KR20040010919A - Method and culture medium for inhibiting cell aging using fk506 or its derivatives - Google Patents

Abstract

PURPOSE: Provided are a method and culture medium for inhibiting cell aging using FK506 or its derivatives, thereby inhibiting cell aging, extending the life cycle of the cell and increasing the growth thereof. CONSTITUTION: A method for inhibiting cell aging, extending the life cycle of the cell and increasing the growth thereof is characterized in that the cell in a culture medium is exposed to an effective amount of FK506 or its derivatives, wherein the FK506 derivative is selected from the group consisting of nor-C9 keto-FK506 derivatives represented by the formulae(I) . In the formula, R and R2 are independently H or hydroxy protection group easily scavenged.

Description

Method and Culture Medium For Inhibiting Cell Aging Using FK506 Or Its Derivatives

The present invention relates to the use of FK506 or a derivative thereof for use in inhibiting aging and / or extending lifespan of cultured cells. More specifically, the present invention utilizes FK506 or derivatives thereof to inhibit aging of cells in culture, prolong cell life, increase cell growth, increase cell viability and / or adapt cells to culture. It relates to a method and culture medium.

In vitro culture of cells isolated from humans leads to a state in which they can no longer proliferate after a certain number of cell divisions. Cells have a limited ability to divide and are called senescence after a certain number of cell divisions. Enters a terminally non-dividing state. Aged cells stop further growth and do not enter the S phase of the cell cycle even if they are stimulated by physiological cell growth mitogen, and expression of some genes necessary for cell growth is suppressed. However, it has been reported that aged cells still retain their metabolic activity and have protein or DNA synthesis for some time (Dimri GD et al., 9363-9367, 1995; Saunders NA et al., 46-). 54, 1993).

The underlying cause of aging is known to be the phenomenon of replicative senescence (Wantanabe Y. et al., 2025-2032, 1997). Replication aging is caused by the accumulation of cell division, which is reported to be due to the accumulation of irregular damage or mutations in the process of protein and RNA synthesis or to any genetically programmed process (Sugawara O. et al. , 707-710, 1990). This report suggests that proliferation is limited by the 'cell division clock' (Bodnor AG et al., 349-352, 1998). In addition, cell cycle regulatory proteins and tumor repressors have been reported to affect the replication aging, with increased expression levels of wild-type p53 and p21 _{WAF1 / CIP1} proteins. Has been shown to induce proliferative loss of some cultured cells, thereby causing cell aging (Brown JP et al., 831-834, 1997; Gallimore PH et al.,, 763-771, 1997; Sugrue M Mm. Et al., 9648-9653, 1997).

It is estimated that aged cells accumulate in vivo and alter the phenotype expressed from these cells, causing various aging-related pathologies (Dimri G.D. et al., 9363-9367, 1995). Therefore, many studies have recently been made as a substance that inhibits the aging of cells can be applied not only to the treatment of diseases related to aging of the cell but also variously useful in the fields of biology and medicine such as cell transplantation including tissue culture. Is actively underway.

The inventors of the present invention, by mixing FK506 or a derivative thereof in a culture medium of the cell and culturing the cell, increase the growth and lifespan of the cell, and in addition, the FK506 or a derivative thereof may be aged during the aging process induced by the cell by p53 gene expression. And based on this, FK506 or its derivatives can be mixed with the culture medium to inhibit the aging of cells in the culture, to prolong the life of the cells, and to increase the growth of the cells, thereby obtaining many useful cells. Could.

In one aspect, the present invention provides a method of inhibiting aging of cells in culture, characterized by exposing the cells in the culture medium to an amount of FK506 or a derivative thereof effective to inhibit cell aging.

In another aspect, the present invention provides a method of extending the life of a cell in a culture, characterized by exposing the cells in the culture medium to an amount of FK506 or a derivative thereof effective to prolong cell life.

In another aspect, the present invention provides a method of enhancing the growth of cells in a culture, characterized by exposing the cells in the culture medium to an amount of FK506 or a derivative thereof effective to enhance cell growth.

In another aspect, the present invention provides a method of increasing the viability of a cell in a culture, characterized by exposing the cells in the culture medium to an amount of FK506 or a derivative thereof effective to increase viability.

In another aspect, the present invention provides a method of adapting a cell to a culture, wherein the cell in the culture medium is exposed to FK506 or a derivative thereof in an amount effective to adapt the cell to the culture.

In another aspect, the present invention provides a cell culture medium useful for inhibiting aging of cells in culture, characterized in that it comprises an amount of FK506 or a derivative thereof effective to inhibit aging of the cell.

In another aspect, the present invention provides a cell culture medium useful for prolonging the life of cells in culture, characterized in that it comprises an amount of FK506 or a derivative thereof effective to prolong cell life.

In another aspect, the present invention provides a cell culture medium useful for enhancing the growth of cells in culture, characterized in that it comprises an amount of FK506 or a derivative thereof effective to enhance cell growth.

In another aspect, the present invention provides a cell culture medium useful for extending the viability of cells in culture, characterized in that it comprises an amount of FK506 or derivative thereof effective to enhance cell viability.

In another aspect, the present invention provides a cell culture medium useful for adapting cells to the culture, characterized in that it comprises an amount of FK506 or a derivative thereof effective to adapt the cells to the culture.

1 is a diagram showing the efficacy of FK506 to inhibit the aging of cells induced by the P53 gene. Human bladder cancer cell line EJ cells were infected with ΔE1 expressing adenovirus (Ad-ΔE1) or adenovirus (10 MOI) expressing p53 (Ad-p53), and 3 hours later, 0.1 μM of FK506 was added thereto, and cultured for 6 days. Afterwards, SA-β-gal activity was examined to show cells showing SA-β-gal activity in percentage (%).

2 shows the efficacy of FK506 on the growth and senescence of chondrocytes. Continuously culturing chondrocytes isolated from the joints of two-week-old young rabbits showed the effect of FK506 on the growth and aging of chondrocytes. The X axis represents the day number and the Y axis represents the total cell number.

3 shows the efficacy of FK506 to prolong cell division recovery of chondrocytes in young rabbits. The X axis shows the dose of FK506 treated, and the Y axis shows the number of cell divisions.

Figure 4 shows the efficacy of FK506 on the growth and aging of chondrocytes extracted from young rabbits. The X axis represents the number of days and the Y axis represents the total cell number.

5 shows the efficacy of prolonging the recovery of cell division of chondrocytes extracted from young rabbits of FK506. The X axis shows the dose (μM) of the treated FK506, and the Y axis shows the number of cell divisions.

Figure 6 shows the efficacy of FK506 on the growth of chondrocytes extracted from the joints of old rabbits. The X axis represents the number of days and the Y axis represents the total cell number.

Figure 7 shows the efficacy of prolonging cell division recovery of chondrocytes extracted from the joints of old rabbits of FK506. The X axis shows the dose (μM) of the treated FK506, and the Y axis shows the number of cell divisions.

8 shows the efficacy of FK506 on the growth of chondrocytes extracted from the joints of old rabbits. The X axis represents the number of days and the Y axis represents the total cell number.

Figure 9 shows the efficacy of prolonging the recovery of cell division of chondrocytes extracted from the joints of old rabbits of FK506. The X axis shows the dose (μM) of the treated FK506, and the Y axis shows the number of cell divisions.

Figure 10 shows the effect on the efficacy of FK506 on the growth of chondrocytes extracted from the joints of old rabbits. The X axis represents the number of days and the Y axis represents the total cell number.

Figure 11 shows the efficacy of prolonging the recovery of cell division of chondrocytes extracted from the joints of old rabbits of FK506. The X axis shows the dose (μM) of the treated FK506, and the Y axis shows the number of cell divisions.

The present invention provides a novel use of cyclosporin A or its derivatives for use in inhibiting aging of cells in cells, extending cell life, increasing cell growth, increasing cell viability and / or adapting cells to culture. It is about.

FK506 was isolated in 1984 as a metabolite of the fungus Streptomyces Tsukabaensis by Fujisawa's team in Japan. In 1989 a clinical trial using FK506 for liver transplantation was initiated. KF506 is 50-100 times more potent than cyclosporin A, but is almost equal to cyclosporin A in terms of toxicity and side effects. Merck, USA, has been working to modify its structure to reduce the toxicity of FK506, and much has been learned about the mechanism and function of FK506 in the process.

FK506 is a 23-membered macrolide represented by the following formula having 14 chiral centers and many microreaction sites:

FK506 can be functionally divided into three regions KFBP-binding region, calcineurin-binding region and Northwest region. The intermolecular interactions between FK506 and FKBP12 are mostly hydrophobic, and only four intermolecular hydrogen bonds are formed between these two molecules. The stronger binding of FK506 and FKBP12 is known to increase their affinity for calcineurin. This increase in affinity leads to stronger immunosuppressive activity. The northwest region of FK506 does not interact with FKBP12 or calcineurin.

FK506 has been used as a treatment for rejection of organ or tissue transplants, graft-to-host response, atopic dermatitis, allergic contact dermatitis, lichen planus mucosae and pyoderma gangrenosum. Recently, other functionalities on FK506 have been revealed. For example, Sulaiman OA, et al., Exp Neurol 2002 May, 175 (1): 127-37, describe that FK506 promotes regeneration of peripheral nerves. However, to date FK506 or a known derivative thereof can be used to inhibit aging of cultured cells, to promote growth of cultured cells, to prolong the life of cultured cells, to increase the viability of cultured cells, or to adapt cells to cultures. No case has been applied.

In the present invention, in order to investigate the aging inhibitory effect of FK506 or its derivatives, the senescence of cells was artificially induced. We have reported an experimental system that induces senescence by expressing the p53 gene (Proc. Natl. Acad. Sci. USA, vol 94, 9648-9653, 1997). This system has been applied to the present invention, and briefly described, aging the EJ bladder cancer cells by inducing expression of the p53 gene by infecting the adenovirus expressing the p53 gene in the EJ bladder cancer cells. As a test for inhibiting aging of FK506 or derivatives thereof according to the present invention, the cells are infected with p53-adenovirus to sensitize EJ cells, and cultured with the addition of FK506 or derivatives thereof. As a result of investigating SA-β-gal (galactosidase) activity as an indicator, it was found that p53-induced cell senescence was suppressed depending on the concentration of FK506 or its derivative added to the culture.

In addition, the inventors conducted an experiment to determine the effect of FK506 or its derivatives on cell division. In other words, experiments were conducted to investigate the effects of FK506 or its derivatives on cell division during continuous culture of cells. Such experiments were also carried out in other individuals of the same species (rabbits), and in young rabbits with active cell division, It was also performed in old rabbits with poor cell division. From these results, it was observed that FK506 or its derivatives exhibited anti-aging effects not only in the cells of young animals but also in the cells of old animals.

Based on these experimental results, we could conclude that FK506 or its derivatives prevent cell aging, prolong cell life, and increase cell growth.

In the present invention, cell aging is defined as a state in which a cell stops cell growth irreversibly without further division after a predetermined number of cell divisions. Aged cells maintain this state for months or years and cannot synthesize DNA, but their metabolism is active. Aged cells exhibit morphological properties that are flattened and grow in size.

Various hypotheses explaining cell aging have been proposed, but active oxygen research and genetic program theory are currently being actively studied in academia. It is hypothesized that cell damage caused by insufficient capacity to remove free radicals from metabolism accumulates for a long time and that the cells age and that the aging process is carried out by a program inherent in genes. As part of gene programmability, the telomere hypothesis has been studied. The human telometer has a structure in which the TTAGGG sequence is repeated. DNA synthase can be synthesized only in the 5 '-> 3' direction, and DNA synthesis starts from 100 to 200 bases of RNA and is removed after the synthesis is completed. It will be as short as size. Therefore, the number of cell division is determined by the length of the telometer. About 80% of cancer cells whose cell aging programs have been destroyed are known to synthesize an enzyme called telomerase, which synthesizes telomeres. It is known that cell changes with aging occur in various ways. These are briefly described as follows:

1. Changes in genomic structure

The cloned DNA is equally isolated from daughter cells. As the number of cell divisions increases, more and more cells are distributed asymmetrically in the amount of DNA.

2. Changes in Genome Stability

Chromosomal aberrations are characteristic of aged cells with or without cell division. The frequency is inversely proportional to lifespan. In non-dividing cells, the methylation, hydroxylation, crosslinking, and strand break of DNA bases increase with age of the cell. The increase in base modifications can be thought of as two cases where base strains accumulate with age and the production of base strains increases with age.

3. Changes in DNA Repair Function

DNA damage is constantly caused by external factors such as ultraviolet radiation or by internal factors such as free radicals that occur during body temperature and energy production. The ability to repair DNA damage is very important for the survival of the individual.

4. Changes in Gene Expression

The activity of proteins changes, which in turn alters the metabolic processes of the cell, thus affecting the function of organs. Changes in protein activity eventually result from changes in gene expression. Gene expression according to aging is very slow due to the characteristics of aging, the degree of change varies depending on the individual, and the type and timing of genes that change in each tissue is different.

5. Changes in the mitochondria

Mitochondria supply the energy needed for cell life through oxidative phosphorylation. Oxygen free radicals are produced because oxygen is the final electron transporter. Oxygen free radicals damage mitochondrial membranes, inhibiting oxidative phosphorylation processes using concentration differences between inside and outside, reducing energy production, inducing mitochondrial DNA mutations and loss, and inhibiting mitochondrial gene expression. Do not recover. Mitochondria are regenerated when cells divide. The change in mitochondria with aging is more pronounced in non-dividing cells. In general, mitochondria of old cells are larger and elongated than young cells. Aged mitochondria are eventually broken down by enzymes of lysosomes and converted into lipofuscin, an aging pigment.

6. Change of cell membrane

Cell membranes regulate the transport of nutrients, metabolites, and ions between cell organelles in and out of cells and within cells, and play an important role in cell-to-cell signaling. The biggest change in aging is the change in lipids, a major component of cell membranes. As aging increases the ratio of cholesterol to phospholipids, this leads to a change in the fluidity of the membrane. Changes in the function of cell membrane proteins, namely Ca: Mg-ATPase activity required for calcium transport, do not change with age, but rather increase in aged cells while Na: K-ATPase activity decreases. In particular, the cell membrane specific enzyme 5'-nuclease activity decreases with age.

7. Changes in Protein Synthesis

Protein synthesis degrades with aging. This is due to decreased gene expression, decreased mRNA transport from the nucleus to the cytoplasm, and poor performance of synthetic machinery.

8. Change by Carbonyl Group

Changes in the carbonyl group of nucleic acids, proteins and lipids are the most common changes in aging cells or degenerative diseases.

Many studies have been attempted to develop derivatives of FK506 to reduce its toxicity (Askin, D. et al., Tetrahedron Lett., 1988, 29, p277; Mills, S. et al., Tetrahedron Lett. , 1988, 29, p281; Desmond, R. et al., Tetrahedron Lett., 1988, 29, p3895; and Askin, D. et al., Tetrahedron Lett., 1988, 29, p4245). U.S. Patent No. 4,980,466 describes a nor-C9 keto-FK506 derivative. EP 0 323 042 A1 also discloses new macrocyclic compounds or their pharmaceutically acceptable salts as derivatives of FK506.

However, all of these derivatives of FK506 are known only for use as immunosuppressive agents and there is no mention that they can be used as cell aging inhibitors according to the present invention. Accordingly, the present invention relates to the use of the FK506 derivatives disclosed in this patent as cell aging inhibitors.

FK506 is known to bind to a protein called cyclophilin to regulate the water solubility of the mitochondrial outer membrane and to regulate the activity of dephosphorylated protein called cytoplasmic calcineurin. Thus, it is clear that all derivatives of FK506 disclosed in this patent can be applied to inhibit the aging of cells according to the present invention, since derivatives of FK506 will also act in conjunction with cyclophilin.

In one embodiment, derivatives of FK506 according to the present invention comprise nor-C9 keto-FK506 derivative compounds represented by formula (I):

(I)

Wherein R 1 and R 2 are independently H or an easily removable hydroxy protecting group.

In another embodiment, the derivative of FK506 according to the present invention comprises a macrocyclic compound represented by the following formula (II) or a pharmaceutically acceptable salt thereof:

(II)

In the above formula,

Each adjacent pair of substituents [R ₁ and R ₂ ], [R ₃ and R ₄ ], [R ₅ and R ₆ ] are independently

(a) represent two adjacent hydrogen atoms, or

(b) form a second bond between adjacent carbon atoms to which they are attached;

R ₂ may be an alkyl group in addition to the above definitions;

R ₇ may represent H, OH or O-alkyl or together with R 1 may represent ═O;

R ₈ and R ₉ independently represent H or OH;

R ₁₀ represents H, alkyl, alkyl substituted by one or more hydroxyl groups, alkenyl, alkenyl substituted by one or more hydroxyl groups or alkyl substituted by ═O;

X represents O, (H, OH), (H, H) or -CH ₂ O-;

Y represents O, (H, OH), (H, H), N-NR ₁₁ R ₁₂ or N-OR ₁₃ ;

R ₁₁ and R ₁₂ independently represent H, alkyl, aryl or tosyl;

R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₂ and R ₂₃ independently represent H or alkyl;

R ₂₀ and R ₂₁ independently represent O, or independently represent (R ₂₀ a, H) and (R ₂₁ a, H), respectively, or OH, O-alkyl or OCH ₂ OCH ₂ CH ₂ OCH ₃ Or they may together represent an oxygen atom in the epoxide ring;

n is 1, 2 or 3;

In addition to the above definitions, Y, R ₁₀ and R ₂₃ may represent a 5- or 6-membered N-, S- or O-containing saturated or unsaturated heterocyclic ring together with the carbon atoms to which they are attached, The ring may be substituted by one or more groups selected from alkyl, hydroxyl, alkyl, O-alkyl, benzyl and -CH ₂ Se (C ₆ H ₅ ) substituted by one or more hydroxyl groups;

Provided that when X and Y both represent O, R ₉ represents OH; R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ and R ₂₂ each represent methyl; R ₂₀ represents OCH ₃ ; R ₈ and R ₂₃ each represent H; R ₂₁ a represents OH; [R ₃ and R ₄ ] and [R ₅ and R ₆ ] each represent a carbon-carbon bond;

(a) when n is 1 and R ₇ represents an OH group and R ₁ and R ₂ are each hydrogen, then R ₁₀ does not represent an allyl group;

(b) when n is 2, R ₇ represents OH and R ₁ and R ₂ each represent hydrogen, then R ₁₀ is not methyl, ethyl, propyl or allyl;

(c) n is 2, R ₇ represents hydrogen, and [R ₁ and R ₂ ] represent a carbon-carbon bond, R ₁₀ does not represent an allyl group.

In the present invention, FK506 or a derivative compound thereof may be converted to an inorganic or organic base addition salt or an inorganic or organic acid addition salt and methods for salt conversion thereof are well known in the field of organic synthesis and pharmaceuticals (Remington's Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, PA, 1985, p1418). Generally, salts are prepared by reacting the stoichiometry of the appropriate base or acid in water or an organic solvent or a mixture of both solvents. Pharmaceutically acceptable salts according to the invention include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids and salts with basic or acidic amino acids. Salts with inorganic bases include, for example, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, aluminum salts and ammonium salts. Salts with organic bases include trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and N, N'- Examples thereof include salts with dibenzylethylenediamine. Examples of salts with inorganic acids include salts with hydrochloric acid, boric acid, nitric acid, sulfuric acid and phosphoric acid. Examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid. Included. Salts with basic amino acids include, for example, arginine, lysine and ornithine. Examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid.

In order to normalize the life history of the cells in culture according to the invention, ie to optimize the growth and maturation of the cells with respect to aging and to adapt cells which are resistant to the culture, the cells are subjected to FK506 or Preference is given to exposure to derivatives thereof. Because cell aging is a gradual process, aging can be prevented even when exposed to FK506 or its derivatives later in cell life cycle, depending on the particular cell type, culture conditions and other factors. However, senescent cells are poor in viability and productivity, meaning that it is not desirable to expose FK506 or its derivatives later in the life cycle of cells. For optimal results, cells may be exposed to FK506 or its derivatives early in life cycle. It is preferable to expose.

According to the present invention, cells which exhibit a short lifespan under in vitro culture conditions, for example, cells which have a lifespan of 2 weeks or less or which undergo a small number of cell divisions, or which do not exhibit normal biological function, for example Cells whose normal production of hormones, enzymes or other biological substances have been inhibited in vivo may be exposed initially by exposing their culture to an adaptive amount of FK506 or a derivative thereof, preferably after mixing the FK506 or a derivative thereof with the culture medium. Inoculation can be adapted to the culture. Examples of cells resistant to culture include lymphoid cells, hepatocytes, pancreatic cells, neurons, thyroid cells and thymic cells of mammals.

FK506 or a derivative thereof may optionally be added to any known culture medium supplemented with protein components such as serum (eg, fetal or newborn calf serum). Examples of badges include, but are not limited to, hams such as Eagle's Basal Medium, Eagle's Minimal Essential Medium, Dulbecco's Modified Eagle's Medium, F10 Badge, or F12 Badge. Medium (Ham's Medium), Fuck's N15 Medium, Fuchs N16 Medium, Waymoth's MB 5421 Medium, McCoy's 5A Medium; RPMI Medium 1603, 1534 or 1640, Leibovitz's L15 Medium, American Type Culture Collection (ATCC) CRCM 30, MCDB Medium 101, 102, 103 or 104, CMRL Medium 1066 or 1415 and Hanks or Earls Equilibrium Salts Solution (Hank's or Earl's Balanced Salt Solution). Basal medium used as known in the art commonly contains essential nutrients to support cell growth in culture, including essential amino acids, fatty acids and carbohydrates. Typically the medium contains additional essential ingredients such as vitamins, cofactors, trace elements and salts in an assimitable amount. Also included are other factors that promote cell growth and maintenance, including compounds and factors necessary for cell survival, function, production and / or proliferation, such as peptide or steroid hormones, growth factors and antibiotics. do. The medium also generally includes buffers, pH adjusters, pH labeling agents and the like.

The medium containing FK506 or its derivatives according to the invention can be applied to various cells, in particular eukaryotic cells. The FK506 or derivative-containing medium of the present invention is suitable for culturing animals, in particular mammalian cells, including human cells, plant cells, insect cells, microorganisms (eg bacteria, fungi, protozoa) and other antibiotic-producing cells. Do. FK506 or derivatives thereof are useful for enhancing the viability of living cells in various tissue cultures adapted for the culturing of such cells. Examples of cultures include cultures of cloned cells, such as hybridoma cell lines; Cultures of mammalian cells, especially human cells, for the production of cell products, in particular proteins and peptides (eg, hormones, enzymes and immunofactors); Culture of virus infected cells for the production of a vaccine; Cultures of plant cells, such as cultures of meristem or callus; Culture of epithelial cells providing tissue for wound healing; And cultures of resistant cells as therapeutic and prophylactic uses. FK506 or derivatives thereof may also be used in media adapted for the production and preservation of biological organs and transplanted tissue.

Thus, certain cell types useful for culturing in accordance with the present invention include mammalian tissues, organs and glands (eg, brain, heart, lung, stomach, intestine, thyroid, adrenal gland, thymus, parathyroid gland, testes, liver, kidney, bladder, Spleen, pancreas, gallbladder, ovary, uterus, prostate and skin); Germ cells (sperm and egg); Lymph nodes, bone, cartilage and stromal cells; And blood cells including immune cells, macrophages, lymphocytes, white blood cells, red blood cells and platelets. Additional cell types include stem, leaf, pollen and seed of the plant; Microorganisms and viruses described above; And cells derived from insect tissues, organs and glands.

Culture techniques in which FK506 or derivatives thereof are applied in accordance with the present invention include the use of solid supports for anchorage-dependent cells, for example in monolayer or suspension culture. Solid supports include glass, carbon, cellulose, hollow fiber membranes and solid substrate forms such as agarose gels. FK506 or a derivative thereof may be bound within the beads, captured in the matrix, or covalently bound. FK506 or derivatives thereof are useful for preserving cultures and encapsulated cells, such as primary cultures, continuous cultures, secondary cultures, frozen or dried or lyophilized cultures. Cultures can also be transferred from conventional media or by previous techniques known as media containing derivatives thereof.

In accordance with the present invention, the cells may significantly increase the viability or lifespan of the cells or significantly increase the biomass of the cells as measured in an amount effective to promote their in vitro culture, for example compared to untreated cells. Or FK506 or derivatives thereof in an amount that significantly increases the bioproductivity of the cells. For in vitro application, about 1-100 pg per ml of cell culture (based on a density of about 10 ⁵ to about 10 ⁷ cells / ml) can be used. FK506 or a derivative thereof is supplemented to the culture, usually once every 2-4 days, as needed. However, the replenishment period can be varied in accordance with the desired outcome and in some cases weekly replenishment.

The main process of the present invention will be described in more detail.

A. Induction of senescence of cells and measurement of effect of FK506 or its derivatives on cells

In the present invention, FK506 or a derivative thereof inhibits cell senescence, prolongs cell lifespan, increases cell growth, increases cell viability, and / or the degree of adaptation of cells to culture. As an example, cells that exhibit resistance to various aged cells or cultures can be used. It can also be used to induce aging of cells.

Aging of the cells of the present invention is carried out by a method of infecting cells with an adenovirus expressing the p53 gene. This method can be performed according to the inventors' aging induction experimental system (Proc. Natl. Sci. USA. Vol 94, 9648-9653). Specifically, EJ bladder cancer cells are cultured in a culture vessel with a culture medium of DMEM containing 10% fetal bovine serum. After culturing the EJ bladder cancer cells, p53-adenovirus is added to infect the cells.

In general, as cells age, they become morphologically broad and flat. In addition, changes in genomic structure, changes in genomic stability, changes in DNA repair function, changes in gene expression, changes in mitochondria, changes in cell membranes, changes in protein synthesis, or carbonyl groups occur.

At the same time as infection with p53-adenovirus and cultured with the addition of FK506 or its derivatives. After a certain period of time, the aging behavior of the cells is determined by examining SA-β-gal (galactosidase), which is a biochemical marker of morphology and aging.

B. Isolation and Culture of Cells

In order to examine the action of FK506 or its derivatives on aged cells or cells resistant to culture, cells to be tested are isolated and cultured. Isolation of cells can be isolated in vivo or in culture in a variety of ways. Cells isolated from the body or culture medium are cultured in an incubator in DMEM medium containing 5% serum. The cultured cells are then centrifuged, washed with DMEM medium containing 10% fetal bovine serum and incubated in DMEM medium containing streptomycin or penicillin. The cultures are changed every 3 days, one day after the cells start to grow by attaching to the bottom of the culture vessel, the cells are separated from the culture vessel with trypsin-EDTA solution and the cell count is calculated.

After culturing a certain number of cells in a new culture vessel and showing about 90% confluence at the bottom of the culture vessel, the cells are separated again with trypsin-EDTA solution, and the number of cells is measured again. Transfer only the cells to the culture vessel and continue incubating. Repeat this process to create cell growth curves.

C. Measuring the number of cells and the number of cells in culture medium to which FK506 or derivatives thereof are added

To determine the effect of FK506 or its derivatives on aging cells or cells resistant to culture, the number of cells and the number of cell divisions in the culture medium to which FK506 or its derivatives were added was measured to determine the lifespan and aging of cells. Notice the change in.

First, the separation of cells can be separated in vivo or in culture in a variety of ways. Cells isolated from the body or culture medium are cultured in an incubator in DMEM medium containing 5% serum. The cultured cells are then centrifuged, washed with DMEM medium containing 10% fetal bovine serum and incubated in DMEM medium containing streptomycin or penicillin. The culture is changed every 3 days, at which point cells are removed from the culture vessel with trypsin-EDTA solution at the time the cells are attached to the bottom of the culture vessel and begin to grow, and the cell count is calculated. A certain number of cells are placed in a culture vessel, each adding different amounts of 0.01-1 mM FK506 or derivatives thereof and incubating with the culture.

When a certain number of cells grow and exhibit about 90% of the confluence of the culture vessel, the cells are separated again with trypsin-EDTA solution, and then the number of cells is measured. Another amount of 0.01-1 mM FK506 or a derivative thereof is added and continued incubation with the culture. While repeating this process, cell growth curves are prepared from the number of cells before and after culture in each culture vessel and the number of cell divisions is calculated.

Hereinafter, the present invention will be illustrated by examples. These examples are merely illustrative of the present invention and should not be understood as the scope of the present invention being limited by the examples.

<Example 1>

Inhibitory effect of FK506 on cellular senescence induced by p53 gene expression

It was investigated whether the treatment of FK506 inhibited the aging caused by the p53 gene.

First, the method of aging the cells by infecting cancer cells with adenovirus expressing the p53 gene was according to the aging induction experiment system of the present inventors (Proc. Natl. Sci. USA, vol 94, 9648-9653; oncogene Vol 20, 5818 -5825). That is, EJ bladder cancer cells were infected with adenovirus expressing the p53 gene to induce p53 gene expression, thereby rapidly aging the EJ bladder cancer cells within 4 to 6 days. 500,000 EJ bladder cancer cells were incubated for 2 days in a 100 mm culture vessel with 10 ml of culture medium (DMEM containing 10% fetal bovine serum), and then infected with approximately 5,000,000 p53-adenoviruses and simultaneously 1 μM of FK506. After 6 days of incubation, SA-β-galactosidase activity, which is a biochemical marker of morphology and aging, was examined.

As a result, cells cultured without adding FK506 changed to p53 expression, which is characteristic of senescent cells, that is, broad and flat, but the shape of cells cultured with FK506 was not changed.

In addition, 62% of the cells cultured in the culture medium without FK506 showed SA-β-galactosidase activity, whereas 5% of the EJ bladder cancer cells cultured in the culture medium containing 0.1 μM of FK506. Only 8% of the cells showed SA-β-galactosidase activity (FIG. 1).

As described above, as a result of measuring SA-β-galactosidase activity, which is a biochemical indicator of cell morphology and aging, it was found that p53-induced cell senescence was suppressed depending on the concentration of FK506 added to the culture medium. Could.

<Example 2>

Effect of FK506 on the Growth of Chondrocytes

It was investigated whether FK506 inhibited aging, extending the life of normal cells and increasing the number of divisions of cells in in vitro culture.

Cartilage slices of two-week-old young New Zealand white rabbits were enzymatically digested to obtain articular chondrocytes. Enzymatic digestion was performed by placing sterile cartilage slices into 5% serum-containing DMEM medium containing 0.2% collagenase type II (381 U / μg), 37 It was carried out in a carbon dioxide (CO ₂₎ incubator for 6-7 hours, single cells separated by this reaction (single cells) were obtained by centrifugation for 5 minutes at 1000 rpm. The obtained cells were washed once with DMEM medium containing 10% fetal bovine serum, and then DMEM medium containing 50μ / ml streptomycin, 50 units / ml penicilin (Gibco-BRL). Incubated at. The culture was replaced every three days. At the time when the cells were completely attached to the bottom of the culture vessel and started to grow (after about 3 to 6 days), the cells were separated from the culture vessel with 1 ml of trypsin-EDTA solution and the total number of cells was counted. .

500,000 cells were placed in a 60 mm culture vessel and incubated with 6 ml of the culture solution to which 0.1, 0.5, 1 μM of FK506 was added. When the cells grow and show about 90% confluence in a 60 mm culture vessel, the cells are separated with 1 ml of trypsin-EDTA solution, and the number of cells is measured and 500,000 cells are collected again. It was cultured in a 60 mm culture vessel (5 ml of FK506 addition). This process was repeated to create a growth curve of chondrocytes (FIG. 2).

As a result, the chondrocytes (control) of the medium without adding FK506 did not proliferate after about 8 cell divisions as shown in FIGS. 2 and 3. The total number of cells was 1.44 × 10 ⁸ . However, chondrocytes grown in culture medium containing 0.5 μM FK506 had about 14 cell divisions and the total number of cells was 9.0268 × 10 ⁹ (62.7 times of the control). In addition, chondrocytes grown in culture medium containing 0.1 μM FK506 had about 13.3 cell division, and the total number of cells was 4.9049 X 10 ⁹ (34.1 times the control), and cartilage grown in culture medium containing 1.0 μM FK506. The cells had about 8.6 cell divisions and the total number of cells was 1.967 × 10 ⁸ (1.4 times the control).

On the other hand, Figures 4 and 5 show the results obtained by independently performing the same experiment using the chondrocytes isolated from the joints of another two weeks old rabbit. The results were similar to those of FIGS. 2 and 3. That is, the chondrocytes (control) in the medium without adding FK506 did not proliferate after about 8 cell divisions. The total number of cells was 1.298 × 10 ⁷ . However, chondrocytes grown in culture medium containing 0.1 μM FK506 had about 13 cell divisions and the total number of cells was 3.9891 × 10 ⁸ (30.7 times the control). In addition, chondrocytes grown in culture medium containing 0.5 μM FK506 had about 11.7 cell divisions, and the total number of cells was 1.64 × 10 ⁸ (12.7 times the control), and cartilage grown in culture medium containing 1.0 μM FK506. The cells had about 8.3 cell divisions and the total number of cells was 1.45 × 10 ⁷ (1.1 times the control).

The results shown in FIGS. 2, 3, 4, and 5 showed that the added FK506 increased the number of cell divisions and increased the number of cells, that is, the lifespan of the cells.

<Example 3>

Effect of FK506 on the Growth of Chondrocytes from Old Rabbits

We investigated whether the anti-aging effect of FK506 affects the growth and lifespan of chondrocytes derived from young earthenware, as well as the chondrocytes isolated from the joints of old rabbits.

First, chondrocytes were isolated from the joints of rabbits older than 18 months. Enzymatic digestion of isolated chondrocytes was carried out as in Example 2. After about 6 days of adaptation, 50,000 cells were placed in a 24-well culture vessel and incubated with 1 ml of culture solution containing 0.1, 0.5, 1 μM of FK506. When cells grow and show about 90% confluence in a 24-well culture vessel, the cells are separated with 200 μl of trypsin-EDTA solution and the number of cells is measured. -Incubated in well culture vessel (1 ml of FK506 addition). In this process, we developed the growth curve of aged chondrocytes extracted from three different old rabbits.

As a result, in Figures 6 and 7, chondrocytes (control) of the medium without FK506 did not grow any longer after about 1.6 cell division, and the total number of cells was 1.47 X 10 ⁵ . However, the most potent 0.1 μM FK506 was grown through 5.7 PD (population doubling), and the total number of cells obtained through the culture was 2.6 X 10 ⁶ (17.8 times the control). Chondrocytes grown in culture medium containing 0.5 μM FK506 had about 4.3 cell divisions, and the total number of cells was 9.85 X 10 ⁵ (6.7 times the control), and chondrocytes grown in culture medium containing 1.0 μM CSA About 3.1 cell divisions were performed, and the total number of cells was 4.4 × 10 ⁵ (2.99 times the control).

Meanwhile, in FIGS. 8 and 9, the same experiment as described above was independently performed using chondrocytes isolated from 3.0 kg of other rabbits about 2 years old. As a result, the chondrocytes of the culture medium without FK506 were divided 1.8 times, and the total number of cells was 1.7 X 10 ⁵ , whereas about 0.1 cell division was performed in the case of 0.1 μM FK506, and the total number of cells was 4.25 X 10. ⁶ (25 times the control). For 0.5 μM FK506, 1.39 X 10 ⁶ cells (8.2 times the control) were obtained through 6.8 cell divisions, and for 1.0 μM FK506, about 4.9 cell divisions were 8.0 X 10 ⁵ (4.7 for control groups). Embryonic cells were obtained.

In addition, the same experiment as described above was independently performed in FIGS. 10 and 11 using chondrocytes isolated from 3.0 kg of other rabbits, about 2 years old. As a result, the chondrocytes of the culture medium without FK506 were divided 2.2 times, and the total number of cells was 2.3 X 10 ⁵ . In contrast, about 6.5 μM FK506 had about 6.5 cell divisions and showed a total cell number of 4.49 × 10 ⁶ (19.6 times the control). 0.5 μM of FK506 showed 2.14 X 10 ⁶ (9.3 times the control) cell counts through 5.4 divisions, and 1.0 μM of FK506 showed 9.51 X 10 ⁵ (4.8 times the control group) through 4.3 cell divisions. Cell total number).

As described above, FK506 showed the effect of promoting the growth of aged chondrocytes in three experiments. While Example 2 was shown to increase the growth capacity of chondrocytes of young rabbits with excellent proliferative activity, in this Example it was shown to promote the growth capacity of the cells derived from old individuals already reached aging.

According to the present invention FK506 or a derivative thereof is effective in inhibiting aging of cultured cells and extending the life of cultured cells.

Claims (18)

Exposing the cells in the culture medium to an amount of FK506 or a derivative thereof effective to inhibit cell aging, thereby inhibiting aging of the cells in the culture. Exposing the cells in the culture medium to an FK506 or derivative thereof in an amount effective to prolong the cell lifespan, thereby extending the lifespan of the cells in the culture. Exposing the cells in the culture medium to an effective amount of FK506 or a derivative thereof to enhance cell growth. Exposing the cells in the culture medium to an amount of FK506 or derivative thereof effective to increase viability, thereby increasing the viability of the cells in the culture. Exposing the cells in the culture medium to FK506 or a derivative thereof in an amount effective to adapt the cells to the culture. The method according to any one of claims 1 to 5, wherein the derivative of FK506 is selected from the group consisting of nor-C9 keto-FK506 derivative compounds represented by the following formula (I): (I) Wherein R 1 and R 2 are independently H or an easily removable hydroxy protecting group. The method according to any one of claims 1 to 5, wherein the derivative of FK506 is selected from the group consisting of macrocyclic compounds represented by the following formula (II) and pharmaceutically acceptable salts thereof: (II) In the above formula, Each adjacent pair of substituents [R ₁ and R ₂ ], [R ₃ and R ₄ ], [R ₅ and R ₆ ] are independently (a) represent two adjacent hydrogen atoms, or (b) form a second bond between adjacent carbon atoms to which they are attached; R ₂ may be an alkyl group in addition to the above definitions; R ₇ may represent H, OH or O-alkyl or together with R 1 may represent ═O; R ₈ and R ₉ independently represent H or OH; R ₁₀ represents H, alkyl, alkyl substituted by one or more hydroxyl groups, alkenyl, alkenyl substituted by one or more hydroxyl groups or alkyl substituted by ═O; X represents O, (H, OH), (H, H) or -CH ₂ O-; Y represents O, (H, OH), (H, H), N-NR ₁₁ R ₁₂ or N-OR ₁₃ ; R ₁₁ and R ₁₂ independently represent H, alkyl, aryl or tosyl; R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₂ and R ₂₃ independently represent H or alkyl; R ₂₀ and R ₂₁ independently represent O, or independently represent (R ₂₀ a, H) and (R ₂₁ a, H), respectively, or OH, O-alkyl or OCH ₂ OCH ₂ CH ₂ OCH ₃ Or they may together represent an oxygen atom in the epoxide ring; n is 1, 2 or 3; In addition to the above definitions, Y, R ₁₀ and R ₂₃ may represent a 5- or 6-membered N-, S- or O-containing saturated or unsaturated heterocyclic ring together with the carbon atoms to which they are attached, The ring may be substituted by one or more groups selected from alkyl, hydroxyl, alkyl, O-alkyl, benzyl and -CH ₂ Se (C ₆ H ₅ ) substituted by one or more hydroxyl groups; Provided that when X and Y both represent O, R ₉ represents OH; R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ and R ₂₂ each represent methyl; R ₂₀ represents OCH ₃ ; R ₈ and R ₂₃ each represent H; R ₂₁ a represents OH; [R ₃ and R ₄ ] and [R ₅ and R ₆ ] each represent a carbon-carbon bond; (a) when n is 1 and R ₇ represents an OH group and R ₁ and R ₂ are each hydrogen, then R ₁₀ does not represent an allyl group; (b) when n is 2, R ₇ represents OH and R ₁ and R ₂ each represent hydrogen, then R ₁₀ is not methyl, ethyl, propyl or allyl; (c) n is 2, R ₇ represents hydrogen, and [R ₁ and R ₂ ] represent a carbon-carbon bond, R ₁₀ does not represent an allyl group. 6. The cell of claim 1, wherein the cells are mammalian brain, heart, lung, stomach, intestine, thyroid, adrenal gland, thymus, parathyroid gland, testes, liver, kidney, bladder, spleen, pancreas, gallbladder, ovary , Uterine, prostate, skin, sperm, egg, lymph node, bone, cartilage, epilepsy, immune cells, macrophages, lymphocytes, white blood cells, red blood cells and platelets. 6. The method of claim 1, wherein the effective amount of FK506 or derivative thereof is 1 pg to 100 pg per ml cell culture based on a density of 10 ⁵ to 10 ⁷ cells / ml. A cell culture medium useful for inhibiting aging of cells in culture, characterized in that it comprises an amount of FK506 or a derivative thereof effective to inhibit aging of cells. A cell culture medium useful for prolonging the life of a cell in culture, characterized in that it comprises an amount of FK506 or a derivative thereof effective to prolong cell life. A cell culture medium useful for enhancing the growth of cells in a culture, characterized in that it comprises an amount of FK506 or a derivative thereof effective to enhance cell growth. A cell culture medium useful for extending the viability of cells in a culture, characterized in that it comprises an amount of FK506 or a derivative thereof effective to enhance cell viability. A cell culture medium useful for adapting cells to a culture, characterized in that it comprises an amount of FK506 or a derivative thereof effective to adapt the cell to the culture. The culture medium according to any one of claims 10 to 14, wherein the derivative of FK506 is selected from the group consisting of nor-C9 keto-FK506 derivative compounds represented by the following formula (I): (I) Wherein R 1 and R 2 are independently H or an easily removable hydroxy protecting group. The culture medium according to any one of claims 10 to 14, wherein the derivative of FK506 is selected from the group consisting of a macrocyclic compound represented by the following formula (II) and a pharmaceutically acceptable salt thereof: (II) In the above formula, Each adjacent pair of substituents [R ₁ and R ₂ ], [R ₃ and R ₄ ], [R ₅ and R ₆ ] are independently (a) represent two adjacent hydrogen atoms, or (b) form a second bond between adjacent carbon atoms to which they are attached; R ₂ may be an alkyl group in addition to the above definitions; R ₇ may represent H, OH or O-alkyl or together with R 1 may represent ═O; R ₈ and R ₉ independently represent H or OH; R ₁₀ represents H, alkyl, alkyl substituted by one or more hydroxyl groups, alkenyl, alkenyl substituted by one or more hydroxyl groups or alkyl substituted by ═O; X represents O, (H, OH), (H, H) or -CH ₂ O-; Y represents O, (H, OH), (H, H), N-NR ₁₁ R ₁₂ or N-OR ₁₃ ; R ₁₁ and R ₁₂ independently represent H, alkyl, aryl or tosyl; R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ , R ₂₂ and R ₂₃ independently represent H or alkyl; R ₂₀ and R ₂₁ independently represent O, or independently represent (R ₂₀ a, H) and (R ₂₁ a, H), respectively, or OH, O-alkyl or OCH ₂ OCH ₂ CH ₂ OCH ₃ Or they may together represent an oxygen atom in the epoxide ring; n is 1, 2 or 3; In addition to the above definitions, Y, R ₁₀ and R ₂₃ may represent a 5- or 6-membered N-, S- or O-containing saturated or unsaturated heterocyclic ring together with the carbon atoms to which they are attached, The ring may be substituted by one or more groups selected from alkyl, hydroxyl, alkyl, O-alkyl, benzyl and -CH ₂ Se (C ₆ H ₅ ) substituted by one or more hydroxyl groups; Provided that when X and Y both represent O, R ₉ represents OH; R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ and R ₂₂ each represent methyl; R ₂₀ represents OCH ₃ ; R ₈ and R ₂₃ each represent H; R ₂₁ a represents OH; [R ₃ and R ₄ ] and [R ₅ and R ₆ ] each represent a carbon-carbon bond; (a) when n is 1 and R ₇ represents an OH group and R ₁ and R ₂ are each hydrogen, then R ₁₀ does not represent an allyl group; (b) when n is 2, R ₇ represents OH and R ₁ and R ₂ each represent hydrogen, then R ₁₀ is not methyl, ethyl, propyl or allyl; (c) n is 2, R ₇ represents hydrogen, and [R ₁ and R ₂ ] represent a carbon-carbon bond, R ₁₀ does not represent an allyl group. The method of claim 12, wherein the cells are mammalian brain, heart, lung, stomach, intestine, thyroid gland, adrenal gland, thymus, parathyroid gland, testes, liver, kidney, bladder, spleen, pancreas, gallbladder, ovary Culture medium which is a cell selected from the group consisting of, uterus, prostate, skin, sperm, egg, lymph node, bone, cartilage, epilepsy, immune cell, macrophage, lymphocyte, leukocyte, erythrocyte and platelet. The culture medium according to any one of claims 12 to 16, wherein the effective amount of FK506 is 1 pg to 100 pg per ml of cell culture based on the density of 10 ⁵ to 10 ⁷ cells / ml.