WO2022244846A1

WO2022244846A1 - Composition for regulating cell proliferation and regulation method of cell proliferation

Info

Publication number: WO2022244846A1
Application number: PCT/JP2022/020851
Authority: WO
Inventors: 友則木村; 篤部坂; 悠介塚本; 真史三田
Original assignee: Kagami株式会社; 国立研究開発法人医薬基盤・健康・栄養研究所
Priority date: 2021-05-19
Filing date: 2022-05-19
Publication date: 2022-11-24
Also published as: JPWO2022244846A1

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

Compositions and methods for modulating cell proliferation

The present invention relates to a composition for modulating cell proliferation and a method for modulating cell proliferation.

Cells are generally recognized as the most basic building blocks of living organisms. They have organs responsible for metabolism such as the glycolytic system and the citric acid cycle for constant life activities, and contain genetic information for self-renewal and replication. It has the ability to express it. Humans are composed of more than 30 trillion somatic and germ cells. There are about 200 types of somatic cells that account for most of them, and a proliferating dividing cell group that constantly divides and proliferates (e.g., myeloblasts, basal cells, etc.), a differentiated dividing cell group that divides and proliferates while differentiating (e.g., myeloid cells, neuroblasts, myoblasts, etc.), reversible postmitotic cell groups that do not normally proliferate (e.g., stem cells, smooth muscle cells, lymphocytes, etc.), fixed postmitotic cell groups that have lost their ability to divide (nerve cells, myocardial cells, etc.) cells, red blood cells, etc.).

In physiologically regenerative tissue, cells that repeat regeneration, function, and death under normal conditions (e.g., epithelial cells, blood cells, etc.). Cells that proliferate and regenerate under special circumstances (e.g., organ parenchymal cells, vascular endothelial cells, connective tissue, etc.), and highly organized and differentiated cells that do not have the ability to spontaneously generate and proliferate in non-regenerative tissues. There are cell types such as (eg, nerve cells, skeletal muscle, cardiomyocytes, etc.).

Cells enter a state of cell death when they can no longer proliferate due to the mitotic lifespan and differentiation lifespan caused by telomeres, and the sum of these events is considered to be the aging of an individual. In regenerative cells, the total number of cells decreases due to apoptosis, and as the burden per cell increases, further apoptosis is promoted, and the number of parenchymal cells in tissues and organs decreases (atrophy), resulting in decreased function. . A tumor is a cell that autonomously proliferates against normal control due to some abnormality in the gene of the cell, and a malignant tumor (cancer) is a tumor that invasively proliferates and metastasizes. Cell fusion, gene introduction into cells, and cell culture are techniques for controlling and modifying genetic and physiological functions by artificially manipulating cell mechanisms and abnormalities. It is used for research and development of regenerative medicine and pharmaceuticals. In cell/tissue culture, differentiation/proliferation is controlled by setting conditions such as temperature, atmosphere, and medium components (glucose, growth factors (e.g., epidermal growth factor, insulin-like growth factor, etc.), nutrients, etc.). , Non-Patent Document 1 describes that the metabolism and regulation of amino acids regulates the proliferative potential of human pluripotent stem cells.

In recent years, due to the advancement of technology for identifying and analyzing chiral amino acids, quantitative research on trace amounts of D-amino acids and L-amino acids in living organisms such as mammals has progressed. The existence and functions of some D-amino acids that have been treated as total amino acids (D-amino acids + L-amino acids) or L-amino acids for convenience have been clarified. D-amino acids are affected by ingestion, symbiotic bacteria, metabolism (decomposition, synthesis), transportation, excretion, etc. (Non-Patent Documents 2 to 6), and the amount in living organisms, tissues, cells, and body fluids changes, and kidney disease , showing a chiral amino acid profile characteristic of diseases such as heart disease and diabetes (Patent Document 1), furthermore, the involvement of D-amino acids in intestinal immunity (Non-Patent Document 7) and protecting kidney-derived cells A phenomenon has been reported (Non-Patent Document 3). There is also a report that carbohydrate metabolism is involved in D-serine biosynthesis in nerve cells (Non-Patent Document 8).

It has been disclosed that the amounts of D-serine and D-alanine in the blood vary in renal cancer, and the amounts of D-alanine, D-proline and D-aspartic acid in the blood vary in diabetes (Patent Document 1. , Patent Document 2). It has also been reported that D-alanine is localized in cells containing insulin in pancreatic islets of Langerhans and 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 elucidated.

WO2013/140785 JP 2017-207490 A WO2020/196436

There is a strong need for methods and drugs to more efficiently proliferate cells, restore, maintain, and enhance functions by regenerating abnormal cells, tissues, and organs inside and outside the body.

The inventors adjusted the amount of D-amino acid and L-amino acid in the medium in primary or subcultures of renal tubular cells and fibroblasts, and found that D-amino acids promoted cell proliferation more than L-amino acids. I discovered that it works. As a result of intensive research on its effect and mechanism, a method for adjusting the growth and regeneration of cells, tissues and organs inside and outside the body, especially conditionally regenerative tissues, was found by increasing or decreasing the amount of D-amino acids. Arrived. That is, the present invention includes the following inventions.

[1] A composition for regulating cell growth, comprising an agent for controlling the amount of D-amino acids.
[2] The composition according to item 1, wherein the control agent is an agent for controlling the amount of D-amino acids in the subject's living body.
[3] The composition according to

item

1 or 2, wherein the cell proliferation is cell proliferation in a living tissue and/or organ, thereby adjusting the size of the living tissue and/or organ.
[4] The composition according to item 2, wherein the subject is a subject with kidney disease.
[5] The composition according to item 2, wherein the subject is a kidney transplant donor and/or recipient.
[6] The composition according to item 2, which improves renal function.
[7] The composition according to item 6, wherein the renal function is glomerular filtration rate.
[8] A composition according to item 1 for modulating the growth of isolated cells.
[9] The composition according to any one of items 1 to 8, wherein the control agent is a D-amino acid or a derivative thereof.
[10] The composition according to 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 control agent is an agent that modulates protein activity related to absorption, transport, distribution, metabolism or excretion of D-amino acids.
[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 that modulates the activity of the protein is an agent that regulates 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] Item 15, wherein the D-amino acid transporter protein is one or more selected from the group consisting of SMCT family, GLUT5, CAT1, THTR2, SNAT2, ASCT family, Asc1, PAT1 and ATB ^0,+ Composition.
[17] The composition according to any one of items 1 to 16, which is a pharmaceutical.
[18] The composition according to any one of Items 1 to 16, which is a food.
[19] The composition according to item 18, wherein the food is a food with health claims or a dietary supplement.
[20] The composition according to item 19, wherein the food with health claims is a food for specified health uses or a food with nutrient claims.
[21] The composition according to any one of items 1 to 20, which activates an mTOR-related pathway.

[22-1] A composition that activates an mTOR-associated pathway, comprising a regulator of the amount of D-amino acids.
[22-2] The composition according to item 22-1, wherein the control agent is an agent for controlling the amount of D-amino acid 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 living tissue and/or organ, thereby adjusting the size of the living tissue and/or organ. thing.
[22-4] The composition of item 22-2, wherein the subject is a subject with renal 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 of item 22-2, which improves renal function.
[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 for modulating growth of isolated cells.
[22-9] The composition according to any one of items 22-1 to 22-8, wherein the control agent 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] any one of items 22-1 to 22-8, wherein the control agent is an agent that modulates protein activity related to D-amino acid absorption, transport, distribution, metabolism or excretion; The described composition.
[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 modulates the activity of the protein is an agent that regulates 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] Item 22-, wherein the D-amino acid transporter protein is one or more selected from the group consisting of SMCT family, GLUT5, CAT1, THTR2, SNAT2, ASCT family, Asc1, PAT1 and ATB ^0,+ 16. The composition according to 15.
[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, wherein the food is a food with health claims or a dietary supplement.
[22-20] The composition according to item 22-19, wherein the food with health claims is a food for specified health uses or a food with nutrient claims.

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

[24] A method of modulating cell proliferation in vitro or ex vivo, comprising:
A method comprising applying a D-amino acid amount controlling agent to a cell, biological tissue or organ and culturing.

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

According to the present invention, by controlling the amount of D-amino acids, it is possible to regulate cell proliferation. It becomes possible to treat or evaluate.

FIG. 1 shows the effect of D, L-amino acids (10 μM) on cell growth in culture. FIG. 2 shows the effect of D, L-amino acids (1 μM) on cell growth in culture. FIG. 3 shows the effects of glycylserine isomers (10 μM) on cell growth in culture. FIG. 4 shows administration of D, L-amino acids and uptake into each organ. Ditto. FIG. 5 shows the effect of D-serine administration on increasing kidney size. n=6-7. Statistical analysis was performed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Data are shown as mean ± SEM. FIG. 6 shows the results of pathway analysis on the effects of D-serine administration. Ditto. FIG. 7 shows the results of D-serine administration and cell cycle-related gene expression analysis. (A) Relative mRNA expression levels. n=6-7. Statistical analysis was performed with an 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 by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. FIG. 8 shows Ki67 positive nuclei analysis by D-serine administration. (A) Representative images of renal cortex stained with anti-Ki67 antibody. The lower left figure in each figure is an enlarged view of the area enclosed by the rectangle in the figure. Scale bar: 50 μm. (B) Number of Ki67-positive nuclei in proximal tubules 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 by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Data are shown as mean ± SEM. FIG. 9 shows the effect of D-serine administration on proliferation of proximal tubule cells. Lectin-positive proximal tubule: green, Ki67: red, DAPI (nucleus): blue. Scale bar: 50 μm. FIG. 10 shows the effect of D-serine administration on glomerular cell proliferation. Relative glomerular area (%) is shown. Statistical analysis was performed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Data are shown as mean ± SEM. FIG. 11 shows the proliferative effect of D-serine administration on renal tubular cells and HK-2 cells. (A) Normal human primary renal proximal tubule cells (RPTEC) or (B) HK-2 cells cultured with 10 μM D-serine relative proliferation rate (%) over time. (C) Normal human primary renal proximal tubule cells (RPTEC) or (D) HK-2 cells cultured with various concentrations of D-serine relative proliferation rate (%). (E) Normal human primary renal proximal tubule cells (RPTEC) or (F) HK-2 cells cultured with each concentration of D-serine or L-serine relative proliferation rate (%). Statistical analysis was performed by one-way ANOVA using Dunnett's post-hoc test. NS: not significantly different, *p<0.05, **p<0.01, ***p<0.001. FIG. 12 shows the results of phosphorylation analysis of HK-2 cells by D-serine administration. (A) Western blot results of phosphorylation of S6K at Thr389 (phospho-S6K (p-S6K)) of HK-2 cells treated with 5 μM D- or L-serine for 10 min in serine-free medium. show. (B) Plot of the relative index (RI) of five independent experiments. Statistical analysis was performed by one-way ANOVA using Dunnett's post-hoc test. NS: not significantly different, *p<0.05, **p<0.01, ***p<0.001. FIG. 13 shows the results of phosphorylation analysis of mouse kidney cells by administration of D-serine. (A) Fed on a serine-free diet and water with or without 0.1% D-serine for 1 week and sacrificed 2 days after unilateral nephrectomy (UNX) or not. Immunoblot of phospho-S6 ribosomal protein (p-S6RP) at Ser235/236 from renal cortex of week-old C57 BL/6 male mice. (B) Plotted the relative index (RI) of three independent experiments. Statistical analysis was performed by one-way ANOVA using Dunnett's post-hoc test. NS: not significantly different, *p<0.05, **p<0.01, ***p<0.001. FIG. 14 shows the effect of D-serine administration on cell proliferation in p18-deficient strains. (A) p18-deficient mouse embryonic fibroblasts (MEFs) and their revertants (Rev) treated with the indicated concentrations of D-serine for 6 hours using serine-free medium. and relative growth rates of the corresponding cell lines treated with vehicle. N=6. Statistical analysis was performed by two-way ANOVA. ***p<0.001. (B) Immunoblot of p18 proteins of p18-deficient MEFs and their revertants. FIG. 15 shows the effect of D-serine administration on cell proliferation in Rheb-deficient strains. (A) Relative growth rates of Rheb-deficient or wild-type MEFs treated with the concentrations of D-serine indicated in the figure for 6 h using serine-free medium, as well as that of the vehicle-treated corresponding cell lines. relative growth rate. N=12. Statistical analysis was performed by two-way ANOVA. ***p<0.001. (B) Rheb immunoblot of Rheb-deficient- and wild-type MEFs. FIG. 16 shows cell proliferation effects by administration of D-serine and rapamycin or PI3 inhibitors. (A) Relative growth rates of HK-2 cells treated with or without rapamycin for 24 hours using serine-free medium and treated with the indicated concentrations of D-serine for 6 hours, and D - Relative growth rate of corresponding cell lines not treated with serine. N=21-24. Statistical analysis was performed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. (B) Relative growth rates of HK-2 cells treated simultaneously with or without the indicated concentrations of D-serine and Ly294002 for 6 hours using serine-free medium and D-serine untreated. relative growth rate of the corresponding cell lines. N=21-24. Statistical analysis was performed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. FIG. 17 shows the results of AKT phosphorylation analysis by D-serine administration. (A) Fed on a serine-free diet and water with or without 0.1% D-serine for 1 week and sacrificed 2 days after unilateral nephrectomy (UNX) or not. Immunoblot results for phospho-AKT (p-AKT) at Ser473 from renal cortex of week-old C57 BL/6 male mice are shown (left). (B) Plotted the relative index (RI) of three independent experiments. Statistical analysis was performed by one-way ANOVA using Dunnett's post-hoc test. NS: not significantly different, *p<0.05, **p<0.01, ***p<0.001. FIG. 18 shows localization analysis of mTOR lysosomes by D-serine administration. (A) High-content microscopy quantification of co-localization of mTOR and LAMP2 in HK-2 cells that were incubated in culture medium followed by amino acid starvation in the presence of 5 μM D- or L-serine for 10 minutes. Mask overlay, primary object (algorithm-defined cell boundaries based on CellMask (red pseudocolor)), and internal secondary object (computationally defined co-localization between mTOR (green) and LAMP2 (yellow)). Statistical analysis was performed by one-way ANOVA using Bonferroni's post-hoc test. NS: not significantly different, *p<0.05, **p<0.01, ***p<0.001. (B) Quantification by high-content microscopy of co-localization of mTOR and LAMP1 in HK-2 cells that were incubated in culture medium followed by amino acid starvation in the presence of 5 μM D- or L-serine for 10 minutes. Mask overlay, primary object (algorithm-defined cell boundaries based on CellMask (red pseudocolor)), and internal secondary object (computationally defined co-localization between mTOR (green) and LAMP1 (yellow)). Statistical analysis was performed by one-way ANOVA using Bonferroni's post-hoc test. NS: not significantly different, *p<0.05, **p<0.01, ***p<0.001. FIG. 19 shows a schematic representation of the activation scheme of mTOR-related pathways by D-serine. (A) Schematic showing that D-serine activates cell proliferation through the mTOR-related pathway (mTOR/p18/Rheb). D-serine can mediate renal remodeling after unilateral nephrectomy. (B) Schematic representation of activation of mTORC1 signaling by D-serine. D-serine enhances the signal from L-amino acids to mTORC1 activation. D-serine also activates mTROC1 through the phosphoinositide 3-kinase (PI3K)/Rheb pathway. FIG. 20 shows the effect of administration of D-serine on increasing glomerular filtration rate (GFR). FIG. 21 shows the results of examining the cell proliferation ability of D-amino acids in B cells.

Embodiments for carrying out the present invention will be described below, but the technical scope of the present invention is not limited to the following embodiments. It should be noted that the prior art documents cited herein are hereby incorporated by reference.

One embodiment of the present invention provides a composition for modulating cell growth, comprising an agent for controlling the amount of D-amino acids.

Also, one embodiment of the present invention provides a composition that activates the mTOR-related pathway, comprising a regulator of the amount of D-amino acids.

Also, one embodiment of the present invention provides a method for regulating cell proliferation, a method for regulating tissue/organ size, or a tissue/organ function, comprising regulating the amount of D-amino acids in a subject. Provide a way to improve.

As used herein, "controlling the amount of D-amino acids" refers to intentionally increasing or decreasing the amount of D-amino acids inside or outside cells, tissues and/or organs, and in body fluids. If there is a target amount and concentration, the D-amino acid in the sample and D-amino acid clearance may be evaluated by monitoring as appropriate.

As used herein, "cell proliferation" may be cell proliferation in a living tissue and/or organ, and may thereby adjust the size of the living tissue and/or organ.

As used herein, the term "adjustment of size of biological tissue and/or organ" means that the amount, morphology, abnormalities, excesses, deficiencies, etc. of cells in a biological tissue and/or organ are brought into a balanced state or in a correct state. It means to Size can be expressed in units of measurement such as physical size, weight, and function, and may be corrected for weight and the like. When there is a desired effect, for example, when the purpose is to grow cultured cells, the effect may be evaluated by performing metabolome analysis of the cell count or medium. In addition, markers in urine tests (urine protein, hematuria, creatinine amount, etc.) and blood tests (creatinine, cystatin C, urea nitrogen: BUN, etc.) for the purpose of adjusting kidney cell proliferation, size, and function Appropriate monitoring, renal function test (renal blood flow: RPF, glomerular filtration rate: GFR), X-ray examination, angiography, ultrasonography, CT, MRI, nuclear medicine examination, endoscopy, renal biopsy The effect may be evaluated by performing (pathological examination) or the like.

As used herein, the term “D-amino acid amount control agent” (also referred to as “D-amino acid amount control agent”) means that it is applied (eg, administered) to a subject in vivo. (eg, in a cell, tissue, organ, or body fluid), or an agent capable of increasing or decreasing the amount of D-amino acids in an isolated cell. As used herein, "regulating the amount of D-amino acids in cells" means increasing or decreasing the amount of D-amino acids in cells by applying a D-amino acid amount controlling agent, It means adjusting the amount of amino acid to an arbitrary range. As used herein, “regulating the amount of D-amino acids in tissues” means that the amount of D-amino acids in tissues (e.g., renal tubules, glomeruli, etc.) is It means adjusting the amount of D-amino acids to any range by increasing or decreasing the amount. As used herein, “regulating the amount of D-amino acids in an organ” means that the amount of D-amino acids in an organ (for example, kidney, heart, etc.) is controlled by applying a D-amino acid amount controlling agent. It means adjusting the amount of D-amino acids to any range by increasing or decreasing the amount. As used herein, "regulating the amount of D-amino acids in body fluids" means that the amount of D-amino acids in body fluids (e.g., blood, urine, etc.) is reduced by applying a D-amino acid amount controlling agent. It means adjusting the amount of D-amino acids to any range by increasing or decreasing the amount.

As used herein, the term "D-amino acid" is meant to include "D-form" proteinogenic amino acids, which are stereoisomers of "L-form" proteinogenic amino acids, and glycine without stereoisomers. Specifically, 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 What it contains. Since D-cysteine contained in a biological sample is oxidized and changed to D-cystine in vitro, in one embodiment of the present invention, D-cysteine can be measured instead of D-cysteine. The amount of D-cysteine contained in the biological sample can be calculated.

The amount of D-amino acids and/or the amount of L-amino acids can be measured by any method, such as chiral column chromatography, measurement using an enzymatic method, and monoclonal antibodies that distinguish optical isomers of amino acids. can be quantified by immunological techniques using Measurement of the amount of D-amino acid and/or the amount of L-amino acid in the sample in the present invention may be carried out using 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 method (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), 45 37. 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.).

The separation and analysis system for optical isomers in the present invention may combine multiple separation analyses. More specifically, a step of passing a sample containing components having optical isomers through a first column packing material as a stationary phase together with a first liquid as a mobile phase to separate said components of said sample; holding each of said components of said sample individually in a multi-loop unit, each of said components of said sample held individually in said multi-loop unit as a stationary phase, with a second liquid as a mobile phase; and resolving the optical isomers contained in each of the components of the sample by feeding through a channel to a second column packing material having an optically active center of The amount of D-amino acids and/or the amount of L-amino acids in a sample can be measured by using a method for analyzing optical isomers, which is characterized by including a step of detecting isomers (Japanese Patent No. 4291628). For HPLC analysis, D- and L-amino acids were previously derivatized with fluorescent reagents such as o-phthalaldehyde (OPA) and 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). or diastereomerization using N-tert-butyloxycarbonyl-L-cysteine (Boc-L-Cys) (Kenji Hamase and Kiyoshi Zaitsu, Analytical Chemistry, Vol. 53, 677-690 ( 2004)). Alternatively, D-amino acids or L-amino acids are measured by an immunological technique using monoclonal antibodies that distinguish optical isomers of amino acids, such as monoclonal antibodies that specifically bind to D-amino acids or L-amino acids. can do. In addition, when the total amount of D- and L-isomers is used as an index, it is not necessary to separate the D- and L-isomers for analysis, and the amino acids can be analyzed without distinguishing between the D- and L-isomers. Also in that case, it can be separated and quantified by an enzymatic method, an antibody method, GC, CE, or HPLC.

As used herein, the term "mTOR-related pathway" refers to a signal pathway involving mTOR (mechanistic target of rapamycin), which is a type of protein kinase (serine-threonine kinase) involved in intracellular signal transduction. It may be a signal pathway containing factors involved in the activation of complex 1 (mTORC1) or mTOR complex 2 (mTORC2), such as Akt, Rheb, and the like. The present invention integrates intracellular and extracellular environmental information such as insulin, growth factors, nutritional/energy status, redox status, etc., by activating mTOR-related pathways, and through transcription, translation, etc., cells respond accordingly. Regulates size, division, survival, etc. Their physiology may regulate cell proliferation.

In the present invention, the amount of biomolecules such as D-amino acids, creatinine, proteins, and drugs is not limited to mere mass, weight, and amount of substance (mol), but also mass per unit of tissue, cell, organ, molecule, volume, or weight. , weight, amount of substance (mol), mass in a liquid such as blood or urine, weight, amount of substance (mol), concentration, specific gravity, density, or any other physical quantity that can be measured.

As one aspect of the present invention, administration of D-amino acids from the outside, addition or removal of D-amino acids in foods, addition or removal of D-amino acids in culture media, and , and drugs or foods that can increase or decrease the amount of D-amino acids in body fluids. For example, by drinking an aqueous solution containing D-amino acids, it is possible to increase the concentration of D-amino acids in blood and cells/tissues (Non-Patent Document 2). can reduce the D-amino acid concentration in For example, D-serine may be directed to the kidney by oral or intravenous administration to control D-serine levels in the kidney (see, eg, Example 2). The D-amino acid used here may contain modifications or derivatives of D-amino acids, or pharmaceutically acceptable salts thereof, as long as the amount of D-amino acid can be increased or decreased, or a pharmacologically acceptable may contain carriers, diluents or excipients that may be used, and may be in the form of a prodrug. In addition, it may contain a target organ function improving agent and the like. The drug in the present invention can be formulated by selecting a dosage form suitable for its administration route. For oral administration, dosage forms such as tablets, capsules, liquids, powders, granules, chewing agents, etc., and for parenteral administration, injections, powders, infusion preparations, etc. can be designed. In addition, these formulations contain various adjuvants used for pharmaceutical purposes, namely, carriers and other adjuvants such as stabilizers, preservatives, soothing agents, flavoring agents, corrigents, fragrances, emulsifiers and fillers. , a pH adjuster, etc., and can be blended within a range that does not impair the effects of the agent (composition) of the present invention. The optical purity of D-amino acids used as drugs and raw materials is preferably 50% or higher, more preferably 90% or higher, but any optical purity can be selected within the range in which the effect is exhibited, It is not limited.

In one embodiment, the D-amino acid amount controlling agent that can be used in the present invention is selected from the group consisting of D-serine, D-asparagine and D-glutamine, and modifications and derivatives thereof. It may be

The present invention may utilize any physiological mechanism to vary the amount of target D-amino acids. As one aspect, proteins related to absorption, transport, distribution, metabolism (synthesis/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, and receptor expression (stimulation, suppression, etc.) and activity (action, inhibition, stimulation, etc.) regulate the amount of target D-amino acids. It becomes possible. 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, and the D-amino acid transporter is an inhibitor/activator. increases or decreases the amount of D-amino acids in the transport source/destination. Agents that act on proteins such as enzymes and transporters may not be effective directly. - It may vary in the amount of amino acids. In that case, the effect can be evaluated by measuring the amount of D-amino acid in body fluids, cells, and tissues at the site of action. In addition, such evaluations can be used to screen drug candidates.

By applying the present invention, any physiological mechanism can be used to vary the amount of D-amino acids in the body, and as a result, it is possible to regulate cell proliferation in the subject. As one aspect, the expression (promotion, suppression) of proteins related to D-amino acid absorption, transport, distribution, metabolism (synthesis and / or degradation), excretion, action, etc., or D-amino acid transporters or receptors etc.) and/or activity (action, inhibition, stimulation, etc.) can control the amount of D-amino acids in the body.

Therefore, the D-amino acid amount control agent that can be used in the present invention directly or indirectly promotes the gene expression of proteins involved in the absorption, transport, distribution, metabolism or excretion of D-amino acids. For example, it may be the protein or a vector that expresses it, or it may be a factor that promotes the upstream activity of the cascade that promotes the expression of the protein, or a vector that expresses it.

In addition, for example, the D-amino acid amount control agent that can be used in the present invention is one that directly or indirectly suppresses gene expression of proteins related to absorption, transport, distribution, metabolism or excretion of D-amino acids. from, for example, small molecules, aptamers, antibodies, antibody fragments, and antisense RNA or DNA molecules, RNAi-inducing nucleic acids, microRNAs (miRNAs), ribozymes, genome-editing nucleic acids and their expression vectors. may be selected.

As used herein, proteins related to absorption, transport, distribution, metabolism (synthesis and/or degradation), excretion, action, etc. of D-amino acids may be enzymes, for example, D-amino acid oxidation It may be an enzyme (DAO), D-aspartate oxidase (DDO), serine isomerase (SRR), DPP-4, and the like. 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. obtain.

In addition, since the D-amino acid transporter can increase or decrease the amount of D-amino acid in the source/destination, agents that act directly or indirectly on the D-amino acid transporter can also be applied to the present invention. .

Although not intended to be limiting, Non-Patent Document 5 describes that as D-amino acid transporter proteins, the SMCT family, the ASCT family, etc. expressed in the brain and kidneys increase the localized amount of D-amino acids by agonists/inhibitors. It is disclosed to change Since these transporters are affected by co-transport substances (e.g., sodium ions) and coordination/competition through scaffolds, D-amino acid transport activity is reduced even by sodium/glucose co-transporter (SGLT2) inhibitors. can be controlled. In addition, Patent Document 3 discloses that angiotensin 2 receptor blockers (ARBs) change the amount of D-amino acids in blood. For example, by measuring the amount of D-amino acid in culture media, cells, tissues, and body fluids before and after administration of antihypertensive agents and antidiabetic nephropathy drugs, drugs and candidates that can control the amount of D-amino acids in the body can be identified. Screening is possible.

As used herein, "aptamers" refer to synthetic DNA or RNA molecules and peptidic molecules that have the ability to specifically bind to target substances, and can be chemically synthesized in vitro in a short period of time. Aptamers used in the present invention can bind to, for example, proteins involved in absorption, transport, distribution, metabolism or excretion of D-amino acids and inhibit their activity. Aptamers used in the present invention can be obtained, for example, by repeatedly selecting bindings to various molecular targets such as small molecules, proteins, and nucleic acids in vitro using the SELEX method (Tuerk C., Gold L., Science, 1990, 249(4968), 505-510; Ellington AD, Szostak JW., Nature, 1990, 346(6287):818-822; No. 5,567,588; U.S. Pat. No. 6,699,843).

As used herein, the term "antibody fragment" refers to a portion of a full-length antibody that maintains antigen-binding activity, generally including its antigen-binding domain or variable domain. Examples of antibody fragments include F(ab')2, Fab', Fab or Fv antibody fragments (including scFv antibody fragments), and the like. Antibody fragments also include fragments obtained by treating an antibody with a protease enzyme and optionally reducing it. Antibodies or antibody fragments used in the present invention may be any of human-derived antibodies, mouse-derived antibodies, rat-derived antibodies, rabbit-derived antibodies, camelid-derived antibodies such as llamas, or goat-derived antibodies. Antibodies may be monoclonal, complete or truncated (eg, F(ab')2, Fab', Fab or Fv fragments), chimerized, humanized or fully human.

As used herein, the term "antisense RNA or DNA molecule" means a base sequence complementary to RNA (sense RNA) having a certain function, such as messenger RNA (mRNA), and forms a double strand with the sense RNA. In other words, it refers to a molecule that has the function of inhibiting protein synthesis that the sense RNA should be responsible for. In the present invention, antisense oligonucleotides, including antisense RNA or DNA molecules, inhibit translation into proteins by binding to mRNAs of proteins involved in absorption, transport, distribution, metabolism or excretion of D-amino acids. do. Thereby, it is possible to reduce the expression level of proteins involved in the absorption, transport, distribution, metabolism or excretion of D-amino acids and inhibit their activity. Methods for synthesizing antisense RNA or DNA molecules are well known in the art and can be used in the present invention.

As used herein, the term "RNAi-inducing nucleic acid" refers to a polynucleotide capable of inducing RNA interference (RNAi) when introduced into a cell, usually 19-30 nucleotides, preferably 19-25 nucleotides. , more preferably RNA, DNA, or chimeric molecules of RNA and DNA containing 19-23 nucleotides, optionally modified. RNAi may occur on mRNA or on post-transcriptional RNA before processing, i.e. RNA of nucleotide sequences comprising exons, introns, 3' untranslated regions and 5' untranslated regions. The RNAi method that can be used in the present invention includes (1) direct introduction of short double-stranded RNA (siRNA) into cells, or (2) incorporation of small hairpin RNA (shRNA) into various expression vectors, or (3) constructing a vector that expresses siRNA by inserting a short double-stranded DNA corresponding to the siRNA into a vector having two promoters arranged in opposite directions between the promoters, and RNAi may be induced by techniques such as introduction into The RNAi-inducing nucleic acid may include siRNA, shRNA, or miRNA that allows 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. Alternatively, they may be introduced using an expression vector that directs these RNAi nucleic acids.

In one embodiment, the RNAi-inducing nucleic acid for a protein associated with D-amino acid absorption, transport, distribution, metabolism or excretion used in the present invention is Any nucleic acid that exhibits a biological effect of inhibiting or significantly inhibiting protein expression can be synthesized by those skilled in the art with reference to the base sequence of the protein. For example, it is chemically synthesized using a DNA (/RNA) automatic synthesizer that utilizes DNA synthesis technology such as the solid-phase phosphoramidite method, or by an siRNA-related contract synthesis company (such as Life Technologies). It is also possible to consign and synthesize. In one embodiment, the siRNA used in the present invention is derived from its precursor, short-hairpin double-stranded RNA (shRNA), through processing by the intracellular RNase Dicer. There may be.

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

As used herein, "ribozyme" is a generic term for enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and M1 RNA contained in RNase P, but there are also hammerhead-type and hairpin-type ribozymes that have an active domain of about 40 nucleotides. (See, for example, Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 2191).

For example, the self-cleaving domain of the hammerhead ribozyme cleaves the sequence G13U14C15 at the 3' side of C15, but base pairing between U14 and A9 is important for its activity. (See, for example, Koizumi, M. et al., FEBS Lett, 1988, 228, 228.). By designing a ribozyme whose substrate-binding site is complementary to the RNA sequence near the target site, it is possible to obtain a restriction enzyme-like RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA. Manufacturers can refer to the following document: Koizumi, M.; et al. , FEBS Lett, 1988, 239, 285. Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 2191. Koizumi, M.; et al. , Nucl. Acids Res. , 1989, 17, 7059. .

Hairpin-type ribozymes can also be used in the present invention. This ribozyme is 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 (for example, Kikuchi, Y. & Sasaki, N., Nucl. Acids. Res., 1991, 19, 6751; Kikuchi Hiroshi, Chemistry and Biology, 1992, 30, 112.). Expression of the D-serine transporter protein can be inhibited by specifically cleaving the transcript of the gene encoding the D-serine transporter protein using a ribozyme.

As used herein, a genome-editing nucleic acid refers to a nucleic acid used to edit a desired gene in a nuclease-based system used for gene targeting. Nucleases used for gene targeting include known nucleases as well as new nucleases that will be used for gene targeting in the future. For example, known nucleases include CRISPR/Cas9 (Ran, FA, 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.

As one aspect, taking advantage of the fact that symbiotic bacteria such as intestinal bacteria are one of the resources of D-amino acids in the body, administration of antibiotics, intestinal regulators, oligosaccharides, probiotics, microbial transplantation, fecal transplantation It is possible to increase or decrease the amount of D-amino acids in the living body by changing the microflora or the growth environment by means such as improvement of dysbiosis. As a non-limiting example of a probiotic, ingestion of yogurt containing 1073R-1 lactobacillus is known to increase fecal D-serine and decrease D-lysine. However, foods known to contain D-amino acids, such as black vinegar, yogurt, microbial fermentation products such as cheese, and bacterial cells or bacterial cell extracts, have many active ingredient candidates in addition to D-amino acids. Therefore, when used as the D-amino acid control agent of the present invention, it is essential to use an amount that allows confirmation of an increase or decrease in the amount of D-amino acid at the site of action upon ingestion. In particular, since L-amino acids have various physiological activities different from those of D-amino acids, foods for which the standards (optical purity, amount), etc. of active ingredients that increase or decrease the amount of D-amino acids have not been set, are not suitable for the composition of the present invention. not included in the range of

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

In this specification, the term "pharmaceuticals" is used to include pharmaceuticals and quasi-drugs.

In this specification, "food" means food in general, but in addition to general food including so-called health food, it also includes food with health claims such as food for specified health use and food with nutrient function claims. Supplements (supplements, dietary supplements), feeds, food additives and the like are also included in the food of the present invention.

Methods of administering drugs include local administration (cutaneous, inhalation, enema, eye drops, ear drops, nasal, intravaginal, etc.), enteral administration, parenteral administration (intravenous, transarterial, transdermal, intramuscular, etc.). enteral administration is preferred. Enteral administration includes oral administration, tube administration, and enema administration. Tube administration includes administration through a nasogastric tube, gastrostomy, or duodenal fistula. Enema administration includes administration using suppositories and enemas. In any case, the dosage form of the drug is not particularly limited, and may be liquid or solid, and can be produced according to the common technical knowledge of those skilled in the art. The specific administration method is also not particularly limited, and administration can be suitably performed according to the common technical knowledge of those skilled in the art.

Targets to which the present invention can be applied include cultured cells, tissues, and organoids in any environment different from in vivo, and patients with cell proliferation, abnormal organ function, or organ atrophy due to disease or injury, or abnormal include patients with suspected Organ transplantation can be performed with the liver, pancreas, kidney, digestive tract, heart, eyeball, etc. In many cases, both the donor from whom the organ is removed and the recipient who receives the organ are transplanted before and after surgery. The present invention can be applied to such subjects because organ damage and functional deterioration accompanied by abnormal cell proliferation appear. Regarding organ injury, for example, the kidney is classified as type I (subcapsular injury), type II (superficial injury), type III (deep injury), etc. by the Japanese Society of Traumatology (Non-Patent Document 11). The present invention can be applied to any subject. In kidney transplantation, the donor loses one of two kidneys, i.e., total kidney size is approximately halved and kidney function is halved, but the size of the remaining kidney increases to compensate for the loss of function. It is known to do, and it is known that the size and function of the kidneys of patients undergoing dialysis treatment are reduced compared to when they are healthy, but when the present invention is applied, D- By increasing the amount of amino acids, regeneration of cells, tissues, and organs can be assisted, and glomerular filtration rate, which is one of renal functions, can be improved. Thus, in one aspect, subjects to which the present invention can be applied may be subjects with kidney disease, kidney transplant donors and/or recipients, and/or dialysis patients, or patients undergoing renal replacement therapy. In addition, animals in which abnormal cell proliferation is induced by genetic modification or drugs (e.g., uninephrectomized mice, etc.) and cultured cells/tissues (e.g., cancer model cells/tissues, stem cells, differentiated cells/tissues, etc.) , can be the object to which the present invention is applied.

One embodiment of the present invention is that the amount of D-amino acids in a medium for culturing cells, and the increase or decrease in the amount of D-amino acids in target cells, tissues and/or body fluids affect cell proliferation. , by measuring the amount of D-amino acids thereof, it can be used as an index of the state of abnormal cell proliferation and the effect of treatment. For example, in the kidney, by monitoring the amount of D-amino acids in the body and the amount of glomerular filtration, it is possible to diagnose and evaluate cell proliferation and functional abnormalities, analyze the mechanism of action of drugs, screen effects and toxicity, and select treatment methods and drugs. , it is possible to assist in determining the dosage, period, etc. Since the amount of D-amino acids in body fluids is affected by other diseases, for the purpose of distinguishing them, the values corrected by renal function markers such as creatinine and other markers are analyzed. may be used for

One embodiment of the present invention is a method of modulating cell proliferation in a subject, comprising:
A method comprising administering to a subject in need thereof an agent for controlling the amount of D-amino acids in vivo is provided.

Also, one embodiment of the present invention is a method for modulating cell proliferation in vitro or ex vivo, comprising applying a D-amino acid amount controlling agent to a cell, biological tissue or organ and culturing it. I will provide a. For example, in an in vitro or ex vivo environment, by culturing a cell, biological tissue, or organ using a buffer solution (e.g., medium) or physiological saline containing the above-mentioned D-amino acid amount regulator, Cell proliferation can be regulated. 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, living tissue or organs.

Also, one embodiment of the present invention provides the use of an agent for controlling the amount of D-amino acids for the manufacture of a pharmaceutical composition for modulating cell proliferation.

As described above, the method of 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 biological tissue and organ abnormalities.

The present invention will be described in more detail below based on examples, but these are not intended to limit the present invention in any way. In the examples, the following reagents were used unless otherwise specified.

- 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)

Example 1. Cell Proliferative Potential of D-Amino Acids in TIG-1 Cells

TIG-1 cells isolated from fetal tissue were plated on a 96-well plate so that the number of viable cells per well was 1.5×10 ⁴ cells. The cells were cultured in a DMEM high glucose + 10% FBS medium overnight at 37°C in 5% CO ₂ , the medium was removed, and the cells were washed with 150 µl of PBS. It was cultured in DMEM amino acid free medium + 0.5% dialyzed FBS at 37°C under 5% CO ₂ for 18 hours. 90 μl of DMEM amino acid free medium + 0.5% dialyzed FBS was added to the wells, 10 μl of each concentration of D-amino acid, L-amino acid, and conjugated amino acid solutions dissolved in PBS was added, and incubated at 37° C. in 5% CO ₂ . Incubated for 6 hours. Subsequently, 10 μl of Cell Counting Kit-8 was added, cultured at 37° C. in 5% CO ₂ for 6 hours, and absorbance was measured at a wavelength of 450 nm.

When 10 μM of each amino acid was added to the 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) were found to have a similar significant proliferative effect on DL bodies. rice field. D-methionine (D-Met) had no cell proliferation effect, but L-methionine (L-Met) had a significant cell proliferation effect (Fig. 1).

When 1 μM amino acid was added to the amino acid-free medium, L-Asn and L-Gln showed no cell growth effect, but D-Asn and D-Gln showed a significant cell growth effect (Fig. 2). .

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) exhibited a cell proliferation effect (Fig. 3).

Example 2. Amino acid administration and distribution to organs

12-week-old Balb/c male mice (SLC, Tokyo) were intravenously injected with ³ H-labeled D-serine and L-serine solutions, and 5, 30, 80, 180 minutes later blood, spleen, heart, kidney, Each tissue of lung, pancreas, liver, brain, and intestine was collected, and after homogenization, radioactivity was counted (cps) using Ecoscint XR (National Diagnostics, Atlanta, GA) and a scintillation counter LSC 5100 (Hitachi, Tokyo, Japan). : count per second), and radioactivity concentrations were converted to kBq/g units of measure using a cross-9 calibration factor, corrected for injection volume (MBq) and body weight to obtain Standardized uptake values (SUV ), statistically processed (n=4-5, mean±SEM, *p<0.05, **p<0.01, ***p<0.001). It was shown that the administered D-serine and L-serine are distributed to each organ due to their specific pharmacokinetics, and that the amount of amino acids can be changed over time (Fig. 4).

Example 3. D-serine administration and kidney size in uninephrectomized mice

In 10-week-old C57BL/6 male mice (SLC, Tokyo) fed a serine-free diet and water with (D-Ser) or without (Vehicle) 0.1% D-serine, one kidney Excision (UNX) and sham surgery (Sham) were performed. For nephrectomy, the renal pedicle of the left kidney of an anesthetized mouse was exposed through a dorsal incision and ligated with a silk thread. Soc Nephrol 22, 902-913 (2011)). Two days later, the kidney weight per body weight and the amount of D-serine and L-serine in plasma were measured and statistically processed (n = 6 to 7; two-way ANOVA, * p < 0.05, ** p<0.01, ***p<0.001). In the D-Ser administration group, the level of D-serine in plasma was higher than in the vehicle group, and the kidney size (kidney weight per body weight) was increased (Fig. 5). This will serve as a model for cell proliferation/tissue regeneration and functional recovery of the remnant kidney of a donor in renal transplantation.

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

10-week-old C57BL/6 male mice given a diet containing no serine and water containing (D-Ser) or not containing (Vehicle) 0.1% D-serine in the same manner as in Example 3 At (SLC, Tokyo), unilateral nephrectomy (UNX) and sham surgery (Sham) were performed, and 2 days later the processed specimens were subjected to RNAseq analysis (n=3). RNA was extracted using TRIzol (15596018, Thermo Fisher Scientific, Waltham, USA). Library preparation was performed with the TruSeq stranded mRNA sample prep kit (Illumina, San Diego, Calif.). Sequencing was performed on an Illumina HiSeq2500 platform in 75 base single-ended mode and base calling using Illumina Casava 1.8.2 software. The sequence read is Top Hat Ver. 2.0.13 to Bowtie2 Ver. 2.2.3 and SAMtools Ver. 0.1.19, and mapped to the mouse reference genome sequence (mm10), fragments per exon kilobase per million mapped fragments (FPKM) were obtained from Cufflinks Ver. 2.2.1 was used for calculation.

RNAseq data uses TargetMine (YA Chen, et al, PLoS One 6, e17844 (2011)), an integrated warehouse of human and mouse biological data from data sources such as Reactome and KEGG. Then, pathway analysis and gene ontology analysis were performed. Genes that were upregulated 2-fold change over the mean in D-serine treated mice compared to Vehicle treated mice were uploaded to TargetMine. Enrichment of Reactome pathways and gene ontology (GO) (M. Ashburner, et al, Nat Genet 25, 25-29 (2000)) was assessed by hypergeometric distributions and estimated p-values, and further by Benajmini and Hochberg Multiple-test correction was performed to suppress false positives using the method (W. S. Noble, et al., Nat Biotechnol 27, 1135-1137 (2009).). Heatmaps were drawn using R.

Analysis data showed activation of cell cycle-related pathways by D-serine administration (Fig. 6), and upregulated genes included cyclins, cell cycle activation family proteins, and mitotic exit upon degradation of cyclins. These results were reproduced by qPCR (Fig. 7). It was found that an increase in the amount of D-serine is associated with physiological activity that promotes and regulates cell proliferation.

Example 5. D-serine administration and kidney cell proliferation

10-week-old C57BL/6 male mice given a diet containing no serine and water containing (D-Ser) or not containing (Vehicle) 0.1% D-serine in the same manner as in Example 3 (SLC, Tokyo), unilateral nephrectomy (UNX) and sham surgery (Sham) were performed, and 2 days later the processed specimens were subjected to histological analysis.

For histological analysis, mice were perfused with saline, excised kidneys were sectioned, fixed with 4% paraformaldehyde, embedded in paraffin, and sections were stained with periodic acid Schiff (PAS). , or immunostained with anti-Ki67 antibody and Histofine Simple Stain Rat MAX PO (R) (414181F, Nichirei Biosciences) (T. Kimura, et al, Y. J Am Soc Nephrol 22, 902-913 (2011)). To count Ki67-positive cells per image, at least 10 fields (x200) were reviewed by a nephrologist blinded to the experimental conditions. For immunofluorescence, paraffin sections were incubated with Ki67 antibody and Alexa594-labeled secondary antibody. FITC-binding lectin from Triticum vulgaris (L4895-5MG, Sigma-Aldrich) was used to stain proximal tubules 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).

Analysis of PAS-stained kidneys showed that the number of Ki67-positive nuclei in renal tubules of UNX mice treated with D-serine was increased compared to those treated with Vehicle, indicating that D-serine It was shown to promote cell proliferation. (Fig. 8). Cell proliferation was observed in the proximal renal tubule of the kidney, which is the major region of D-serine reabsorption (Fig. 9), and the glomerular region tended to expand with D-serine administration (Fig. 10).

Example 6. Addition of D-serine and proliferation of cultured cells, mTOR-related metabolic regulation

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

For proliferation assays, cells were seeded in 96-well plates using medium. The next day, the medium was changed to 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]), was added). 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). Experiments using MEFs used type I collagen-coated microplates (4860-010, AGC Techno Glass, Haibara, Japan).

Immunoblots were performed by lysing cells for 1 hour on ice with RIPA buffer (89901, Thermo Fisher) supplemented with protease inhibitor cocktail (4693132001, Roshe) with or without phosphatase inhibitors (4906837001, Roshe, Basel, Switzerland). and centrifuged at 15000G for 10 minutes. The supernatant was boiled in SDS-PAGE gel loading buffer for 3 minutes, 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)). .

Compared to the serine-free medium group, the group supplemented with D-serine promoted cell proliferation at a low concentration in human renal tubular cell lines and primary cells (Fig. 11). Addition of D-serine enhanced Thr389 phosphorylation of S6K in HK-2 cells (FIG. 12). D-serine addition also induced Ser235/236-phosphorylation of S6RP in the kidney of UNX mice (FIG. 13). Thus, D-serine was shown to enhance the signal for activation of mTORC1. The effect of D-serine on the mTOR pathway was also confirmed by the suppression of D-serine-induced cell proliferation in p18-deficient strains (Fig. 14) (R. Yonehara, et al Nat Commun 8, 1625 ( 2017)). Since the Rheb-deficient strain suppressed the cell proliferation effect of D-serine, it was suggested that the effect of D-serine was also mediated via insulin/PI3K signaling (Fig. 15) (RA 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 (FIG. 16). Treatment with D-serine induced Ser473-phosphorylation of AKT in the kidney of UNX mice (see Example 3) (Figure 17). Furthermore, treatment with D-serine inhibited the dissociation of mTOR from lysosomes (Fig. 18), suggesting that D-serine assists lysosomal localization of mTOR. From the above results, it was revealed that D-serine and mTOR are cooperating and coordinating in cell proliferation (Fig. 19).

Example 7. D-serine administration and glomerular filtration rate

Eighteen-week-old serine racemase knockout rats were given water containing (D-Ser) or not containing 0.1-0.5% D-serine (Vehicle) ad libitum for one month, followed by FITC-sinistrin static. Glomerular filtration rate (GFR) was measured by clearance measurements of note. In the D-serine administration group (n=8), GFR increased by about 15% on average compared to the vehicle group (n=2) (FIG. 20).

Example 8. Cell Proliferative Potential of D-Amino Acids in B Cells

For the B-cell line proliferation assay, IL-3-dependent mouse pro-B-cell line Ba/F3 cells were seeded in 96-well plates using culture medium. The medium is 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) and 100 ng/10 μM D - Cells were cultured in the presence or absence of alanine for 48 hours, relative cell numbers were determined using WST-8 kit (CK04, Dojindo Laboratories, Kumamoto, Japan) and T-test was performed.

The presence of D-alanine was shown to significantly proliferate B cells (Fig. 21).

Claims

A composition for regulating cell growth, comprising an agent for controlling the amount of D-amino acids.
The composition according to claim 1, wherein the control agent is an agent for controlling the amount of D-amino acids in a subject's body.
The composition according to claim 1 or 2, wherein the cell proliferation is cell proliferation of a living tissue and/or organ, thereby adjusting the size of the living tissue and/or organ.
The composition of claim 2, wherein the subject is a subject with kidney disease.
The composition according to claim 2, wherein the subject is a kidney transplant donor and/or recipient.
The composition according to claim 2, which improves renal function.
The composition according to claim 6, wherein the renal function is glomerular filtration rate.
The composition according to claim 1 for modulating growth of isolated cells.
The composition according to any one of claims 1 to 8, wherein the control agent is a D-amino acid or derivative thereof.
The composition according to claim 9, wherein said D-amino acid is selected from the group consisting of D-serine, D-asparagine and D-glutamine.
The composition according to any one of claims 1 to 8, wherein the control agent is an agent that modulates protein activity related to absorption, transport, distribution, metabolism or excretion of D-amino acids.
The composition according to claim 11, wherein said metabolism is degradation or synthesis.
The composition according to claim 11 or 12, wherein the agent that modulates the activity of the protein is an agent that regulates gene expression of the protein.
The composition according to any one of claims 11 to 13, wherein the protein is selected from the group consisting of D-aspartate oxidase and serine isomerase.
The composition according to any one of claims 11 to 13, wherein said protein is a D-amino acid transporter protein.
16. The composition according to claim 15, wherein said D-amino acid transporter protein is one or more selected from the group consisting of SMCT family, GLUT5, CAT1, THTR2, SNAT2, ASCT family, Asc1, PAT1 and ATB 0,+ . .
The composition according to any one of claims 1 to 16, which is a pharmaceutical.
The composition according to any one of claims 1 to 16, which is a food.
The composition according to claim 18, wherein the food is a food with health claims or a dietary supplement.
The composition according to claim 19, wherein the food with health claims is a food for specified health uses or a food with nutrient claims.
The composition according to any one of claims 1 to 20, which activates mTOR-related pathways.
A composition that activates an mTOR-related pathway, comprising a regulatory agent in the amount of D-amino acids.
A method of modulating cell proliferation in a subject, comprising:
A method comprising administering to a subject in need thereof an agent for controlling the amount of D-amino acids in vivo.
A method of modulating cell proliferation in vitro or ex vivo, comprising:
A method comprising applying a D-amino acid amount controlling agent to a cell, biological tissue or organ and culturing.
　Use of an agent for controlling the amount of D-amino acids for the manufacture of a pharmaceutical composition for the regulation of cell proliferation.