JP2012060976A - Cytostatic agent, and preservation solution for cell or organ - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide new and useful uses of a yamamarin derivative, and to provide particularly a preservation solution for a cell and an organ preventing the occurrence of post-ischemic reperfusion disorders of a transplanted organ and further exhibiting sufficient preservation effects even without regulating the preservation temperature of the organ to a low temperature.SOLUTION: The preservation solution includes C6-DILRG-NHand C10-DILRG-NHas active ingredients.

Description

The present invention relates to a cell growth inhibitor, a preservation solution for cells or organs.

The present inventors have found that, in their research on the silkworm (Antheraea yamamai) of the family Saturniidae, they have an amino acid sequence, aspartic acid-isoleucine-leucine-arginine-glycine (DILRG), and the C-terminal is amidated. , A new peptide having a molecular weight of 570.959 was found, and a patent was obtained by clarifying the dormancy control action and the cancer cell growth inhibition action of this peptide (Patent Documents 1 and 2). In addition, this peptide is also named “Yamamarin” by the present inventors.

In addition, the present inventors have advanced research on peptide derivatives in order to increase the cell permeability and cell growth inhibitory activity of the peptides, and have been named as a conjugate with palmitic acid (“C16-yamamarine”). ) Has been reported (Non-patent document 1).

However, in Non-Patent Document 1, a cytostatic effect has not been confirmed for peptide derivatives (C2, C8) other than C16-yamamulin. Therefore, it was difficult to say that the useful use of the peptide derivative having a carbon number of C16 or less was established.

On the other hand, with the advancement of medical technology, organ transplant surgery has been widely performed. When the damage to the organs of the victim is so great that recovery from normal treatment cannot be expected, the donor's organs are transplanted to the recipient and treated. A transplanted organ removed from an organ donor (donor) for transplant surgery has a blood flow disruption and no oxygen supply via the blood flow (ischemic state), and may take several minutes to several hours before transplantation. Saved. At this time, storage conditions such as storage temperature and storage solution may not be properly selected, or it may take a long time before transplantation. In such cases, when the blood flow in the transplanted organ is restored by transplantation (during reperfusion), the transplanted organ may have a matrix or functional disorder. For example, in liver transplantation, there is a case in which graft dysfunction is observed after the transplantation due to microcirculatory disorder accompanied by increased coagulation activity and thrombus formation. Such an injury is generally called post-ischemic reperfusion injury of the transplanted organ.

Studies have been conducted on methods for preventing such symptoms and preserving the organs for transplantation in a physiologically favorable state. The organ preservation methods that have been reported so far are roughly classified into 1) perfusion method and 2) simple immersion preservation method.

In the perfusion method, organs are perfused during the preservation period to replenish cells with necessary oxygen and nutrients and to remove waste products, thereby maintaining organ metabolism and extending the preservation period. .. However, since the organ preservation by the perfusion method involves various factors such as perfusion temperature, perfusion pressure, perfusion rate, and composition of perfusate, detailed optimal conditions have not been established.

On the other hand, the simple immersion preservation method is a method of preventing tissue damage due to oxygen deficiency by keeping organs at a low temperature and suppressing cell metabolism, and is widely used in clinical settings as a simple and effective method. ing. Specifically, before or after extraction, it is common to wash the blood components from the inflowing blood vessels and the like from the vascular bed of the organ using a low temperature preservation solution, and then immerse the excised organ in the preservation solution.

Well-known organ preservation solutions that have been put into practical use are Eurocollins solution containing glucose and various electrolytes and Wisconsin solution containing impermeable component, oncotic pressure component, energy metabolism promoting component and hormone, respectively. Has been. However, although Euro-Collins solution is effective for highly viable kidneys, it is said that it does not have sufficient tissue/cell protection effects on organs other than kidneys, and Wisconsin solution is not suitable as a formulation. It is said that it is stable and has to be stored at low temperature after preparation.

In order to overcome such drawbacks, various organ preservation solutions have been proposed (for example, Patent Documents 3, 4, and 5). However, some of these organ preservation solutions also have problems such as difficulty in preparation of preparations and maintenance of homeostasis, and low solubility in water.

Further, in any of the above organ preservation solutions, there is a fundamental problem that the function recovery of the organ after transplantation is hindered because it is necessary to keep the organ at a low temperature. Further, it has been pointed out that the fact that a cooling device or the like is required and the burden on the transportation of organs is large should be improved.

Patent No. 3023790 Patent No. 3579711 JP 2000-191401 A JP 2002-60301 JP 2005-306749 JP

Yang et al. A Palmitonyl Conjugate of an Insect Pentapeptide Causes Growth Arrest in Mammalian Cells and Mimics the Action of Diapause Hormone (2007).

Then, from the background described above, the present inventor has found new findings regarding the cell growth inhibitory effect of peptide derivatives other than C16-yamamulin.

The present invention provides a novel and useful application of peptide derivatives other than C16-Yamamarine, in particular, prevents the occurrence of post-ischemic reperfusion injury of a transplanted organ, and further, even if the storage temperature of the organ is not low, An object is to provide a cell and organ preservation solution that exhibits a sufficient preservation effect.

The cell growth inhibitor of the present invention is characterized by containing a compound represented by the following formula as an active ingredient.

(R in the formula represents an acyl group having 6 or 10 carbon atoms)
Furthermore, the preservation solution for cells or organs of the present invention is characterized by containing a compound represented by the following formula as an active ingredient.

(R in the formula represents an acyl group having 6 or 10 carbon atoms)
The method for preserving cells or organs of the present invention is characterized by immersing the cells or organs in a preservation solution for cells or organs containing a compound represented by the following formula as an active ingredient.

(R in the formula represents an acyl group having 6 or 10 carbon atoms)
In the method for preserving cells or organs of the present invention, the temperature of the preservation solution is preferably 5 to 37°C.

Furthermore, the novel compound of the present invention is represented by the following formula.

(R in the formula represents an acyl group having 6 or 10 carbon atoms)

The cell growth inhibitor of the present invention can markedly suppress cell growth. Therefore, the subculture interval can be changed flexibly, and the labor of a cell culture worker or the like can be significantly reduced. Moreover, the preservation solution of the present invention can effectively preserve cells or organs.

The cell growth inhibitor of the present invention contains the following compounds as active ingredients.

R in the formula in this compound represents an acyl group (hereinafter, referred to as “N-terminally acylated DILRG-NH ₂ ”), and this N-terminally acylated DILRG-NH ₂ has hitherto been studied by the present inventors. Of the "peptide having aspartic acid-isoleucine-leucine-arginine-glycine and having an amidated C-terminal" (hereinafter referred to as "DILRG-NH ₂ "), which was found by It can be synthesized by doing. As DILRG-NH ₂ , for example, those isolated and purified from larvae of silkworm (Antheraea yamamai) can be used, or those produced by a known peptide synthesis method can also be used. Although it can be obtained by other methods, the peptide synthesis method is preferable in consideration of economical efficiency and mass productivity.

The introduction of the acyl group can be carried out by a known method, and the carbon number of the acyl group in the N-terminal acylated DILRG-NH ₂ is determined by the cell permeability, the effect of suppressing the oxygen consumption of cells, and the effect of suppressing the cell growth. From the viewpoint, it can be 6 or 10.

Cells targeted by the cell growth inhibitor of the present invention include stem cells, skin cells, mucosal cells, hepatocytes, pancreatic islet cells, nerve cells, chondrocytes, endothelial cells, epithelium isolated from human or non-human animal tissues. It includes cells, bone cells and muscle cells, and further includes sperms, eggs or fertilized eggs of animals such as livestock and fish, insect cells, plant cells and the like.

Furthermore, the cell growth inhibitor of the present invention can be used, for example, by adding an appropriate amount (for example, a final concentration of about 10 μM to 10 mM) to cells cultured in a known medium. Further, the cell growth inhibitor of the present invention can be used, for example, in a state of being dissolved in an organic solvent or the like. The cell growth inhibitor of the present invention can include various other known compositions provided that it does not inhibit its growth inhibitory activity. According to the cell growth inhibitor of the present invention, the subculture interval can be flexibly changed, and the labor of a cell culture worker or the like can be significantly reduced.

The preservation solution of the present invention is based on the novel finding that N-terminally acylated DILRG-NH ₂ can reversibly suppress oxygen consumption of cells and organs. No organ preservation solution using such insect-derived peptide has been known at all.

The reversible effect of suppressing oxygen consumption means that it is useful as a preservative for cells and organs.

That is, the preservation solution of the present invention is prepared by using N-terminally acylated DILRG-NH ₂ as a solution. For example, when the transplanted organ is preserved, the organ is immersed in the preservation solution during the preservation period. By suppressing the oxygen consumption and removing the organ from the preservation solution before transplantation, the oxygen consumption of the organ can be returned to the normal level. Therefore, it is possible to prevent the tissue damage of the organ due to the lack of oxygen.

Furthermore, N-terminally acylated DILRG-NH ₂ has a small number of amino acids serving as the skeleton of the chemical structure, and is an extremely short small molecule, so that it is easy to prepare a stock solution and maintain homeostasis, for example. .. Further, it can be stored at room temperature, has excellent storability and handleability, and can be stored for a long time.

In addition, the preservation solution of the present invention, even in the form of N-terminally acylated DILRG-NH ₂ alone, has N-terminally acylated DILRG-NH ₂ and other components such as glucose, maltose, sucrose, lactose, and raffinose. , Trehalose, mannitol, hydroxyethyl starch, sugars such as pullulan, gluconic acid, lactic acid, acetic acid, propionic acid, β-hydroxybutyric acid, organic acids such as citric acid, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, phosphoric acid Electrolytes such as sodium dihydrogen, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate, potassium carbonate, vitamins such as L-ascorbic acid and vitamin E, glycine, glutamic acid , Amino acids such as lysine, antidiuretic hormones, hormones such as insulin, citric acid, citrate, heparin, anticoagulants such as sodium edetate, calcium antagonists, adrenergic β receptor antagonists, angiotensin converting enzyme inhibitors, etc. Antihypertensive agents, nucleobases such as adenosine phosphate, antifreeze agents such as antifreeze proteins, active oxygen quenchers, cell activating agents, antibiotics, antiplatelet factors, liver injury inhibitors, excipients, binders, disintegration Organ preservation liquids such as agents, dispersants, viscous agents, reabsorption promoters, surfactants, solubilizers, preservatives, preservatives, emulsifiers, isotonic agents, stabilizers, buffers, pH adjusters, etc. It may be in the form of a composition with one or a plurality of the components usually incorporated in the above.

Further, when the preservative solution of the present invention is used by being mixed with a known organ preservative solution such as Euro-Collins solution or Wisconsin solution, their organ preserving ability can be improved.
Furthermore, when cells and organs are preserved by the preservative of the present invention, the concentration of N-terminally acylated DILRG-NH _{2 in} the preservation solution should be appropriately determined according to the types of cells and organs and other conditions. You can Specifically, a range of 10 μM to 10 mM can be exemplified.

The preferred application temperature for using the storage solution of the present invention is 5 to 37°C, particularly preferably 25 to 37°C. Unlike the conventional case, the preservation solution of the present invention does not necessarily need to preserve the organ at a low temperature, so that the function of the organ can be smoothly restored after transplantation. Of course, there is a problem of functional recovery in organ transplantation, but the temperature of the preservation solution can be low (for example, 5° C. or lower), and in this case, cells and organs can be preserved for a longer period of time. ..

Then, the target cells of the preservation solution of the present invention include, for example, stem cells isolated from human or non-human animal tissues, skin cells, mucosal cells, hepatocytes, pancreatic islet cells, nerve cells, chondrocytes, endothelial cells, It includes epithelial cells, osteocytes, muscle cells, and further includes sperm, eggs or fertilized eggs of animals such as livestock and fish, insect cells, plant cells and the like.

Furthermore, the organs to which the preservation solution of the present invention is applied include skin, blood vessel, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta or pancreas.

Examples of the present invention will be described below. The present invention is not limited to the examples below.
<1> Synthesis of N-terminally acylated DILRG-NH _{2 Using} a peptide synthesizer (PSSM-8, manufactured by Shimadzu Corp.), N-terminal free on the resin, a peptide with a protecting group, and aspartic acid were prepared by a conventional method. - isoleucine - leucine - arginine - were synthesized glycine _{-NH 2 (DILRG-NH 2)} .
Then, the peptide resin is suspended in a mixed solvent of dimethylformamide (DMF) and pyridine, and hexanoic acid (C6 carboxylic acid) or decanoic acid (C10 carboxylic acid) and WSCD(1-ethyl-3-(3-dimethylaminoprppyl)- carbodiimide hydrochloride) was added, and the mixture was stirred overnight at room temperature. After the reaction was completed, the resin was collected by filtration and washed with DMF and methanol. The thus obtained acyl peptide resin was cocktail treated cut off in the usual way to give the crude acylated _{DILRG-NH2 (C6-DILRG-} NH 2, C10-DILRG-NH 2). The acylation of the peptide resin with the mixture of hexanoic acid or decanoic acid and WSCD used here can also be achieved by treatment with a corresponding carboxylic acid chloride (hexanoyl chloride or decanoyl chloride). Hereinafter, a C6-DILRG-NH ₂ and C10-DILRG-NH ₂ is described as "C6- Yamamarin, C10-Yamamarin".

The purification was performed by connecting a reversed-phase column Develosil-ODS HG-5 (20 mm×250 mm, Nomura Chemical Co., Ltd.) to an HPLC system (Gulliver Co., Ltd., JASCO Corporation). Elution was carried out at a flow rate of 4 ml/min with a gradient of acetonitrile (0 to 100% in 0 to 120 minutes) in the presence of 0.1% trifluoroacetic acid (TFA) to elute the active fraction. It was Absorbance was measured at 220 nm. The peptide was mixed with an equal amount of matrix (saturated with 40% acetonitrile/0.1% TFAα-CHCA) on a sample plate and then dried, and MALDI-TOF MS (Discovery, Shimadzu Corporation). The structure was confirmed by.

<2> Cell proliferation inhibition test (1) HepG2 cells (human hepatoma cells)
HepG2 cells were seed-cultured in a DMEM medium (manufactured by Sigma) containing 10% FCS (GIBCO, Low-IgG fetal calf serum) at 37° C. under a CO ₂ concentration of 5%. Then, the seed cells were dispensed into a 96-well culture plate with 100 μL of 2.0×10 ⁴ cells/mL cells per well.

After culturing this plate for 1 day at 37°C and CO ₂ concentration of 5% and confirming that the cells adhered to the bottom of the well, the test substance was added to the culture plate under the following 3 conditions. Culturing was carried out for 5 days under conditions of 37° C. and 5% CO ₂ concentration.
1) Add 1.0 μL of PBS per well as Control 2) Add 1.0 μL of 200 mM C6-yamamaline dissolved in DMSO per well (final concentration 2 mM)
3) Add 0.5 μL of 20 mM C10-yamamarine dissolved in DMSO per well (final concentration 100 μM)
The cell number was measured daily before the addition of the test substance, 0 days, and from 1 to 5 days after the addition of the test substance in a proliferation confirmation test by WST-1 assay (Premix WST-1 manufactured by Takara Bio Inc.).

That is, 10 μL of Premix WST-1 was added to each well of the target microplate, the mixture was incubated at 37° C. under a CO ₂ concentration of 5% for 1 hour, and then the absorbance was measured at 450 nm. The number of cells in the WST-1 assay was determined from the WST-1 assay standard curve prepared in advance.

As a result, while the cells became Confrant with Control without addition of C6 Yamamamarin and C10 Yamamamarin, in the case where C6 Yamamamarin and C10 Yamamamarin were added at the start of the culture, the cells did not become confrent even after 5 days of culture. The culture could be continued (Table 1).

(2) Confirmation of the effect of addition of C6 Yamamaline and C10 Yamamaline using various cells 1) K562 cells (CML, chronic myelogenous leukemia) in RPMI medium containing 10% FCS (GIBCO, Low-IgG fetal bovine serum) Seed culture was performed in Nissui Pharmaceutical under the conditions of 37° C. and a CO ₂ concentration of 5%.
2) After thawing NHDF (normal human skin fibroblasts, Kurabo), seed in Medium 106S medium (Kurabo) with LSGS custom growth additive at 37°C and 5% CO ₂ concentration. Culture was performed.
3) Seed HepG2 cells (human hepatoma cells) in DMEM medium (Sigma) containing 10% FCS (GIBCO, Low-IgG fetal bovine serum) at 37°C and 5% CO ₂ concentration. Culture was performed.

Then, for K562 seed cells, 100 μL of 1.1×10 ⁴ cells/mL cells were dispensed per well into a 96-well culture plate. For NHDF seed cells, 100 μL of 2.4×10 ⁴ cells/mL cells were dispensed per well into a 96-well culture plate. For HepG2 seed cells, 100 μL of 2.0×10 ⁴ cells/mL cells were dispensed into a 96-well culture plate.

Furthermore, the test substance was added to the culture plate into which the cells had been dispensed under the following three conditions, and the cells were cultured under the conditions of 37° C. and a CO ₂ concentration of 5%.
1) Add 1.0 μL of PBS per well as Control 2) Add 1.0 μL of 200 mM C6-yamamaline dissolved in DMSO per well (final concentration 2 mM)
3) Add 0.5 μL of 20 mM C10-yamamarine dissolved in DMSO per well (final concentration 100 μM)
The effect of C6 Yamamallin and C10 Yamamallin on each cell was determined on the day of the start of culture and on the 5th day of culture by observing under a microscope whether or not the cells in the visual field were proliferating.

These results (Table 2) show that, although there are some variations depending on the cell type, basically, C6 Yamamamarin and C10 Yamamamarin have a growth inhibitory effect on all cells ([Table 2]). ).

The concentration and timing of addition of C6 Yamamamarin and C10 Yamamamarin can be appropriately set, and it is also possible to culture for a longer period without passage. Therefore, these C6 Yamamarin and C10 Yamamarin can flexibly change the subculture interval and provide a method for significantly reducing the labor of cell culture workers and the like.

In addition, it can be presumed that such cell growth inhibitory effect of C6 Yamamarin and C10 Yamamarin was able to reversibly suppress the oxygen consumption of cells. Therefore, by using C6 Yamamarine and C10 Yamamarine as active ingredients, they can be effectively used as a preservation solution for cells and organs.

Claims (5)

A cell growth inhibitor comprising a compound represented by the following formula as an active ingredient.
(R in the formula represents an acyl group having 6 or 10 carbon atoms) A preservation solution for cells or organs, which contains a compound represented by the following formula as an active ingredient.
(R in the formula represents an acyl group having 6 or 10 carbon atoms) A method for preserving cells or organs, which comprises immersing the cells or organs in a preservative solution for cells or organs containing a compound represented by the following formula as an active ingredient.
(R in the formula represents an acyl group having 6 or 10 carbon atoms) The method for preserving cells or organs according to claim 2, wherein the temperature of the preservation solution is 5 to 37°C. A novel compound represented by the following formula.
(R in the formula represents an acyl group having 6 or 10 carbon atoms)