestion, symbiotic bacteria, metabolism (degradation, synthesis), transport, excretion, etc. (Non-Patent Documents 2-6), and that characteristic chiral amino acid profiles are exhibited in diseases such as kidney disease, heart disease, and diabetes (Patent Document 1). Furthermore, it has been reported that D-amino acids are involved in intestinal immunity (Non-Patent Document 7) and protect kidney-derived cells (Non-Patent Document 3). Furthermore, carbohydrate metabolism has been reported to be involved in the biosynthesis of D-serine in neurons (Non-Patent Document 8).

[0006] It has been disclosed that the levels of D-serine and D-alanine in the blood fluctuate in renal cancer, while the levels of D-alanine, D-proline, and D-aspartic acid in the blood fluctuate in diabetes (Patent Documents 1 and 2). It has also been reported that D-alanine is localized in cells containing insulin in the pancreatic islets of Langerhans and in cells containing adrenocorticotropic hormone in the anterior pituitary gland (Non-Patent Documents 9 and 10). However, the relationship between the presence of D-amino acids and cell proliferation has not yet been clarified.

[0007] International Publication No. 2013 / 140785 Japanese Patent Application Laid-Open No. 2017-207490 International Publication No. 2020 / 196436

[0008] S. Someya, S. Tohyama, K. Kameda, S. Tanosaki, Y. Morita, K. Sasaki, M.-I. Kang, Y. Kishino, M. Okada, H. Tani, Y. Soma, K. Nakajima, T. Umei, O. Sekine, T. Moriwaki, H. Kanazawa, E. Kobayashi, J. Fujita and K. Fukuda, Tryptophan metabolism regulates proliferative capacity of human pluripotent stem cells, iScience, 24 (2021). DOI: 10.1016 / j.isci.2021.102090Y. Miyoshi, R. Konno, J. Sasabe, K. Ueno, Y. Tojo, M. Mita, S. Aiso and K. Hamase, Alteration of intrinsic amounts of D-serine in mice lacking serine racemase and D-amino acid oxidase, Amino Acids, 43, 1919-1931 (2012). DOI: 10.1007 / s00726-012-1398-4Y. Nakade, Y. Iwata, K. Furuichi, M. Mita, K. Hamase, R. Konno, T. Miyake, N. Sakai, S. Kitajima, T. Toyama, Y. Shinozaki, A. Sagara, T. Miyagawa, A. Hara, M. Shimizu, Y. Kamikawa, K. Sato, M. Oshima, S. Yoneda-Nakagawa, Y. Yamamura, S. Kaneko, T. Miyamoto, M. Katane, H. Homma, H. Morita, W. Suda, M. Hattori and T.Wada, Gut microbiota-derived D-serine protects against acute kidney injury, JCI Insight, 3 (2018). DOI: 10.1172 / jci.insight.97957M. Ariyoshi, M. Katane, K. Hamase, Y. Miyoshi, M. Nakane, A.Hoshino, Y. Okawa, Y. Mita, S. Kaimoto, M. Uchida, K. Fukai, K. Ono, S. Tateishi, D. Hato, R. Yamanaka, S. Honda, Y. Fushimura, E. Iwai-Kanai, N. Ishihara, M. Mita, H. Homma and S. Matoba, D-Glutamate is metabolized in the heart mitochondria, Scientific Reports, 7 (2017). DOI: 10.1038 / srep43911P. Wiriyasermkul, S. Moriyama, Y. Tanaka, P. Kongpracha, N. Nakamae, M. Suzuki, T. Kimura, M. Mita, J. Sasabe and S. Nagamori, D-Serine, an emerging biomarker of kidney diseases, is a hidden substrate of sodium-coupled monocarboxylate transporters, bioRxiv preprint. DOI: 10.1101 / 2020.08.10.244822A. Hesaka, S. Sakai, K. Hamase, T. Ikeda, R. Matsui, M. Mita, M. Horio, Y. Isaka and T. Kimura, D-Serine reflects kidney function and diseases, Scientific Reports, 9 (2019). DOI: 10.1038 / s41598-019-41608-0J. Ask, Y.Miyoshi, S. Rakoff-Nahoum, T. Zhang, M. Mita, B.M. Davis, K. Hamase and M.K. Waldor, Interplay between microbial D-amino acids and host D-amino acid oxidase modifies murine mucosal defence and gut microbiota, Nature Microbiology, 1 (2016). DOI: 10.1038 / nmicrobiol.2016.125M. Suzuki, J. Sasabe, Y. Miyoshi, K. Kuwasako, Y. Muto, K. Hamase, M. Matsuoka, N. Imanishi and S. Aiso, Glycolytic flux controls D-serine synthesis through glyceraldehyde-3-phosphate dehydrogenase in astrocytes, Proceedings of the National Academy of Sciences of the United States of America, 112, E2217-2224 (2015). DOI: 10.1073 / pnas.1416117112A. Morikawa, K. Hamase, T. Ohgusu, S. Etoh, H. Tanaka, I. Koshiishi, Y. Shoyama and K. Zaitsu, Immunohistochemical localization of D-alanine to β-cells in rat pancreas, Biochemical and Biophysical Research Communications, 355, 872-876 (2007). DOI: 10.1016 / j.bbrc.2007.02.056S. Etoh, K. Hamase, A. Morikawa, T. Ohgusu and K.Zaitsu, Enantioselective visualization of D-alanine in rat anterior pituitary gland: localization to ACTH-secreting cells, Analytical and Bioanalytical Chemistry, 393, 217-223 (2009). DOI: 10.1007 / s00216-008-2401-5 Japanese Society of Traumatology Organ Injury Classification Committee: Kidney Injury Classification 2008 (Japanese Society of Traumatology). Japanese Journal of Trauma 2008; 22:265.

[0009] There is a strong demand for methods and drugs for more efficient cell proliferation and for restoring, maintaining, and enhancing the function of abnormal cells, tissues, and organs both inside and outside the body by regenerating them.

[0010] The inventors adjusted the amounts of D-amino acids and L-amino acids in the medium for primary or subculture of renal tubular cells and fibroblasts, and discovered that D-amino acids have a greater effect on promoting cell proliferation than L-amino acids. As a result of extensive research into this effect and mechanism, they discovered a method for adjusting the proliferation and regeneration of cells, tissues, and organs, particularly tissues in a conditional regeneration system, both in and out of the body by increasing or decreasing the amount of D-amino acids, leading to the present invention. Specifically, the present invention encompasses the following inventions:

[0011] [1] A composition for regulating cell proliferation, comprising a regulator of D-amino acid levels. [2] The composition of item 1, wherein the regulator is a regulator of D-amino acid levels in the subject's living body. [3] The composition of item 1 or 2, wherein the cell proliferation is cell proliferation in a biological tissue and / or organ, thereby regulating the size of the biological tissue and / or organ. [4] The composition of item 2, wherein the subject is a subject with kidney disease. [5] The composition of item 2, wherein the subject is a kidney transplant donor and / or recipient. [6] The composition of item 2, wherein the composition improves kidney function. [7] The composition of item 6, wherein the kidney function is glomerular filtration rate. [8] The composition of item 1, wherein the composition is for regulating the proliferation of isolated cells. [9] The composition of any one of items 1 to 8, wherein the regulator is a D-amino acid or a derivative thereof.

[10] The composition of item 9, wherein the D-amino acid is selected from the group consisting of D-serine, D-asparagine, and D-glutamine.

[11] The composition according to any one of items 1 to 8, wherein the regulator is an agent for regulating the activity of a protein involved in the absorption, transport, distribution, metabolism, or excretion of a D-amino acid.

[12] The composition according to item 11, wherein the metabolism is degradation or synthesis.

[13] The composition according to item 11 or 12, wherein the agent for regulating the activity of the protein is a regulator of gene expression of the protein.

[14] The composition according to any one of items 11 to 13, wherein the protein is selected from the group consisting of D-aspartate oxidase and serine isomerase.

[15] The composition according to any one of items 11 to 13, wherein the protein is a D-amino acid transporter protein.

[16] The D-amino acid transporter protein is selected from the group consisting of SMCT family, GLUT5, CAT1, THTR2, SNAT2, ASCT family, Asc1, PAT1, and ATB. ０，＋

[17] The composition according to any one of items 1 to 16, which is a pharmaceutical product.

[18] The composition according to any one of items 1 to 16, which is a food product.

[19] The composition according to item 18, which is a food with health claims or a dietary supplement.

[20] The composition according to item 19, which is a food with health claims or a food with nutrient claims.

[21] The composition according to any one of items 1 to 20, which activates an mTOR-related pathway.

[0012] [22-1] A composition that activates an mTOR-related pathway, comprising a regulator of D-amino acid levels. [22-2] The composition according to Item 22-1, wherein the regulator is a regulator of D-amino acid levels in the subject's living body. [22-3] The composition according to Item 22-1 or 22-2, wherein the cell proliferation is cell proliferation in a biological tissue and / or organ, thereby adjusting the size of the biological tissue and / or organ. [22-4] The composition according to Item 22-2, wherein the subject is a subject with kidney disease. [22-5] The composition according to Item 22-2, wherein the subject is a kidney transplant donor and / or recipient. [22-6] The composition according to Item 22-2, wherein the renal function is improved. [22-7] The composition according to Item 22-6, wherein the renal function is glomerular filtration rate. [22-8] The composition according to Item 22-1, wherein the composition is for regulating the proliferation of isolated cells. [22-9] The composition according to any one of items 22-1 to 22-8, wherein the regulator is a D-amino acid or a derivative thereof. [22-10] The composition according to item 22-9, wherein the D-amino acid is selected from the group consisting of D-serine, D-asparagine, and D-glutamine. [22-11] The composition according to any one of items 22-1 to 22-8, wherein the regulator is an agent that regulates the activity of a protein involved in the absorption, transport, distribution, metabolism, or excretion of a D-amino acid. [22-12] The composition according to item 22-11, wherein the metabolism is degradation or synthesis. [22-13] The composition according to item 22-11 or 22-12, wherein the agent that regulates the activity of the protein is a regulator of gene expression of the protein. [22-14] The composition according to any one of items 22-11 to 22-13, wherein the protein is selected from the group consisting of D-aspartate oxidase and serine isomerase. [22-15] The composition according to any one of Items 22-11 to 22-13, wherein the protein is a D-amino acid transporter protein. [22-16] The D-amino acid transporter protein is selected from the group consisting of SMCT family, GLUT5, CAT1, THTR2, SNAT2, ASCT family, Asc1, PAT1, and ATB. ０，＋[22-17] The composition according to any one of items 22-1 to 22-16, which is a pharmaceutical. [22-18] The composition according to any one of items 22-1 to 22-16, which is a food. [22-19] The composition according to item 22-18, which is a food with health claims or a dietary supplement. [22-20] The composition according to item 22-19, which is a food with health claims or a food with nutrient claims.

[0013]

[23] A method for regulating cell proliferation in a subject, comprising administering to a subject in need thereof an agent for regulating the amount of D-amino acids in the body.

[0014]

[24] A method for regulating cell proliferation in vitro or ex vivo, comprising the steps of applying a regulator of the amount of a D-amino acid to a cell, biological tissue, or organ, and culturing the cell.

[0015]

[25] Use of an agent for controlling the amount of a D-amino acid for the manufacture of a pharmaceutical composition for regulating cell proliferation.

[0016] According to the present invention, it is possible to regulate cell proliferation by controlling the amount of D-amino acids, and therefore it is possible to prevent, treat, or evaluate conditions or diseases (e.g., tumors) in which cell proliferation is abnormal.

[0017] Figure 1 shows the effect of D,L-amino acids (10 μM) on the proliferation of cultured cells. Figure 2 shows the effect of D,L-amino acids (1 μM) on the proliferation of cultured cells. Figure 3 shows the effect of glycylserine isomers (10 μM) on the proliferation of cultured cells. Figure 4 shows the administration of D,L-amino acids and their uptake into each organ. As above. Figure 5 shows the effect of D-serine administration on increasing kidney size. n = 6-7. Statistical analysis was performed using two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Data are shown as mean ± SEM. Figure 6 shows the results of pathway analysis on the effects of D-serine administration. As above. Figure 7 shows the results of D-serine administration and cell cycle-related gene expression analysis. (A) Relative mRNA expression level. n = 6-7. Statistical analysis was performed using unpaired two-tailed Student's t-test. *p<0.05. Data are shown as mean ± SEM. (B) Relative mRNA expression levels. n=6-7. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Figure 8 shows Ki67 positive nuclei analysis following D-serine administration. (A) Representative images of renal cortex stained with anti-Ki67 antibody. The images at the bottom left of each figure are enlarged views of the boxed areas in the figures. Scale bar: 50 μm. (B) Number of Ki67-positive nuclei in the proximal tubule per field (n = 6-7), (C) relative tubule area (n = 6-7), (D) number of cells per tubule (n = 6-7). Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Data are shown as mean ± SEM. Figure 9 shows the effect of D-serine administration on proximal tubule cell proliferation. Lectin-positive proximal tubules: green, Ki67: red, DAPI (nuclei): blue. Scale bar: 50 μm. Figure 10 shows the effect of D-serine administration on glomerular cell proliferation. Relative glomerular area (%) is shown. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Data are shown as mean ± SEM. Figure 11 shows the proliferative effect of D-serine administration on renal tubular cells, HK-2 cells. (A) Normal human primary renal proximal tubular cells (RPTEC) or (B) HK-2 cells are cultured with 10 μM D-serine, showing the relative proliferation rate (%) over time.(C) Relative proliferation rate (%) of normal human primary renal proximal tubule cells (RPTEC) or (D) HK-2 cells when cultured with various concentrations of D-serine is shown. (E) Relative proliferation rate (%) of normal human primary renal proximal tubule cells (RPTEC) or (F) HK-2 cells when cultured with various concentrations of D-serine or L-serine is shown. Statistical analysis was performed using one-way ANOVA with Dunnett's post-hoc test. NS: not significant; *p<0.05, **p<0.01, ***p<0.001. Figure 12 shows the results of phosphorylation analysis of HK-2 cells after administration of D-serine. (A) Western blot results for phosphorylation of S6K at Thr389 (phosphorylated-S6K (p-S6K)) in HK-2 cells treated with 5 μM D- or L-serine for 10 minutes in serine-free medium. (B) Relative index (RI) from five independent experiments is plotted. Statistical analysis was performed by one-way ANOVA with Dunnett's post-hoc test. NS: not significant; *p<0.05, **p<0.01, ***p<0.001. Figure 13 shows the results of phosphorylation analysis of mouse kidney cells following administration of D-serine. (A) Immunoblot of phospho-S6 ribosomal protein (p-S6RP) at Ser235 / 236 from the renal cortex of 10-week-old C57BL / 6 male mice fed a serine-free diet and water with or without 0.1% D-serine for one week and sacrificed two days later with or without unilateral nephrectomy (UNX). (B) Relative index (RI) from three independent experiments is plotted. Statistical analysis was performed using one-way ANOVA with Dunnett's post-hoc test. NS: not significant; *p<0.05, **p<0.01, ***p<0.001. Figure 14 shows the effect of D-serine administration on cell proliferation in p18-deficient strains. (A) Relative proliferation rates of p18-deficient mouse embryonic fibroblasts (MEFs) and their revertants (Rev) treated with the indicated concentrations of D-serine for 6 hours in serine-free medium, compared with the corresponding cell lines treated with vehicle. N=6. Statistical analysis was performed by two-way ANOVA. ***p<0.001.(B) Immunoblot of p18 protein in p18-deficient MEFs and their revertants. Figure 15 shows the effect of D-serine administration on cell proliferation in Rheb-deficient lines. (A) Relative proliferation rates of Rheb-deficient or wild-type MEFs treated with the indicated concentrations of D-serine for 6 hours in serine-free medium, as well as the relative proliferation rates of the corresponding vehicle-treated cell lines. N=12. Statistical analysis was performed using two-way ANOVA. ***p<0.001. (B) Immunoblot of Rheb in Rheb-deficient and wild-type MEFs. Figure 16 shows the effect of D-serine administration and rapamycin or a PI3 inhibitor on cell proliferation. (A) Relative proliferation rates of HK-2 cells treated with or without rapamycin for 24 hours in serine-free medium and then treated with D-serine at the indicated concentrations for 6 hours, as well as the relative proliferation rates of the corresponding cell lines not treated with D-serine. N=21-24. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. (B) Relative proliferation rates of HK-2 cells treated with or without D-serine and Ly294002 at the indicated concentrations for 6 hours in serine-free medium, as well as the relative proliferation rates of the corresponding cell lines not treated with D-serine. N=21-24. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Figure 17 shows the results of AKT phosphorylation analysis following D-serine administration. (A) Immunoblot results for phospho-AKT (p-AKT) at Ser473 from the renal cortex of 10-week-old C57BL / 6 male mice were fed a serine-free diet and water with or without 0.1% D-serine for one week and sacrificed two days later with or without unilateral nephrectomy (UNX) (left). (B) Relative index (RI) values ​​from three independent experiments are plotted. Statistical analysis was performed using one-way ANOVA with Dunnett's post-hoc test. NS: not significant; *p<0.05, **p<0.01, ***p<0.001. Figure 18 shows the results of analysis of mTOR lysosomal localization following D-serine administration.(A) High-content microscopy quantification of mTOR and LAMP2 colocalization in HK-2 cells incubated in culture medium followed by amino acid starvation for 10 minutes in the presence of 5 μM D- or L-serine. Mask overlay, primary object (algorithmically defined cell boundary based on CellMask (red pseudocolor)), and internal secondary object (computationally defined colocalization between mTOR (green) and LAMP2 (yellow)). Statistical analysis was performed by one-way ANOVA with Bonferroni's post-hoc test. NS: not significant; *p<0.05; **p<0.01; ***p<0.001. (B) High-content microscopy quantification of mTOR and LAMP1 colocalization in HK-2 cells incubated in culture medium followed by amino acid starvation for 10 minutes in the presence of 5 μM D- or L-serine. Mask overlay, primary object (algorithmically defined cell boundary based on CellMask (red pseudocolor)), and internal secondary object (computationally defined colocalization between mTOR (green) and LAMP1 (yellow)). Statistical analysis was performed by one-way ANOVA with Bonferroni's post-hoc test. NS: not significant; *p<0.05, **p<0.01, ***p<0.001. Figure 19 shows a schematic diagram of the activation scheme of mTOR-related pathways by D-serine. (A) A schematic diagram showing that D-serine activates cell proliferation via the mTOR-related pathway (mTOR / p18 / Rheb). This shows that D-serine can mediate renal remodeling after unilateral nephrectomy. (B) A schematic diagram showing the activation of mTORC1 signaling by D-serine. D-serine enhances the signal from L-amino acids to activate mTORC1. D-serine also activates mTROC1 via the phosphoinositide 3-kinase (PI3K) / Rheb pathway. Figure 20 shows the effect of D-serine administration on increasing glomerular filtration rate (GFR). Figure 21 shows the results of examining the cell proliferation potential of D-amino acids in B cells.

[0018] Hereinafter, embodiments for carrying out the present invention will be described, but the technical scope of the present invention is not limited to the following embodiments. Note that the prior art documents cited in this specification are incorporated herein by reference.

[0019] One embodiment of the present invention provides a composition for regulating cell proliferation, comprising a regulator of the amount of a D-amino acid.

[0020] Additionally, one embodiment of the present invention provides a composition for activating an mTOR-related pathway, comprising a regulator of the amount of a D-amino acid.

[0021] Furthermore, one embodiment of the present invention provides a method for regulating cell proliferation, regulating tissue / organ size, or enhancing tissue / organ function, which comprises controlling the amount of D-amino acids in a subject.

[0022] As used herein, "controlling the amount of a D-amino acid" refers to intentionally increasing or decreasing the amount of a D-amino acid inside or outside a cell, tissue, and / or organ, as well as in a body fluid. When a target amount or concentration is specified, the D-amino acid in a sample and D-amino acid clearance may be appropriately monitored for evaluation.

[0023] As used herein, "cell proliferation" may refer to cell proliferation in biological tissues and / or organs, thereby adjusting the size of the biological tissues and / or organs.

[0024] As used herein, "adjusting the size of biological tissues and / or organs" refers to balancing or correcting abnormalities, excesses, or deficiencies in cell mass, morphology, or function in biological tissues and / or organs. Size can be expressed in units of measurement, such as morphological size, weight, or function, and may be corrected by body weight, etc. If a desired effect is achieved, for example, when the purpose is to grow cultured cells, the effect may be evaluated by performing metabolomic analysis of cell count or culture medium. Furthermore, when the purpose is to adjust kidney cell growth, size, or function, the effect may be evaluated by appropriately monitoring markers in urine tests (urine protein, hematuria, creatinine level, etc.), blood tests (creatinine, cystatin C, urea nitrogen (BUN), etc.), or by performing renal function tests (renal blood flow (RPF), glomerular filtration rate (GFR)), X-ray examination, angiography, ultrasound examination, CT, MRI, nuclear medicine examination, endoscopic examination, renal biopsy (pathological examination), etc.

[0025] As used herein, the term "D-amino acid amount regulator" (also referred to as "D-amino acid amount controller") refers to an agent that, when applied (e.g., administered), can increase or decrease the amount of a D-amino acid in a subject's living body (e.g., in a cell, tissue, organ, or body fluid) or in isolated cells. As used herein, "regulating the amount of a D-amino acid in a cell" means that, by applying a D-amino acid amount controller, the amount of a D-amino acid in the cell is increased or decreased, thereby adjusting the amount of the D-amino acid to a desired range. As used herein, "regulating the amount of a D-amino acid in a tissue" means that, by applying a D-amino acid amount controller, the amount of a D-amino acid in a tissue (e.g., renal tubule, glomerulus, etc.) is increased or decreased, thereby adjusting the amount of the D-amino acid to a desired range. As used herein, "regulating the amount of a D-amino acid in an organ" means that, by applying a D-amino acid amount controller, the amount of a D-amino acid in an organ (e.g., kidney, heart, etc.) is increased or decreased, thereby adjusting the amount of the D-amino acid to a desired range. As used herein, "regulating the amount of D-amino acids in body fluids" means adjusting the amount of D-amino acids within a desired range by increasing or decreasing the amount of D-amino acids in body fluids (e.g., blood, urine, etc.) through the application of a D-amino acid amount regulator.

[0026] As used herein, the term "D-amino acid" refers to a proteinogenic amino acid in the "D-form," which is a stereoisomer of an "L-form" proteinogenic amino acid, as well as glycine, which does not have a stereoisomer. Specifically, the term refers to glycine, D-alanine, D-histidine, D-isoleucine, D-allo-isoleucine, D-leucine, D-lysine, D-methionine, D-phenylalanine, D-threonine, D-allo-threonine, D-tryptophan, D-valine, D-arginine, D-cysteine, D-glutamine, D-proline, D-tyrosine, D-aspartic acid, D-asparagine, D-glutamic acid, and D-serine. Since D-cysteine ​​contained in a biological sample is oxidized to D-cystine outside the body, in one embodiment of the present invention, the amount of D-cysteine ​​contained in a biological sample can be calculated by measuring D-cystine instead of D-cysteine.

[0027] The amount of D-amino acids and / or L-amino acids can be measured by any method, such as chiral column chromatography, enzymatic methods, or immunological methods using monoclonal antibodies that distinguish optical isomers of amino acids. The amount of D-amino acids and / or L-amino acids in a sample in the present invention can be measured by any method known to those skilled in the art. For example, chromatographic methods and enzymatic methods (Y. Nagata et al., Clinical Science, 73 (1987), 105. Analytical Biochemistry, 150 (1985), 238., A. D'Aniello et al., Comparative Biochemistry and Physiology Part B, 66 (1980), 319. Journal of Neurochemistry, 29 (1977), 1053., A. Berneman et al., Journal of Microbial & Biochemical Technology, 2 (2010), 139., W. G. Gutheil et al., Analytical Biochemistry, 287 (2000), 196., G. Molla et al., Methods in Molecular Biology, 794 (2012), 273., T. Ito et al., Analytical Biochemistry, 371 (2007), 167., etc.), antibody methods (T. Ohgusu et al., Analytical Biochemistry, 357 (2006), 15. etc.), gas chromatography (GC) (H. Hasegawa et al., Journal of Mass Spectrometry, 46 (2011), 502., M.C. Waldhier et al., Analytical and Bioanalytical Chemistry, 394 (2009), 695., A. Hashimoto, T. Nishikawa et al., FEBS Letters, 296 (1992), 33., H. Bruckner and A. Schieber, Biomedical Chromatography, 15 (2001), 166., M. Junge et al., Chirality, 19 (2007), 228., M. C. Waldhier et al., Journal of Chromatography A, 1218 (2011), 4537., etc.), capillary electrophoresis (CE) (H. Miao et al., Analytical Chemistry, 77 (2005), 7190., D. L. Kirschner et al., Analytical Chemistry, 79 (2007), 736., F. Kitagawa, K. Otsuka, Journal of Chromatography B, 879 (2011), 3078., G. Thorsen and J. Bergquist, Journal of Chromatography B, 745 (2000), 389., etc.), high performance liquid chromatography (HPLC) (N. Nimura and T. Kinoshita, Journal of Chromatography, 352 (1986), 169., A. Hashimoto et al., Journal of Chromatography, 582 (1992), 41., H. Bruckner et al., Journal of Chromatography A, 666 (1994), 259., N. Nimura et al., Analytical Biochemistry, 315 (2003), 262., C. Muller et al., Journal of Chromatography A, 1324 (2014), 109., S. Einarsson et al., Analytical Chemistry, 59 (1987), 1191., E. Okuma and H. Abe, Journal of Chromatography B, 660 (1994), 243., Y. Gogami et al., Journal of Chromatography B, 879 (2011), 3259., Y. Nagata et al., Journal of Chromatography, 575 (1992), 147., S. A. Fuchs et al., Clinical Chemistry, 54 (2008), 1443., D. Gordes et al., Amino Acids, 40 (2011), 553., D. Jin et al., Analytical Biochemistry, 269 (1999), 124., J. Z. Min et al., Journal of Chromatography B, 879 (2011), 3220., T. Sakamoto et al., Analytical and Bioanalytical Chemistry, 408 (2016), 517., W. F. Visser et al., Journal of Chromatography A, 1218 (2011), 7130., Y. Xing et al., Analytical and Bioanalytical Chemistry, 408 (2016), 141., K. Imai et al., Biomedical Chromatography, 9 (1995), 106., T. Fukushima et al., Biomedical Chromatography, 9 (1995), 10., R. J. Reischl et al., Journal of Chromatography A, 1218 (2011), 8379., R. J. Reischl and W. Lindner, Journal of Chromatography A, 1269 (2012), 262., S. Karakawa et al., Journal of Pharmaceutical and Biomedical Analysis, 115 (2015), 123., Hamase K, et al., Chromatography 39 (2018) 147-152, etc.).

[0028] The optical isomer separation and analysis system of the present invention may combine multiple separation and analysis steps. More specifically, the amount of D-amino acids and / or L-amino acids in a sample can be measured by using a method for analyzing optical isomers, which includes the steps of passing a sample containing components having optical isomers together with a first liquid as a mobile phase through a first column packing material as a stationary phase to separate the components of the sample, individually retaining each of the components of the sample in a multi-loop unit, supplying each of the components of the sample individually retained in the multi-loop unit together with a second liquid as a mobile phase through a flow path to a second column packing material having an optically active center as a stationary phase to resolve the optical isomers contained in each of the components of the sample, and detecting the optical isomers contained in each of the components of the sample (Japanese Patent No. 4291628). In HPLC analysis, D- and L-amino acids may be derivatized in advance with fluorescent reagents such as o-phthalaldehyde (OPA) or 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), or diastereomerized using N-tert-butyloxycarbonyl-L-cysteine ​​(Boc-L-Cys) or the like (Kenji Hamase and Kiyoshi Zaitsu, Analytical Chemistry, Vol. 53, pp. 677-690 (2004)). Alternatively, D- or L-amino acids can be measured by immunological techniques using monoclonal antibodies that distinguish optical isomers of amino acids, for example, monoclonal antibodies that specifically bind to D- or L-amino acids. Furthermore, when the total amount of D- and L-isomers is used as an indicator, it is not necessary to separate and analyze the D- and L-isomers; amino acids can also be analyzed without distinguishing between the D- and L-isomers. In this case, separation and quantification can be performed using enzymatic methods, antibody methods, GC, CE, or HPLC.

[0029] As used herein, the term "mTOR-associated pathway" refers to a signaling pathway involving mTOR (mechanistic target of rapamycin), a type of protein kinase (serine-threonine kinase) involved in intracellular signal transduction, and may be, for example, a signaling pathway including factors involved in the activation of mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2), such as Akt and Rheb. By activating the mTOR-associated pathway, the present invention integrates intracellular and extracellular environmental information, such as insulin, growth factors, nutritional and energy status, and redox status, and regulates cell size, division, survival, and the like in response to these information through transcription, translation, and the like. These physiological mechanisms can regulate cell proliferation.

[0030] In the present invention, the amount of a biomolecule or drug, such as a D-amino acid, creatinine, or protein, is expressed not only in terms of simple mass, weight, or amount of substance (mol), but also in any measurable physical quantity, such as the mass, weight, or amount of substance (mol) per tissue, cell, organ, or molecular unit, or per volume or weight, or the mass, weight, amount of substance (mol), concentration, specific gravity, or density in a liquid such as blood or urine.

[0031] One aspect of the present invention may be a drug or food that can increase or decrease the amount of D-amino acids inside or outside cells, tissues, organs, and body fluids by administering D-amino acids externally, adding or removing D-amino acids to foods, or adding or removing D-amino acids from culture media. For example, drinking an aqueous solution containing D-amino acids can increase D-amino acid concentrations in blood and cells / tissues (Non-Patent Document 2), and ingesting foods from which D-amino acids have been removed can decrease D-amino acid concentrations in blood. For example, D-serine, which is administered orally or intravenously, can be targeted to the kidney, thereby controlling D-serine levels in the kidney (see, for example, Example 2). The D-amino acids used herein may contain modified or derivative D-amino acids, or pharmaceutically acceptable salts thereof, as long as they can increase or decrease the amount of D-amino acids. They may also contain pharmacologically acceptable carriers, diluents, or excipients, or may take the form of prodrugs. They may also contain agents for improving the function of the target organ. The drugs of the present invention can be formulated in a dosage form appropriate for the administration route. For oral administration, dosage forms such as tablets, capsules, liquids, powders, granules, chewable preparations, etc. can be designed; for parenteral administration, dosage forms such as injections, powders, and infusions can be designed. These preparations may also contain various pharmaceutical adjuvants, i.e., carriers and other auxiliary agents, such as stabilizers, preservatives, soothing agents, flavorings, corrigents, fragrances, emulsifiers, fillers, and pH adjusters, which can be incorporated within a range that does not impair the effects of the drug (composition) of the present invention. The optical purity of the drug and the D-amino acid used as a raw material is preferably 50% or higher, more preferably 90% or higher; however, any optical purity can be selected within the effective range, and is not limited thereto.

[0032] In one embodiment, the D-amino acid dosage regulator that can be used in the present invention may be selected from the group consisting of D-serine, D-asparagine, and D-glutamine, as well as modified forms and derivatives thereof.

[0033] The present invention may utilize any physiological mechanism to vary the amount of a target D-amino acid. In one embodiment, the amount of a target D-amino acid can be controlled by the expression (promotion, suppression, etc.) or activity (agonism, inhibition, stimulation, etc.) of proteins involved in the absorption, transport, distribution, metabolism (synthesis and degradation), excretion, action, etc. of D-amino acids, such as enzymes (D-amino acid oxidase (DAO), D-aspartate oxidase (DDO), serine isomerase (SRR), DPP-4, etc.), transporters, or receptors. DAO inhibitors (e.g., sodium benzoate, chlorpromazine, risperidone, etc.) suppress the oxidation of D-amino acids, thereby increasing the amount of D-amino acids at the site of action, while inhibitors and activators of D-amino acid transporters increase or decrease the amount of D-amino acids at the source and destination of transport. Drugs that act on proteins such as enzymes and transporters do not necessarily have a direct effect; they may indirectly change the amount of D-amino acids, for example, through competitive reactions between substrates or agonists / antagonists, or through scaffold sharing. In such cases, the effect can be evaluated by measuring the amount of D-amino acids in body fluids, cells, or tissues at the site of action. Furthermore, such evaluations can be used to screen drug candidates.

[0034] By applying the present invention, it is possible to vary the amount of D-amino acids in a living body by utilizing any physiological mechanism, thereby regulating cell proliferation in a subject. In one embodiment, the amount of D-amino acids in a living body can be controlled by adjusting the expression (promotion, inhibition, etc.) and / or activity (activation, inhibition, stimulation, etc.) of proteins involved in the absorption, transport, distribution, metabolism (synthesis and / or degradation), excretion, or action of D-amino acids, or of D-amino acid transporters or receptors.

[0035] Therefore, the regulator of the amount of D-amino acids that can be used in the present invention may directly or indirectly promote the gene expression of a protein involved in the absorption, transport, distribution, metabolism, or excretion of D-amino acids, and may be, for example, the protein or a vector that expresses it, or a factor that promotes the activity upstream of the cascade that promotes the expression of the protein or a vector that expresses it.

[0036] Furthermore, for example, the D-amino acid amount regulator that can be used in the present invention may directly or indirectly suppress the gene expression of a protein associated with the absorption, transport, distribution, metabolism, or excretion of D-amino acids, and may be selected from, for example, a small molecule compound, an aptamer, an antibody, an antibody fragment, an antisense RNA or DNA molecule, an RNAi-inducing nucleic acid, a microRNA (miRNA), a ribozyme, a genome-editing nucleic acid, and an expression vector thereof.

[0037] Herein, proteins involved in the absorption, transport, distribution, metabolism (synthesis and / or degradation), excretion, action, or the like of D-amino acids may be, for example, enzymes, such as D-amino acid oxidase (DAO), D-aspartate oxidase (DDO), serine isomerase (SRR), DPP-4, etc. For example, DAO inhibitors can increase the amount of D-amino acids at the site of action by suppressing the oxidation of D-amino acids, and therefore can be used in the present invention as agents for controlling the amount of D-amino acids.

[0038] Furthermore, since the D-amino acid transporter can increase or decrease the amount of D-amino acid at the transport source or destination, agents that act directly or indirectly on the D-amino acid transporter can also be applied to the present invention.

[0039] Although not intended to be limiting, Non-Patent Document 5 discloses that D-amino acid transporter proteins, such as the SMCT family and ASCT family expressed in the brain and kidney, can change the localized amount of D-amino acids by agonists / inhibitors. Because these transporters are affected by cooperation / competition through cotransporters (e.g., sodium ions) and scaffolds, the transport activity of D-amino acids can also be controlled by, for example, sodium / glucose cotransporter (SGLT2) inhibitors. Furthermore, Patent Document 3 discloses that angiotensin 2 receptor antagonists (ARBs) change the amount of D-amino acids in the blood. For example, by measuring the amount of D-amino acids in culture media, cells, tissues, and body fluids before and after administration of antihypertensive drugs or diabetic nephropathy therapeutic drugs, it is possible to screen for drugs and candidates that can control the amount of D-amino acids in the body.

[0040] As used herein, the term "aptamer" refers to a synthetic DNA or RNA molecule or a peptide molecule that has the ability to specifically bind to a target substance, and can be chemically synthesized in a test tube in a short period of time. The aptamer used in the present invention can bind to, for example, a protein involved in the absorption, transport, distribution, metabolism, or excretion of D-amino acids, and inhibit their activity. The aptamers used in the present invention can be obtained, for example, by using the SELEX method to repeatedly select in vitro the binding to various molecular targets such as small molecules, proteins, and nucleic acids (see Tuerk C., Gold L., Science, 1990, 249(4968), 505-510; Ellington AD, Szostak JW., Nature, 1990, 346(6287):818-822; U.S. Patent No. 6,867,289; U.S. Patent No. 5,567,588; and U.S. Patent No. 6,699,843).

[0041] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody that retains its antigen-binding activity, and generally includes the antigen-binding domain or variable domain. Examples of antibody fragments include F(ab')2, Fab', Fab, or Fv antibody fragments (including scFv antibody fragments). Fragments obtained by treating an antibody with a protease enzyme and optionally reducing it are also included in the antibody fragment category. The antibody or antibody fragment used in the present invention may be any of human-derived antibodies, mouse-derived antibodies, rat-derived antibodies, rabbit-derived antibodies, antibodies from camelids such as llamas, and goat-derived antibodies. Furthermore, these antibodies or antibody fragments may be polyclonal or monoclonal antibodies, complete or truncated antibodies (e.g., F(ab')2, Fab', Fab, or Fv fragments), chimeric antibodies, humanized antibodies, or fully human antibodies.

[0042] As used herein, the term "antisense RNA or DNA molecule" refers to a molecule that has a base sequence complementary to a functional RNA (sense RNA), such as messenger RNA (mRNA), and that, by forming a duplex with the sense RNA, inhibits the synthesis of the protein that the sense RNA is intended to control. In the present invention, antisense oligonucleotides containing antisense RNA or DNA molecules bind to the mRNA of proteins involved in the absorption, transport, distribution, metabolism, or excretion of D-amino acids, thereby inhibiting their translation into protein. This reduces the expression level of proteins involved in the absorption, transport, distribution, metabolism, or excretion of D-amino acids, thereby inhibiting their activity. Methods for synthesizing antisense RNA or DNA molecules are well known in the art and can be used in the present invention.

[0043] As used herein, the term "RNAi-inducing nucleic acid" refers to a polynucleotide capable of inducing RNA interference (RNAi) when introduced into a cell. The polynucleotide is typically an RNA, DNA, or chimeric molecule of RNA and DNA containing 19 to 30 nucleotides, preferably 19 to 25 nucleotides, and more preferably 19 to 23 nucleotides, and is optionally modified. RNAi may occur in mRNA, or in RNA immediately after transcription before processing, i.e., RNA with a nucleotide sequence containing exons, introns, a 3' untranslated region, and a 5' untranslated region. RNAi methods that can be used in the present invention include (1) directly introducing short double-stranded RNA (siRNA) into cells, (2) incorporating small hairpin RNA (shRNA) into various expression vectors and introducing the vector into cells, or (3) creating a vector that expresses siRNA by inserting a short double-stranded DNA corresponding to the siRNA between two promoters arranged in opposing directions, and then introducing the vector into cells. The RNAi-inducing nucleic acid may include siRNA, shRNA, or miRNA that enables cleavage of the RNA of the D-serine transporter protein or suppression of its function, and these RNAi nucleic acids may be directly introduced using liposomes or the like, or may be introduced using an expression vector that induces these RNAi nucleic acids.

[0044] In one embodiment, the RNAi-inducing nucleic acid used in the present invention for a protein associated with D-amino acid absorption, transport, distribution, metabolism, or excretion may be any nucleic acid that exhibits the biological effect of inhibiting or significantly suppressing the expression of a protein associated with D-amino acid absorption, transport, distribution, metabolism, or excretion, and can be synthesized by a person skilled in the art with reference to the base sequence of the protein. For example, the nucleic acid can be chemically synthesized using an automated DNA / RNA synthesizer utilizing DNA synthesis techniques such as the solid-phase phosphoramidite method, or can be synthesized by an siRNA-related contract synthesis company (e.g., Life Technologies, Inc.). In one embodiment, the siRNA used in the present invention may be derived from its precursor, short-hairpin double-stranded RNA (shRNA), via processing by the intracellular RNase Dicer.

[0045] As used herein, "microRNA (miRNA)" refers to a single-stranded RNA molecule 21 to 25 bases long that is involved in post-transcriptional regulation of gene expression in eukaryotes. miRNAs generally recognize the 3'UTR of mRNA to suppress translation of target mRNA and inhibit protein production. Therefore, miRNAs that can directly and / or indirectly reduce the expression level of D-serine transporter proteins are also included within the scope of the present invention.

[0046] As used herein, the term "ribozyme" refers to a general term for an enzymatic RNA molecule capable of catalyzing the specific cleavage of RNA. Ribozymes include those with a size of 400 nucleotides or more, such as group I intron-type ribozymes and M1 RNA contained in RNase P, but there are also those with an active domain of about 40 nucleotides, called hammerhead or hairpin types (see, for example, Koizumi M. and Otsuka E., Protein, Nucleic Acid, Enzymes, 1990, 35, 2191).

[0047] For example, the self-cleaving domain of a hammerhead ribozyme cleaves the 3' side of C15 in the sequence G13U14C15. However, base pairing between U14 and A9 is considered important for its activity, and it has been shown that cleavage can also be achieved with A15 or U15 instead of C15 (see, for example, Koizumi, M. et al., FEBS Lett, 1988, 228, 228). By designing a ribozyme whose substrate binding site is complementary to an RNA sequence near the target site, it is possible to obtain an RNA-cleaving ribozyme that recognizes the UC, UU, or UA sequence in the target RNA like a restriction enzyme, and one skilled in the art can prepare such a ribozyme with reference to the following literature: Koizumi, M. et al., FEBS Lett, 1988, 239, 285; Koizumi, M. and Otsuka, Eiko, Protein, Nucleic Acid, and Enzymes, 1990, 35, 2191. ;Koizumi,M. et al. , Nucl. Acids Res. , 1989, 17, 7059. .

[0048] Hairpin ribozymes can also be used in the present invention. These ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ringspot virus (Buzayan, JM., Nature, 1986, 323, 349). It has been shown that target-specific RNA-cleaving ribozymes can also be produced from hairpin ribozymes (see, for example, Kikuchi, Y. & Sasaki, N., Nucl. Acids. Res., 1991, 19, 6751; Kikuchi, Hiroshi, Chemistry and Biology, 1992, 30, 112). By using ribozymes to specifically cleave the transcript of a gene encoding a D-serine transporter protein, the expression of the D-serine transporter protein can be inhibited.

[0049] As used herein, the term "genome editing nucleic acid" refers to a nucleic acid used to edit a desired gene in a system utilizing a nuclease used in gene targeting. Nucleases used in gene targeting include not only known nucleases but also new nucleases that will be used for gene targeting in the future. For example, known nucleases include CRISPR / Cas9 (Ran, F.A., et al., Cell, 2013, 154, 1380-1389), TALEN (Mahfouz, M., et al., PNAS, 2011, 108, 2623-2628), ZFN (Urnov, F., et al., Nature, 2005, 435, 646-651), and the like.

[0050] In one embodiment, commensal bacteria, including enterobacteria, are one of the living organism's sources of D-amino acids. By utilizing these methods, such as the administration of antibiotics, intestinal regulators, and oligosaccharides, or by using probiotics, microbial transplants, fecal transplants, and amelioration of dysbiosis, the microflora and growth environment can be altered to increase or decrease the amount of D-amino acids in the living organism. While not intended to be limiting, as an example of probiotics, the ingestion of yogurt containing 1073R-1 lactic acid bacteria is known to increase D-serine and decrease D-lysine in feces. However, foods known to contain D-amino acids, such as microbial fermentation products such as black vinegar, yogurt, and cheese, as well as bacterial cells or bacterial cell extracts, contain many potential active ingredients in addition to D-amino acids. Therefore, when used as a D-amino acid amount regulator of the present invention, it is essential to use an amount that allows the increase or decrease in D-amino acid amount at the site of action to be confirmed upon ingestion. In particular, since L-amino acids have various physiological activities different from those of D-amino acids, foods for which specifications (optical purity, amount, etc.) for the active ingredient that increases or decreases D-amino acid amount are not established are not included in the scope of the composition of the present invention.

[0051] As described above, drugs or foods that can increase or decrease the amount of D-amino acids in cells, tissues, organs, or body fluids of a subject, or in a medium for culturing cells, regardless of the mechanism, can be used as any means for controlling the amount of D-amino acids applicable to the present invention.

[0052] In this specification, the term "drug" is used to include pharmaceutical products and quasi-drugs.

[0053] In this specification, "food" refers to food in general, but also includes general foods including so-called health foods, as well as health functional foods such as foods for specified health uses and foods with nutrient functions, and further includes dietary supplements (supplements, nutritional supplements), feed, food additives, etc., in the food of the present invention.

[0054] The administration method of a drug or the like may be provided in a dosage form suitable for local administration (on the skin, inhalation, enema, eye drop, ear drop, nasal administration, intravaginal administration, etc.), enteral administration, or parenteral administration (intravenous, intraarterial, transdermal, intramuscular injection, etc.), but enteral administration is preferred. Enteral administration includes oral administration, enteral tube administration, and enema administration. Enteral administration includes administration via a nasogastric tube, gastrostomy, or duodenal fistula. Enema administration includes administration using a suppository or enema. In either case, the dosage form of the drug is not particularly limited, and may be liquid or solid, and can be prepared according to the common general technical knowledge of those skilled in the art. The specific administration method is also not particularly limited, and can be suitably administered according to the common general technical knowledge of those skilled in the art.

[0055] Subjects to which the present invention can be applied include cultured cells, tissues, and organoids in any environment different from that in vivo, as well as patients with or suspected of having cell proliferation, abnormal organ function, or organ atrophy due to disease or injury. Organ transplants can be performed on the liver, pancreas, kidney, digestive tract, heart, eyeball, etc., but in many cases, both the donor from which the organ is removed and the recipient of the organ experience organ damage and functional impairment accompanied by abnormal cell proliferation before and after surgery, so the present invention can be applied to such subjects. Organ damage, for example, in the kidney, is classified by the Japanese Association for the Surgery of Trauma into type I (subcapsular injury), type II (superficial injury), and type III (deep injury) (Non-Patent Document 11), and the present invention can be applied to any of these subjects. In kidney transplants, the donor loses one of their two kidneys, reducing total kidney size by approximately half and reducing renal function by half. However, the remaining kidney increases in size to compensate for the lost function. It is known that the kidney size and function of patients undergoing dialysis treatment are reduced compared to healthy individuals. By increasing the amount of D-amino acids, the present invention can support the regeneration of cells, tissues, and organs, and also improve the glomerular filtration rate, one of the renal functions. Therefore, in one aspect, subjects to which the present invention can be applied include subjects with kidney disease, kidney transplant donors and / or recipients, and / or dialysis patients, or patients undergoing renal replacement therapy. Furthermore, the present invention can also be applied to animals in which abnormalities in cell proliferation have been induced by genetic modification or drugs (e.g., unilaterally nephrectomized mice, etc.) and cultured cells and tissues (e.g., cancer model cells and tissues, stem cells, differentiated cells and tissues, etc.).

[0056] In one embodiment of the present invention, the amount of D-amino acids in a cell culture medium or the amount of D-amino acids in target cells, tissues, and / or body fluids affects cell proliferation. Therefore, measuring these D-amino acid amounts can serve as an indicator of the state of abnormal cell proliferation and the effectiveness of treatment. For example, in the kidney, monitoring the amount of D-amino acids in vivo and the glomerular filtration rate can aid in the diagnosis and evaluation of cell proliferation and functional abnormalities, analysis of drug mechanisms of action and screening of efficacy and toxicity, selection of treatment methods and drugs, and determination of dosage and duration. Since the amount of D-amino acids in body fluids is affected by other diseases, values ​​corrected for D-amino acid clearance or D-amino acid amount using renal function markers such as creatinine or other markers may be used in analysis to distinguish between these diseases.

[0057] One embodiment of the present invention provides a method for modulating cell proliferation in a subject, comprising administering to a subject in need thereof an agent for regulating the amount of D-amino acids in the body.

[0058] Another embodiment of the present invention provides a method for regulating cell proliferation in vitro or ex vivo, comprising the step of applying a D-amino acid level regulator to cells, biological tissues, or organs and culturing them. For example, cell proliferation can be regulated by culturing cells, biological tissues, or organs in an in vitro or ex vivo environment using a buffer (e.g., medium) or physiological saline containing the D-amino acid level regulator. The buffer that can be used in the present invention may be any known buffer that can be used for culturing, protecting, or preserving cells, biological tissues, or organs.

[0059] Furthermore, one embodiment of the present invention provides use of an agent for controlling the amount of a D-amino acid for the manufacture of a pharmaceutical composition for regulating cell proliferation.

[0060] As described above, the method for regulating cell proliferation by controlling the amount of D-amino acids is extremely useful not only for efficient cell culture but also for the prevention, treatment and diagnosis of abnormalities in biological tissues and organs.

[0061] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. In the examples, the following reagents were used unless otherwise specified.

[0062] ・ Anti-phospho-p70 S6 kinase (Thr389) antibody (#9234; 1:2000 IB) (Cell Signaling Technology (Danvers, USA)) ・ Anti-p70 S6 kinase antibody (#2708; 1:2000 IB) (Cell Signaling Technology (Danvers, USA)) ・ Phospho-S6 ribosomal protein (Ser235 / 236) antibody (#4858, 1:2000 IB) (Cell Signaling Technology (Danvers, USA)) ・ Anti-S6 ribosomal protein antibody (#2217, 1:2000 IB) (Cell Signaling Technology (Danvers, USA)) ・ Anti-phospho-AKT (Ser473) antibody (#9271; 1:2000 IB) (Cell Signaling Technology (Danvers, USA)) ・ Anti-AKT antibody (#9272; 1:2000 IB) (Cell Signaling Technology (Danvers, USA)) ・ Anti-p18 LAMTOR1 / C11orf59 antibody (#8975; 1:2000 IB) (Cell Signaling Technology (Danvers, USA)) ・ Anti-mTOR antibody (#2983; 1:1000 IF) (Cell Signaling Technology (Danvers, USA)) ・ HRP-labeled anti-mouse IgG antibody (#7076; 1:5000 IB) (Cell Signaling Technology (Danvers, USA)) ・ HRP-labeled anti-rabbit IgG antibody (#7074; 1:5000 IB (Cell Signaling Technology (Danvers, USA)) ・ Anti-Rheb antibody (sc-271509; 1:2000 IB) (Santa Cruz Biotechnology, Dallas, USA) ・ Anti-LAMP1 antibody (sc-19992; 1:1000 IF) (Santa Cruz Biotechnology, Dallas, USA) ・ Anti anti-β-Actin antibody (A-5316;1:20000 IB) (Sigma-Aldrich, St. Louis, USA) - Anti-Ki67 antibody (#718071; 1:1 IHC) (Nichirei Biosciences, Tokyo, Japan) - Alexa Fluor 488-labeled secondary antibody (A-32790; 1:1000 IF) (Invitrogen, Carlsbad, USA) - Alexa Fluor 594-labeled secondary antibody (A-21207, 1:1000 IHC; and A-11007, 1:1000 IF) (Invitrogen, Carlsbad, USA) - Anti-LAMP2 antibody (ab25631; 1:200 IF) (abcam, Cambridge, UK) - Alexa Fluor 594-labeled secondary antibody (ab150116; 1:1000 IF) (abcam, Cambridge, UK). Insulin 8 (I9278-5ML, Sigma-Aldrich), rapamycin (R0161, LKT Laboratories, St. Paul, USA), Ly294002 (129-04861, Fujifilm Wako Chemicals Corporation, Osaka, Japan), L-serine (#2719) and D-serine (#2818, Peptide Institute, Ibaragi, Japan), L-leucine (124-00852, Fujifilm Wako);

[0063] Example 1. Cell proliferation ability of D-amino acids in TIG-1 cells

[0064] TIG-1 cells isolated from fetal tissue were plated on a 96-well plate at a density of 1.5 x 10 viable cells per well. ４ The cells were seeded in DMEM high glucose + 10% FBS medium overnight in 5% CO ２ The cells were cultured at 37°C for 18 hours in 5% CO with DMEM amino acid-free medium + 0.5% dialyzed FBS. ２90 μl of DMEM amino acid-free medium + 0.5% dialyzed FBS was added to each well, and 10 μl of each concentration of D-amino acid, L-amino acid, and conjugated amino acid solution dissolved in PBS was added. ２ The cells were cultured at 37°C for 6 hours. Then, 10 μl of Cell Counting Kit-8 was added, and the cells were cultured under 5% CO ２ After incubation at 37°C for 6 hours, the absorbance was measured at a wavelength of 450 nm.

[0065] When 10 μM of each amino acid was added to amino acid-free medium, L-serine (L-Ser) had no cell proliferation effect, but D-serine (D-Ser) had a significant cell proliferation effect. Alanine (Ala), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), valine (Val), and lysine (Lys) all had similarly significant proliferation effects in both DL and L forms. D-methionine (D-Met) had no cell proliferation effect, but L-methionine (L-Met) had a significant cell proliferation effect (Figure 1).

[0066] When 1 μM of amino acids was added to an amino acid-free medium, L-Asn and L-Gln had no effect on cell proliferation, but D-Asn and D-Gln had a significant effect on cell proliferation (FIG. 2).

[0067] When 10 μM of conjugated amino acids (dipeptides) were added to the amino acid-free medium, D-serine-glycine (dSG) and glycine-D-serine (GdS) showed cell proliferation effects (FIG. 3).

[0068] Example 2. Amino acid administration and organ distribution

[0069] 12-week-old Balb / c male mice (SLC, Tokyo) ３H-labeled D-serine and L-serine solutions were intravenously injected, and blood, spleen, heart, kidney, lung, pancreas, liver, brain, and intestinal tissues were collected 5, 30, 80, and 180 minutes later. After homogenization, radioactivity was counted (cps: counts per second) using Ecoscint XR (National Diagnostics, Atlanta, GA) and a scintillation counter LSC 5100 (Hitachi, Tokyo, Japan). Radioactivity concentrations were converted to kBq / g measurement units using a cross-9 calibration coefficient, corrected for the injection dose (MBq) and body weight, and calculated as standardized uptake values. The data were statistically analyzed using standardized average values ​​(SUV) (n=4-5, mean ± SEM, *p<0.05, **p<0.01, ***p<0.001). The results showed that the administered D-serine and L-serine were distributed to each organ through specific pharmacokinetics, and that the amino acid levels could be changed over time (Figure 4).

[0070] Example 3. D-serine administration and kidney size in unilaterally nephrectomized mice

[0071] Ten-week-old C57BL / 6 male mice (SLC, Tokyo) were fed a serine-free diet and water containing (D-Ser) or not containing (Vehicle) 0.1% D-serine. Unilateral nephrectomy (UNX) and sham surgery were performed. Nephrectomy was performed by exposing the renal pedicle of the left kidney of anesthetized mice through a dorsal incision and ligating it with silk suture. Sham surgery was performed by simply performing a similar incision (T. Kimura, Y. et al., J Am Soc Nephrol 22, 902-913 (2011)). Two days later, kidney weight per body weight and plasma D-serine and L-serine levels were measured and statistically analyzed (n = 6-7; two-way ANOVA, *p<0.05, **p<0.01, ***p<0.001). Plasma D-serine levels were higher in the D-Ser administration group compared to the vehicle group, and kidney size (kidney weight per body weight) increased (Figure 5). This provides a model for cell proliferation, tissue regeneration, and functional recovery of the donor's remaining kidney in kidney transplantation.

[0072] Example 4. D-serine administration and expression of cell cycle-related genes

[0073] Ten-week-old C57BL / 6 male mice (SLC, Tokyo) were fed a serine-free diet and water containing (D-Ser) or not containing (Vehicle) 0.1% D-serine. Unilateral nephrectomy (UNX) and sham surgery were performed. Two days later, samples were subjected to RNA-seq analysis (n=3). RNA was extracted using TRIzol (15596018, Thermo Fisher Scientific, Waltham, USA). Library preparation was performed using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA). Sequencing was performed on an Illumina HiSeq2500 platform in 75-base single-end mode, and base calling was performed using Illumina Casava 1.8.2 software. Sequencing reads were mapped to the mouse reference genome sequence (mm10) using TopHat version 2.0.13 in combination with Bowtie2 version 2.2.3 and SAMtools version 0.1.19, and fragments per kilobase of exon per million mapped (FPKM) were calculated using Cufflinks version 2.2.1.

[0074] RNA-seq data was analyzed using TargetMine (Y. A. Chen, et al., PLoS One 6, e17844 (2011)), an integrated warehouse of human and mouse biological data from sources such as Reactome and KEGG, and pathway and gene ontology analyses were performed. Genes that were upregulated by more than two-fold (2-fold change) in D-serine-treated mice compared to vehicle-treated mice were uploaded to TargetMine. Reactome pathway and Gene Ontology (GO) (M. Ashburner, et al., Nat Genet 25, 25-29 (2000)) enrichment was assessed by hypergeometric distribution and estimated p-values, and multiple testing correction was performed to suppress false positives using the Benajmini and Hochberg method (W. S. Noble, et al., Nat Biotechnol 27, 1135-1137 (2009)). Heatmaps were plotted using R.

[0075] The analysis data showed that D-serine administration activated cell cycle-related pathways (Figure 6). Upregulated genes included cyclins, cell cycle activation family proteins, and CDC20, a key regulator of mitotic exit via cyclin degradation. These results were reproduced by qPCR (Figure 7). Increased D-serine levels were associated with physiological activities that promote and regulate cell proliferation.

[0076] Example 5. D-serine administration and kidney cell proliferation

[0077] In the same manner as in Example 3, 10-week-old C57BL / 6 male mice (SLC, Tokyo) were fed a serine-free diet and water containing (D-Ser) or not containing (Vehicle) 0.1% D-serine. They underwent uninephrectomy (UNX) and sham surgery (Sham). Two days later, the processed specimens were subjected to histological analysis.

[0078] For histological analysis, mice were perfused with saline, and the excised kidneys were sectioned, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections were stained with periodic acid-Schiff (PAS) staining or immunostained with anti-Ki67 antibody and Histofine Simple Stain Rat MAX PO® (414181F, Nichirei Biosciences) (T. Kimura, et al., Y. J Am Soc Nephrol 22, 902-913 (2011)). At least 10 fields (x200) were reviewed by a nephrologist blinded to the experimental conditions to count Ki67-positive cells per image. For immunofluorescence, paraffin sections were incubated with Ki67 antibody and Alexa594-conjugated secondary antibody. Proximal tubules were stained using FITC-conjugated lectin from Triticum vulgaris (L4895-5MG, Sigma-Aldrich), and nuclei were counterstained with DAPI. Images were captured using a fluorescence microscope (Axio Observer) and a digital camera (AxioCam506 Mono and AxioCamMRc, ZEISS, Oberkochen, Germany). All images were processed using ZEN 2 pro software (ZEISS) and Image J (NIH).

[0079] Analysis of PAS-stained kidneys showed that the number of Ki67-positive nuclei in the renal tubules of UNX mice treated with D-serine was increased compared with those treated with vehicle, indicating that D-serine promoted cell proliferation (Figure 8). Cell proliferation was observed in the proximal tubules of the kidney, which are the main area of ​​D-serine reabsorption (Figure 9), and the glomerular area tended to expand with D-serine administration (Figure 10).

[0080] Example 6. D-serine addition, cultured cell proliferation, and mTOR-related metabolic regulation

[0081] HK-2 (CRL-2190, ATCC, Manassas, USA) and human primary renal proximal tubule cells (RPTEC; CC-2553, Lonza, Basel, Switzerland) were cultured in the recommended medium. HeLa cells (JCRB9004, JCRB Cell Bank, Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM, 08458-16, Nacalai Tesque, Kyoto, Japan) containing 10% FCS (10270-106, Gibco, Carlsbad, USA). p18-deficient MEFs (p18 KO) and their revertant mutants (Rev), as well as p18-expressing Rheb and p18-double-deficient MEFs (Rheb KO) were cultured as described in S. See S. Nada, et al., EMBO J 28, 477-489 (2009), and S. Nada, et al., J Biochem, (2020). In experiments using Rheb KO cells, a p18 revertant was used as a control (RhebWT).

[0082] For proliferation assays, cells were seeded in 96-well plates using the medium. The next day, the medium was replaced with 0.5% dialyzed FCS (26400-036, Gibco) and serine-free medium (amino acid-free DMEM [048-33575] supplemented with MEM essential amino acids [132-15641], glycine [073-00732], and GlutaMax (L-alanyl-L-glutamine) [016-21841, Fujifilm Wako]). After overnight incubation, D-serine and L-serine were added, respectively, and relative cell numbers were measured over time using a WST-8 kit (CK04, Dojindo Laboratories, Kumamoto, Japan). In experiments using MEFs, type I collagen-coated microplates (4860-010, AGC Technoglass, Haibara, Japan) were used.

[0083] For immunoblotting, cells were lysed on ice for 1 hour in RIPA buffer (89901, Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (4693132001, Roshe) with or without phosphatase inhibitors (4906837001, Roshe, Basel, Switzerland), and then centrifuged at 15,000 xg for 10 minutes. The supernatant was boiled for 3 minutes in SDS-PAGE gel loading buffer, separated by SDS-PAGE, transferred to a PVDF membrane, and subjected to Western blot analysis (T. Kimura, et al., EMBO J 36, 42-60 (2017)).

[0084] Compared to the serine-free medium group, the addition of D-serine promoted cell proliferation at low concentrations in human renal tubular cell lines and primary cells (Figure 11). Addition of D-serine enhanced Thr389 phosphorylation of S6K in HK-2 cells (Figure 12). Furthermore, addition of D-serine induced Ser235 / 236 phosphorylation of S6RP in the kidneys of UNX mice (Figure 13). Thus, D-serine enhanced the signal leading to mTORC1 activation. The effect of D-serine on the mTOR pathway was also confirmed by the suppression of D-serine-induced cell proliferation in p18-deficient cells (Figure 14) (R. Yonehara, et al. Nat Commun 8, 1625 (2017)). The cell proliferation effect of D-serine was suppressed in Rheb-deficient cells, suggesting that the effect of D-serine is also mediated through insulin / PI3K signaling (Figure 15) (R. A. Saxton, et al. Metabolism, and Disease. Cell 168, 960-976 (2017)). Inhibition of mTOR using rapamycin or the PI3K inhibitor Ly294002 also suppressed D-serine-induced cell proliferation (Figure 16). Treatment with D-serine induced Ser473-phosphorylation of AKT in the kidneys of UNX mice (see Example 3) (Figure 17). Furthermore, treatment with D-serine suppressed the dissociation of mTOR from lysosomes (Figure 18), suggesting that D-serine supports the lysosomal localization of mTOR. These results demonstrate that D-serine and mTOR cooperate and cooperate in cell proliferation (FIG. 19).

[0085] Example 7. D-serine administration and glomerular filtration rate

[0086] Eighteen-week-old serine racemase knockout rats were given water containing 0.1-0.5% D-serine (D-Ser) or not (Vehicle) ad libitum for one month, after which glomerular filtration rate (GFR) was measured by measuring the clearance of intravenously administered FITC-sinistrin. The D-serine group (n = 8) showed an average increase of approximately 15% in GFR compared with the Vehicle group (n = 2) (Figure 20).

[0087] Example 8. Cell proliferation effect of D-amino acids in B cells

[0088] For B cell line proliferation assays, IL-3-dependent murine pro-B cell line Ba / F3 cells were seeded in 96-well plates using alanine-free medium (amino acid-free RPMI, MEM essential amino acids, GlutaMax (l-alanyl-l-glutamine), and 5% (v / v) dialyzed FCS (26400-036, Gibco)) in the presence or absence of 100 ng / 10 μM D-alanine. Cells were cultured for 48 hours. Relative cell numbers were measured using a WST-8 kit (CK04, Dojindo Laboratories, Kumamoto, Japan), and a t-test was performed.

[0089] The presence of D-alanine was shown to significantly proliferate B cells (Figure 21).

Claims

1. A composition for regulating cell growth, comprising an agent for controlling the amount of D - amino acid.

2. The composition according to claim 1, wherein the controlling agent is an agent for controlling the amount of D - amino acid in a subject's living body.

3. The composition according to claim 1 or 2, wherein the cell growth is cell growth of a living tissue and / or organ, thereby adjusting the size of the living tissue and / or organ.

4. The composition according to claim 2, wherein the subject is a subject having kidney disease.

5. The composition according to claim 2, wherein the subject is a donor and / or recipient of a kidney transplant.

6. The composition according to claim 2, which improves kidney function.

7. The composition according to claim 6, wherein the kidney function is glomerular filtration rate.

8. The composition according to claim 1, for regulating the growth of isolated cells.

9. The composition according to claim 1 or 2, wherein the controlling agent is D - amino acid or its derivative.

10. The composition according to claim 9, wherein the D - amino acid is selected from the group consisting of D - serine, D - asparagine, and D - glutamine.

11. The composition according to claim 1 or 2, wherein the controlling agent is an agent for adjusting the activity of a protein related to the absorption, transport, distribution, metabolism, or excretion of D - amino acid.

12. The composition according to claim 11, wherein the metabolism is decomposition or synthesis.

13. The composition according to claim 11, wherein the agent for adjusting the activity of the protein is an agent for controlling the gene expression of the protein.

14. The composition according to claim 11, wherein the protein is selected from the group consisting of D - aspartate oxidase and serine isomerase.

15. The composition according to claim 11, wherein the protein is a D - amino acid transporter protein.

16. The D-amino acid transporter protein is selected from one or more of the group consisting of the SMCT family, GLUT5, CAT1, THTR2, SNAT2, the ASCT family, Asc1, PAT1, and ATB ０，＋ The composition according to claim 15, wherein the D-amino acid transporter protein is selected from one or more of the group consisting of the SMCT family, GLUT5, CAT1, THTR2, SNAT2, the ASCT family, Asc1, PAT1, and ATB

17. The composition according to claim 1 or 2, which is a pharmaceutical.

18. The composition according to claim 1 or 2, which is a food.

19. The composition according to claim 18, wherein the food is a functional food or a dietary supplement.

20. The composition according to claim 19, wherein the functional food is a food for specified health use or a food with nutritional function.

21. The composition according to claim 1 or 2, which activates the mTOR - related pathway.

22. A composition for activating the mTOR - related pathway, comprising an agent for controlling the amount of D - amino acid.

23. A method for regulating cell proliferation in vitro or ex vivo, comprising: applying a controlling agent for the amount of D-amino acid to cells, biological tissues or organs and culturing them; A method comprising the above steps.

24. Use of a controlling agent for the amount of D-amino acid for the manufacture of a pharmaceutical composition for regulating cell proliferation.