JP5771376B2 - Cell growth inhibitor, cell or organ preservation solution - Google Patents

Description

  The present invention relates to a cell growth inhibitor, a cell or organ preservation solution.

  In the research on Stherniidae's Antheraea yamamai, the present inventors have an amino acid sequence, aspartic acid-isoleucine-leucine-arginine-glycine (DILRG), and the C-terminus is amidated. In addition, a novel peptide having a molecular weight of 570.959 was found, and patents were obtained by clarifying the dormancy control action and cancer cell growth inhibitory action of this peptide (Patent Documents 1 and 2). This peptide is also named “Yamamarin” by the present inventors.

  In addition, the present inventors proceeded with research on peptide derivatives in order to increase the cell permeability and cytostatic activity of the peptide, and named a conjugate with palmitic acid (named “C16-Yamamarin”). ) Has been reported (Non-patent Document 1).

  However, in Non-Patent Document 1, no cell inhibitory effect has been confirmed for peptide derivatives (C2, C8) other than C16-yamamarin. Therefore, it has been difficult to say that a useful application of a peptide derivative having a carbon number of C16 or less has been established.

  On the other hand, with the advancement of medical technology, organ transplant surgery has been widely performed. If the organ damage in the patient is very large and recovery by normal treatment cannot be expected, the donor organ is transplanted to the recipient and treated. Transplanted organs removed from organ donors (donors) for transplantation are in a state where blood flow is interrupted and oxygen is not supplied via the blood flow (ischemic state), and it takes several minutes to several hours until transplantation. Saved. At this time, storage conditions such as storage temperature and storage solution may not be appropriately selected, and it may take a long time to transplant. In such a case, when the blood flow in the transplanted organ is restored by transplantation (at the time of reperfusion), a substrate or functional disorder may occur in the transplanted organ. For example, in liver transplantation, graft dysfunction may be observed due to microcirculatory disturbance with increased coagulation activity and thrombus formation after transplantation. Such a disorder is generally called a post-ischemic reperfusion disorder of a transplanted organ.

  Studies have been made on methods for preventing such symptoms and preserving transplanted organs in a physiologically favorable state. The organ preservation methods reported to date are roughly divided into two methods: 1) perfusion method, and 2) simple immersion preservation method.

  The perfusion method performs organ perfusion during the preservation period, supplies the cells with necessary oxygen and nutrients, and removes waste products, thereby maintaining organ metabolism and extending the preservation period. . However, organ preservation by the perfusion method involves various factors such as perfusion temperature, perfusion pressure, perfusion flow rate, and composition of the perfusate, and detailed optimum conditions have not been established.

  On the other hand, the simple immersion preservation method is a method for 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, a method is generally used in which blood components are washed from the blood vessel bed of the organ using a low-temperature preservation solution from an inflowing blood vessel or the like before or after the removal, and the extracted organ is immersed in the preservation solution.

  Well-known organ preservation solutions include Eurocollins solution containing glucose and various electrolytes, and Wisconsin solution containing impermeable components, colloid osmotic pressure components, energy metabolism promoting components and hormones, respectively. It has been. However, Eurocollins solution is effective for highly viable kidneys, but it is said that the protective effect on tissues and cells is not sufficient for organs other than kidneys, and Wisconsin solution is not suitable as a preparation. It is said to be stable and has the disadvantage of having to be stored at low temperatures after preparation.

  In order to overcome such drawbacks, various organ preservation solutions have also been proposed (for example, Patent Documents 3, 4, and 5). However, these organ preservation solutions also have problems such as difficulty in preparation of the preparation 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 recovery of the function of the organ after transplantation is hindered because the organ needs to be kept at a low temperature. Furthermore, it has been pointed out as a point to be improved that a cooling device or the like is required and the burden of transporting the organ is large.

Patent No. 3023790 Patent No. 3571971 JP 2000-191401 A JP 2002-60301 A JP 2005-306749 A

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

  And from the above background, the present inventor has found a new finding regarding the cell growth inhibitory effect of peptide derivatives other than C16-yamamarin.

  The present invention provides a novel and useful use of peptide derivatives other than C16-Yamamarin, in particular, prevents the occurrence of post-ischemic reperfusion injury in transplanted organs, and further, even if the organ preservation temperature is not reduced, It is an object 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.

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

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

(Wherein R represents an acyl group having 6 or 10 carbon atoms)
In the cell or organ preservation method 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.

(Wherein R represents an acyl group having 6 or 10 carbon atoms)

  The cell growth inhibitor of the present invention can remarkably suppress cell growth. Therefore, the subculture interval can be changed flexibly, and the labor of cell culture workers and 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.

In the compound, R in the formula represents an acyl group (hereinafter referred to as “N-terminal acylated DILRG-NH ₂ ”), and this N-terminal acylated DILRG-NH ₂ has been conventionally studied by the inventors. An acyl group was introduced at the N-terminus of “a peptide having aspartic acid-isoleucine-leucine-arginine-glycine and amidated at the C-terminus” (hereinafter referred to as “DILRG-NH ₂ ”). Can be synthesized. DILRG-NH _2, for example, larvae from the isolation of wild silkworm (Antheraea yamamai), can either be used after purification, it is also possible to use those produced by known peptide synthesis methods. Although it can be obtained by other methods, taking into consideration economic efficiency, mass productivity, etc., acquisition by a peptide synthesis method is preferable.

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

  Examples of cells that are targets of the cell growth inhibitor of the present invention include stem cells, skin cells, mucosal cells, hepatocytes, 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 sperm, 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 by adding an appropriate amount (for example, final concentration of about 10 μM to 10 mM) to cells cultured in a known medium, for example. Moreover, the cell growth inhibitor of this invention can be used in the state melt | dissolved in the organic solvent etc., for example. The cell growth inhibitor of the present invention can include various other known compositions on the condition that its growth inhibitory activity is not inhibited. According to the cell growth inhibitor of the present invention, the subculture interval can be changed flexibly, and the labor of cell culture workers and the like can be significantly reduced.

The preservation solution of the present invention is based on the novel finding that N-terminal acylated DILRG-NH ₂ can reversibly suppress oxygen consumption in cells and organs. No organ preservation solution using such insect-derived peptides 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 preparing N-terminal acylated DILRG-NH ₂ as a solution. For example, when storing a transplanted organ, 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 normal again. Therefore, organ damage caused by oxygen deficiency can be prevented.

Furthermore, since N-terminal acylated DILRG-NH ₂ has a small number of 5 amino acids as the skeleton of the chemical structure and is an extremely short small molecule, for example, it is easy to prepare a stock solution and maintain homeostasis. . In addition, it can be stored at room temperature, has excellent storability and handleability, and can be stored for a long time.

Moreover, even if the preservation solution of the present invention is in the form of N-terminal acylated DILRG-NH ₂ alone, N-terminal acylated DILRG-NH ₂ and other, for example, glucose, maltose, sucrose, lactose, raffinose , Carbohydrates such as trehalose, mannitol, hydroxyethyl starch, pullulan, organic acids such as gluconic acid, lactic acid, acetic acid, propionic acid, β-hydroxybutyric acid, citric acid, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, phosphoric acid Sodium dihydrogen, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium carbonate, potassium carbonate and other electrolytes, L-ascorbic acid, vitamin E and other vitamins, glycine, glutamic acid , Amino acids such as lysine Anti-diuretic hormone, hormones such as insulin, anticoagulants such as citric acid, citrate, heparin, sodium edetate, calcium antagonists, adrenergic β receptor antagonists, antihypertensives such as angiotensin converting enzyme inhibitors, adenosine Nucleobase such as acid phosphate, anti-freezing agent such as anti-freezing protein, active oxygen scavenger, cell activator, antibiotic, antiplatelet factor, liver injury inhibitor, excipient, binder, disintegrant, dispersant, Usually used in organ preservation solutions such as viscosity agents, resorption accelerators, surfactants, solubilizers, preservatives, preservatives, emulsifiers, tonicity agents, stabilizers, buffers, pH adjusters It may be in the form of a composition with one or more components.

In addition, when the preservation solution of the present invention is used in a known organ preservation solution such as Eurocollins solution or Wisconsin solution, the organ preservation ability can be improved.
Furthermore, when cells and organs are stored using the preservative of the present invention, the concentration of N-terminal acylated DILRG-NH _{2 in} the storage solution should be determined appropriately according to the type of cells and organs and other conditions. Can do. Specifically, for example, a range of 10 μM to 10 mM can be exemplified.

  And preferable application temperature as conditions on which the preservation | save liquid of this invention is used is 5-37 degreeC, Most preferably, it is 25-37 degreeC. Since the preservation solution of the present invention does not necessarily need to preserve an organ at a low temperature as in the prior art, the function of the organ after transplantation is smoothly restored. Of course, there is a problem of functional recovery in organ transplantation, but the temperature of the preservation solution can be lowered (for example, 5 ° C. or lower), and in this case, cells and organs can be preserved for a longer period of time. .

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

  Further, organs that are the target of the preservation solution of the present invention include skin, blood vessels, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, pancreas, and the like.

Examples of the present invention will be described below. The present invention is not limited to the following examples.
<1> Synthesis of N-terminal acylated DILRG-NH _{2 Using} a peptide synthesizer (PSSM-8, manufactured by Shimadzu Corporation), N-terminal liberation, protected peptide, and aspartic acid on the resin by a conventional method - isoleucine - leucine - arginine - were synthesized glycine _{-NH 2 (DILRG-NH 2)} .
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 stirred overnight at room temperature. After completion of the reaction, 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 the corresponding carboxylic acid chloride (hexanoyl chloride or decanoyl chloride). Hereinafter, C6-DILRG-NH ₂ and C10-DILRG-NH ₂ are referred to as “C6-yamamarine, C10-yamamarin”.

  The purification was carried out by connecting a reverse phase column Develosil-ODS HG-5 (20 mm × 250 mm, manufactured by Nomura Chemical Co., Ltd.) to an HPLC system (Gulliver Corporation JASCO). Elution is carried out at a flow rate of 4 ml / min using an acetonitrile concentration gradient (0 to 100% from 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, dried, and MALDI-TOF MS (Discovery, manufactured by Shimadzu Corporation) The structure was confirmed by

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

This plate is cultured for one day at 37 ° C under 5% CO ₂ concentration. After confirming that the cells adhere to the bottom of the well, a test substance is added to the culture plate under the following three conditions. The cells were cultured for 5 days under the 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-Yamamarin dissolved in DMSO per well (final concentration 2 mM)
3) Add 0.5 μL per well of 20 mM C10-Yamamarin dissolved in DMSO (final concentration 100 μM)
The number of cells was measured every day before the addition of the test substance, on the 0th day, and after the addition of the test substance on the 1st to 5th days in the growth confirmation test using the WST-1 assay (Premix WST-1 manufactured by Takara Bio Inc.).

That is, 10 μL of Premix WST-1 was added per well of the target microplate, 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 by the WST-1 assay was determined from a WST-1 assay calibration curve prepared in advance.

  As a result, cells became confluent with Control without C6 Yamamarine and C10 Yamamarine added, whereas cells with C6 Yamamarine and C10 Yamamarine added at the start of culture did not become confluent even after 5 days of culture. The culture could be continued (Table 1).

(2) Confirmation of addition effect of C6 Yamamarine and C10 Yamamarine using various cells 1) RPMI medium containing 10% FCS (GIBCO, Low-IgG fetal bovine serum) with K562 cells (CML, chronic myelogenous leukemia) In Nissui Pharmaceutical Co., Ltd.), seed culture was performed under conditions of 37 ° C. and CO ₂ concentration of 5%.
2) NHDF (normal human dermal fibroblasts, the Kurabo Co.), after thawing, in medium supplemented with LSGS custom proliferation additives Medium106S (Kurabo), 37 ° C., seed with 5% CO ₂ conditions Culture was performed.
3) HepG2 cells (human hepatoma cells), 10% FCS (GIBCO Inc., in Low-IgG fetal bovine serum) DMEM medium containing (manufactured by Sigma), 37 ° C., seed with 5% CO ₂ conditions Culture was performed.

Thereafter, for K562 seed cells, 100 μL of 1.1 × 10 ⁴ cells / mL cells were dispensed into a 96-well culture plate. For NHDF seed cells, 100 μL of 2.4 × 10 ⁴ cells / mL cells were dispensed 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, a test substance was added to the culture plate into which the cells were dispensed under the following three conditions, and the culture was performed at 37 ° C. under 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-Yamamarin dissolved in DMSO per well (final concentration 2 mM)
3) Add 0.5 μL per well of 20 mM C10-Yamamarin dissolved in DMSO (final concentration 100 μM)
The effect of C6 Yamamarine and C10 Yamamarine on each cell was determined on the start date of culture and on the 5th day of culture by observing whether or not the cells in the visual field were growing under a microscope.

  Although these results (Table 2) have some variations depending on the cell type, basically, it was found that C6 yamamarin and C10 yamamarin have a growth inhibitory effect on any cell ([Table 2] ).

  In addition, about the addition density | concentration and addition timing of C6 yamamarine and C10 yamamarin, it can set suitably and it can also culture | cultivate without a subculture for a long period of time. Therefore, these C6 Yamamarine and C10 Yamamarine can flexibly change the subculture interval, and provide a method for significantly reducing labor of cell culture workers and the like.

  Moreover, it can be estimated that such cell growth suppression effect by C6 Yamamarine and C10 Yamamarine was able to reversibly suppress the oxygen consumption of cells. Therefore, by using C6 yamamarin and C10 yamamarin as active ingredients, it can be effectively used as a preservation solution for cells and organs.

Claims (4)

An animal cell or organ preservation solution comprising a compound represented by the following formula as an active ingredient.
(Wherein R represents an acyl group having 6 or 10 carbon atoms) An animal cell or organ preservation method comprising immersing an animal cell or organ in an animal cell or organ preservation solution containing a compound represented by the following formula as an active ingredient .
(Wherein R represents an acyl group having 6 or 10 carbon atoms) The method for preserving animal cells or organs according to claim 1, wherein the temperature of the preservation solution is 5 to 37 ° C. A novel compound represented by the following formula:
(Wherein R represents an acyl group having 6 or 10 carbon atoms)