Method for exposing poorly water-soluble test substance to cultured cells

Abstract

To provide a method for exposing a poorly water-soluble test substance to cultured cells.SOLUTION: Provided is a method for exposing a poorly water-soluble test substance to cultured cells is provided, wherein an emulsified product comprising (A) the poorly water-soluble test substance, (B) polyoxyethylene sorbitan fatty acid ester, and (C) a medium is obtained, and then the emulsified product is exposed to the cultured cells for a predetermined period of time, the method using a cell survival rate of the cultured cells as an indicator to evaluate the presence or absence of cytotoxicity or single-dose toxicity of the poorly water-soluble test substance.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for exposing a poorly water-soluble test substance to cultured cells.

Background Art

[0002] When exposing a test substance to cultured cells, there are methods of directly adding the test substance to the culture medium or dissolving the test substance in a water-soluble solvent such as water or physiological saline and then adding it to the culture medium. Further, when the test substance is poorly water-soluble, there is a method of dissolving the test substance in an organic solvent such as dimethyl sulfoxide (DMSO) or ethanol and adding it to the culture medium. However, since these organic solvents exhibit cytotoxicity when their concentration in the culture medium becomes high, it is necessary to dissolve the test substance in the organic solvent in advance so that the concentration becomes 100 to 1000 times the final concentration and add it so that it becomes 0.1 wt% to 1.0 wt% with respect to the total amount of the culture solution. Therefore, when the solubility of the test substance in the organic solvent is low, the final concentration cannot be set high, and there are cases where the toxicity of a test substance that originally has toxicity cannot be correctly evaluated.

[0003] Further, when the test substance is insoluble in both water and an organic solvent, there is a method of dispersing the test substance in the culture medium. However, since the test substance floats on the upper part of the culture medium, it may not be clear whether the test substance is in contact with the cells. Further, when it precipitates at the lower part of the culture medium, the test substance may physically contact the cells and have an adverse effect on the cells. Therefore, a technique for uniformly dispersing the test substance in the culture medium has been desired.

[0004] As methods for exposing poorly water-soluble test substances to cultured cells, methods using additives for media containing zwitterions having both positive and negative charges within one molecule (Patent Document 1), methods of embedding cells in a biocompatible gel that can be reversibly gelled and bringing the gel containing the cells into contact with the poorly water-soluble test substance (Patent Document 2), methods using copolymers of 2-methacryloyloxyethyl phosphorylcholine and n-stearyl methacrylate / n-lauryl methacrylate (Patent Document 3), etc. have been proposed. However, in the method of Patent Document 1, the zwitterions may affect the evaluation results by affecting the cells. Also, in the method of Patent Document 2, the poorly water-soluble test substance may affect the gelation of the medium and thus affect the evaluation results. Furthermore, in the method of Patent Document 3, in the case of a test substance having a charge, it may adversely affect the dispersibility of the copolymer.

[0005] Single-dose toxicity refers to a toxic reaction that indicates changes in the general condition including lethality caused by a single administration of a test substance, and is generally evaluated using rodents such as mice and rats. Recently, from the perspective of reducing animal experiments due to animal welfare, a single-dose toxicity evaluation method using cultured cells as an alternative to animal experiments has also been proposed (Non-Patent Document 1). However, the single-dose toxicity evaluation method using cultured cells can be carried out when the test substance dissolves or is uniformly dispersed in the medium, but cannot be carried out for test substances that do not meet these conditions.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a method for exposing a poorly water-soluble test substance to cultured cells.

Means for Solving the Problems

[0009] That is, the present invention provides a method for exposing a poorly water-soluble test substance to cultured cells, comprising: obtaining an emulsion containing (A) a poorly water-soluble test substance, (B) a polyoxyethylene sorbitan fatty acid ester, and (C) a culture medium, and then exposing the emulsion to cultured cells for a certain period of time.

[0010] The present invention also provides a method for exposing a poorly water-soluble test substance to cultured cells, characterized in that (A) the octanol / water partition coefficient of the poorly water-soluble test substance is 3.5 or more.

[0011] The present invention also provides a method for exposing a poorly water-soluble test substance to cultured cells, characterized in that (B) the polyoxyethylene sorbitan fatty acid ester is polysorbate 65.

[0012] The present invention also provides a method for evaluating the presence or absence of cytotoxicity of a poorly water-soluble test substance, characterized by using the above exposure method and using the cell viability of cultured cells as an index.

[0013] The present invention also provides a method for evaluating the presence or absence of single-dose toxicity of a poorly water-soluble test substance, characterized by using the above exposure method and using the cell viability of cultured cells as an index.

[0014] The present invention also provides an evaluation kit for carrying out the above-described evaluation.

Advantages of the Invention

[0015] According to the present invention, it becomes possible to expose a poorly water-soluble test substance, which has been difficult heretofore, to cultured cells, and it becomes possible to appropriately evaluate the cytotoxicity and single-dose toxicity of the poorly water-soluble test substance.

Modes for Carrying Out the Invention

[0016] The cultured cells in the present invention refer to cells artificially cultured in vitro. The origin thereof is not particularly limited, but cells derived from humans or animals other than humans can be used. Further, it may be primary cultured cells just collected from a living body or established cultured cells whose proliferative ability is maintained by subculture. Furthermore, it may be adherent cultured cells that grow while adhering to a culture vessel, or floating cultured cells that grow in a suspended state in a medium.

[0017] The (A) poorly water-soluble test substance in the present invention refers to a test substance that does not dissolve in water or in organic solvents such as DMSO or ethanol. The present invention is particularly useful when the octanol / water partition coefficient of the poorly water-soluble test substance is 3.5 or more, and examples thereof include mineral oil, corn oil, 1-octanethiol, and the like. Note that the octanol / water partition coefficient is the ratio of the distribution degrees of a certain chemical substance in n-octanol and water (the ratio of the concentrations in the n-octanol phase and the water phase), and can be calculated, for example, by the flask shaking method defined in OECD Guideline 107, the HPLC method defined in OECD Guideline 117, or quantum chemical calculations or molecular dynamics simulations.

[0018] The (B) polyoxyethylene sorbitan fatty acid ester in the present invention is a kind of nonionic surfactant, which is obtained by adding a polyoxyethylene chain to sorbitan fatty acid ester. For example, polysorbate 20, polysorbate 60, polysorbate 65, polysorbate 80, etc. can be mentioned. Among these, polysorbate 65 is preferably used in view of the ease of emulsification and the stability of the obtained emulsion, etc.

[0019] The (C) medium in the present invention refers to a medium commonly used to maintain the growth property of cultured cells. In adherent cultured cells, for example, Eagle's minimum essential medium (EMEM), Dulbecco's modified Eagle's medium (DMEM), etc. can be used, and in suspension cultured cells, for example, RPMI-1640 medium, etc. can be used. Also, if necessary, serum, growth factors, antibiotics, etc. can be added to these media and used.

[0020] The emulsion in the present invention refers to an oil-in-water (O / W) emulsion having an internal phase of (A) poorly water-soluble test substance and an external phase of (C) medium. The method for preparing the emulsion is not particularly limited, and examples include a method of obtaining an emulsion by mechanical stirring or ultrasonic treatment. Also, if necessary, an emulsion can be obtained by heating (A) poorly water-soluble test substance, (B) polyoxyethylene sorbitan fatty acid ester, and (C) medium to 60°C or higher and then mixing them. Further, for the purpose of enhancing the stability of the emulsion, polymers, alcohols, etc. can be contained in the emulsion if necessary.

[0021] The "certain period of time" in the present invention refers to a time sufficient for efficiently contacting the poorly water-soluble test substance with the cultured cells, and is not particularly limited, and examples include 24 hours, 48 hours, 72 hours, etc.

[0022] The method for evaluating the presence or absence of cytotoxicity in the present invention is not particularly limited. For example, the MTT method, crystal violet staining method (CV staining method), neutral red uptake method (NRU method), protein quantification method, cell count method, etc. can be mentioned. As an evaluation method, for example, the concentration of a poorly water-soluble test substance is set in several steps, the cell viability at each concentration is calculated, and the presence or absence of cytotoxicity can be evaluated by comparing it with a negative control. Further, if necessary, the test substance concentration (IC50) at which the cell viability becomes 50% can be calculated to evaluate the presence or absence of cytotoxicity.

[0023] The method for evaluating the presence or absence of single-dose toxicity in the present invention does not refer to a method using animals such as mice or rats, but refers to an evaluation method using cultured cells as an alternative to animal experiments. The cultured cells to be used are not particularly limited. For example, mouse-derived fibroblasts (3T3 cells), rabbit corneal-derived cells (SIRC cells), etc. can be used. Regarding the presence or absence of single-dose toxicity, for example, when the IC50 exceeds 2,000 μg / mL, it can be evaluated that the single-dose toxicity is low, and when the IC50 is 2,000 μg / mL or less, it can be excluded from the evaluation target (it cannot be said that the single-dose toxicity is low).

[0024] The evaluation kit in the present invention refers to an evaluation kit for exposing an emulsion containing (A) a poorly water-soluble test substance, (B) a polyoxyethylene sorbitan fatty acid ester, and (C) a medium to cultured cells and evaluating the presence or absence of cytotoxicity or single-dose toxicity of (A) the poorly water-soluble test substance using the cell viability of the cultured cells after a certain period as an index. The content of the kit is not particularly limited. For example, a container for weighing (A) the poorly water-soluble test substance containing a predetermined amount of (B) the polyoxyethylene sorbitan fatty acid ester and a container for weighing (C) the medium are respectively set, and a kit that can easily obtain an emulsion of the poorly water-soluble test substance by mixing these, or a kit having a stirring function necessary for mixing these, etc. can be mentioned.

Example

[0025] Examples are given below to specifically describe the present invention, but the technical scope of the present invention is not limited thereto.

[0026] 1. Poorly water-soluble test substance As the poorly water-soluble test substances, mineral oil (Sigma-Aldrich) and corn oil (FUJIFILM Wako Pure Chemical Corporation), which are known to have no toxicity in a single-dose toxicity test using animals, and 1-octanethiol (Tokyo Chemical Industry Co., Ltd.), which is known to have toxicity in a single-dose toxicity test using animals, were used respectively.

[0027] 2. Preparation of emulsion After mixing 0.2 g of the poorly water-soluble test substance and 0.02 g of polyoxyethylene sorbitan fatty acid ester, it was heated to 70°C. Then, DMEM medium heated to 70°C was added to make the total amount 50 g, and then ultrasonic treatment was performed for 2 minutes to prepare an emulsion of the poorly water-soluble test substance.

[0028] 3. Evaluation of emulsion stability The stability of the emulsion immediately after emulsification and 24 hours after emulsification was visually evaluated according to the following criteria. <Criteria> ◎: No oil floating, maintaining the emulsified state 〇: Slight oil floating, but maintaining the emulsified state ×: Not emulsified and separated

[0029]

Table 1

[0030] As shown in Table 1, in Examples 1 to 3 using polyoxyethylene sorbitan fatty acid esters, the stability of the emulsion was good. In particular, in Example 2 using polysorbate 65, excellent stability was shown even after 24 hours. On the other hand, in Comparative Example 1 using polyglyceryl-10 isostearate instead of polyoxyethylene sorbitan fatty acid ester, the emulsified state could not be maintained. From these facts, it became clear that an emulsion excellent in stability can be obtained by using the method of the present invention.

[0031] 4. Evaluation of single-dose toxicity using cultured cells 3T3 cells cultured using DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin were seeded at 3.0×10 3 cells / well in a 96-well multiplate, and then cultured at 37°C in a 5% CO 2 2 environment for 24 hours. After diluting the emulsion prepared above to 8 concentrations with a common ratio of 2, half of the diluted emulsion was added to a 96-well multiplate and further cultured for 48 hours. The highest concentration of each test substance was 2,000 μg / mL. The cell viability at each concentration was calculated by the NRU method, and the IC50 of each test substance was calculated.

[0032] <Judgment criteria> When the IC50 exceeded 2,000 μg / mL, the single-dose toxicity was evaluated as low, and when the IC50 was 2,000 μg / mL or less, it was excluded from the evaluation target.

[0033]

Table 2

[0034] As shown in Table 2, since the IC50 of mineral oil and corn oil exceeded 2,000 μg / mL, it was evaluated that the single-dose toxicity was low. On the other hand, the IC50 of 1-octanethiol was 2,000 μg / mL or less, and it was excluded from the evaluation target. From these facts, it became clear that the single-dose toxicity of a poorly water-soluble test substance can be appropriately evaluated by using the method of the present invention.

Industrial Applicability

[0035] According to the present invention, since the cytotoxicity and single-dose toxicity of a poorly water-soluble test substance can be appropriately carried out and the contribution to an alternative test method for animal experiments is also high, it is applicable not only to cosmetics and foods but also to pharmaceuticals and the like.

Claims

1. A method for exposing a poorly water-soluble test substance to cultured cells, comprising: obtaining an emulsion containing (A) a poorly water-soluble test substance, (B) a polyoxyethylene sorbitan fatty acid ester, and (C) a culture medium, and then exposing the emulsion to the cultured cells for a certain period of time. A method for exposing a poorly water-soluble test substance to cultured cells, characterized by the above.

2. The method for exposing a poorly water-soluble test substance to cultured cells according to Claim 1, characterized in that (A) the octanol / water partition coefficient of the poorly water-soluble test substance is 3.5 or more.

3. The method for exposing a poorly water-soluble test substance to cultured cells according to Claim 1 or 2, characterized in that (B) the polyoxyethylene sorbitan fatty acid ester is polysorbate 65.

4. A method for evaluating the presence or absence of cytotoxicity of a poorly water-soluble test substance, characterized by using the exposure method according to Claim 1 and using the cell viability of cultured cells as an index.

5. A method for evaluating the presence or absence of single-dose toxicity of a poorly water-soluble test substance, characterized by using the exposure method according to Claim 1 and using the cell viability of cultured cells as an index.

6. An evaluation kit for carrying out the evaluation according to Claim 4 or 5.

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