Solubilizers and solubilizing solutions

Abstract

The present invention provides a solubilizing agent for poorly water-soluble substances, comprising a compound represented by formula (1) (the symbols in the following formula are defined as described in the specification). #imgabs0#

Description

Technical Field

[0001] The present invention relates to a solubilizing agent for poorly water-soluble substances and a solubilizing solution for poorly water-soluble substances. The solubilizing agent and the solubilizing solution of the present invention can be used to dissolve poorly water-soluble substances present in samples such as plasma, serum and blood in water or an aqueous solution (such as a buffer solution). Background Art

[0002] To detect diseases early, assays utilizing immune responses are widely used in clinical testing and pharmaceutical diagnostics. Among these, there is a desire to expand the scope of assays and improve assay sensitivity, with the detection of poorly water-soluble substances being a key challenge.

[0003] In assays utilizing immune reactions, substances (e.g., peptides, proteins, nucleic acids, antibodies, antigens, etc.) contained in samples such as serum, plasma, cell extracts, and urine are detected. However, when the test targets are poorly water-soluble substances such as lipids, fat-soluble vitamins, and steroids, their low solubility in water can hinder the immune response by causing antibodies or enzymes to fail to react, or produce precipitation or white turbidity during measurement, making their detection difficult.

[0004] All along, when improving the solubility of these poorly water-soluble substances, widely use surfactant.Surfactant is divided into anionic surfactant, cationic surfactant, amphoteric surfactant and nonionic surfactant, and its kind is also very many.In this regard, as described in patent documentation 1, when nonionic surfactant and anionic surfactant are used in combination, because the kind of surfactant is many, the research of its combination becomes very complicated.And, in the diagnostic medicine of application immune response or enzyme reaction, along with the protein denaturation that surfactant causes, the activity of antibody or enzyme reduces, therefore, when selecting spendable surfactant and researching its addition concentration, need expend great labor.

[0005] Patent Documents 2 and 3 describe methods for solubilizing membrane proteins or poorly water-soluble substances using 2-methacryloyloxyethyl phosphorylcholine polymers. However, when 2-methacryloyloxyethyl phosphorylcholine polymers are added to a solution, the viscosity of the solution may increase depending on the concentration of the polymer, potentially reducing operability.

[0006] Non-Patent Document 1 describes the synthesis of a phosphorylcholine surfactant. However, Non-Patent Document 1 does not disclose a solubilization test of a poorly water-soluble substance using the phosphorylcholine surfactant.

[0007]

Prior art literature

[0008] [Patent Literature]

[0009] Patent Document 1: Japanese Patent Application Laid-Open No. 5-312807

[0010] Patent Document 2: Japanese Patent Application Laid-Open No. 2007-314526

[0011] Patent Document 3: Japanese Patent Application Laid-Open No. 2003-137816

[0012]

Non-patent literature

[0013] Non-Patent Document 1: Ryosuke Matsumoto, Kimiaki Takami, Kazuo Ishihara, "Simple Synthesis of a Zwitterionic Surfactants via Michael-Type Addition of Mathacrylate and Alkane Thiol Compounds", Langmuir Letters, Vol. 26, No. 16, pp. 13028-13032, July 6, 2010. Summary of the Invention

[0014] [Problems solved by the invention]

[0015] The methods disclosed in Patent Documents 1 to 3 can dissolve poorly water-soluble substances to some extent, but are currently insufficient. In addition, the polymers described in Patent Documents 2 and 3 are high molecular weight, and therefore, depending on their concentration, the viscosity of the resulting solution may increase, potentially reducing the operability of the solution. Therefore, the present invention aims to provide a solubilizing agent that can dissolve poorly water-soluble substances such as fat-soluble vitamins and fatty acids in water or an aqueous solution (e.g., a buffer solution).

[0016]

Methods to solve the problem

[0017] The inventors of the present application conducted intensive studies on the above-mentioned problems and found that the compound represented by the following formula (1) (hereinafter sometimes simply referred to as "compound (1)") is useful as a solubilizer for poorly water-soluble substances such as fat-soluble vitamins, thereby completing the present invention.

[0018] Based on the above findings, the present invention is as follows.

[0019] [1] A solubilizing agent for a poorly water-soluble substance, comprising a compound represented by formula (1),

[0020] Formula (1):

[0021]

[0022] In the formula, X represents a hydrogen atom or a methyl group, and n represents an integer of 5 to 15.

[0023] [2] The solubilizing agent according to [1] above, wherein the poorly water-soluble substance is at least one selected from fat-soluble vitamins, steroids, and fatty acids.

[0024] [3] A solubilizing solution for a poorly water-soluble substance, comprising the solubilizing agent described in [1] or [2] above and water.

[0025] Effects of the invention

[0026] Compound (1), the active ingredient of the solubilizing agent of the present invention, can improve the solubility of poorly water-soluble substances in water. Furthermore, since compound (1) has a low molecular weight, it is possible to suppress the increase in viscosity of a solution containing compound (1), thereby enabling the preparation of a solubilizing solution for poorly water-soluble substances with good operability. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below. In the present invention, a "poorly water-soluble substance" refers to a substance whose water solubility meets any of the categories of "slightly poorly soluble", "poorly soluble", "extremely poorly soluble", and "almost insoluble" as described in the 17th revision of the Japanese Pharmacopoeia. It should be noted that the water solubility in the 17th revision of the Japanese Pharmacopoeia is determined according to the following criteria. That is, when the object is solid, after being made into a powder, it is placed in water and shaken vigorously for 30 seconds every 5 minutes at 20±5°C. The degree of solubility within 30 minutes is studied. The substance is judged as "slightly poorly soluble" if the amount of water required to dissolve 1g or 1mL of the object is 30mL or more and less than 100mL, if the amount of water required to dissolve 1g or 1mL of the object is 100mL or more and less than 1000mL, if the amount of water required to dissolve 1g or 1mL of the object is 1000mL or more and less than 10000mL, if the amount of water required to dissolve 1g or 1mL of the object is 1000mL or more and less than 10000mL, if the amount of water required to dissolve 1g or 1mL of the object is 30mL or more and less than 10000mL, if the amount of water required to dissolve 1g or 1mL of the object is 10 ...0mL, if the amount of water required to dissolve 1g or 1mL of the object is 10000mL or more and less than 100000mL, if the amount of water required to Furthermore, the "solubilizing solution for a poorly water-soluble substance" refers to a solution for dissolving a poorly water-soluble substance.

[0028] The solubilizing agent of the present invention can be used in immunological detection methods using enzyme reactions or antigen-antibody reactions. Specifically, the solubilizing agent of the present invention can be used in known radioimmunoassays (RIA), enzyme immunoassays (EIA), fluorescence immunoassays (FIA), latex turbidimetry, etc., and is particularly preferably used in enzyme immunoassays (EIA), fluorescence immunoassays (FIA), latex turbidimetry, and Western blotting.

[0029] The active ingredient of the solubilizing agent of the present invention is a compound represented by the following formula (1).

[0030]

[0031] X in formula (1) represents a hydrogen atom or a methyl group, preferably a methyl group. n in formula (1) represents an integer of 5 to 15. If n is less than 5, the solubilizing performance of compound (1) may be reduced, and if it exceeds 15, the water solubility of compound (1) itself may be reduced. n is preferably 7 to 15.

[0032] The content of the compound (1) in the solubilizing agent of the present invention is preferably 50 to 100% by mass relative to the total mass of the solubilizing agent. The solubilizing agent of the present invention is more preferably composed of the compound (1).

[0033] Compound (1) can be synthesized, for example, by reacting 2-(meth)acryloyloxyethyl-2-trimethylammonioethyl phosphate and 1-alkanethiol in an alcohol solvent using an amine catalyst such as diisopropylamine at room temperature for 10 to 50 hours. The 1-alkanethiol is preferably one having 6 to 16 carbon atoms.

[0034] The solubilizing solution of the present invention contains the solubilizing agent of the present invention (i.e., the solubilizing agent containing compound (1)) and water. The solubilizing solution of the present invention may also contain components other than compound (1) and water (hereinafter referred to as "other components"). Examples of other components include amino acids and salts thereof such as glycine, alanine, serine, threonine, glutamic acid, aspartic acid (aspartic acid), glutamine, asparagine (asparagine), lysine, and histidine; peptides such as glycylglycine; inorganic acids and salts thereof such as phosphoric acid and its salts, boric acid and its salts, hydrochloric acid and its salts (e.g., sodium chloride, salt of hydrochloric acid and trishydroxymethylaminomethane), and sulfates; flavins; organic acids and salts thereof such as acetic acid and its salts, citric acid and its salts, malic acid and its salts, maleic acid and its salts, and gluconic acid and its salts; sugars such as glucose, fructose, sucrose, and lactose; alcohols such as methanol, ethanol, and isopropanol; reagents for measurement, and the like.

[0035] As water used in the solubilization solution of the present invention, for example, purified water, pure water, ion exchange water, etc. can be mentioned. In addition, the solubilization solution of the present invention can be a buffer solution (for example, a buffer solution that can be used for immunological assays). Specific examples of the buffer solution include phosphate buffer, acetate buffer, carbonate buffer, citrate buffer, Tris buffer, and HEPES buffer.

[0036] From the viewpoint of solubilizing performance, the content (concentration) of the compound (1) in the solubilizing solution of the present invention is preferably 0.01% by mass or more relative to the solubilizing solution as a whole, more preferably 0.025% by mass or more, further preferably 0.05% by mass or more, particularly preferably 0.10% by mass or more. The upper limit of the content of compound (1) is not particularly limited, as long as compound (1) is dissolved in the solubilizing solution. The content of the compound in the solubilizing solution of the present invention is preferably 20% by mass or less, more preferably 10% by mass or less, further preferably 5% by mass or less, particularly preferably 1% by mass or less.

[0037] From the viewpoint of maintaining the activity of proteins such as antibodies and enzymes or preventing denaturation, the water content in the solubilization solution of the present invention is preferably 80% by mass or more, more preferably 90% by mass or more, further preferably 95% by mass or more, particularly preferably 99% by mass or more, preferably 99.99% by mass or less, more preferably 99.975% by mass or less, further preferably 99.95% by mass or less, and particularly preferably 99.90% by mass or less.

[0038] Next, the poorly water-soluble substances are described. Examples of poorly water-soluble substances include fat-soluble vitamins (vitamin D, vitamin A, vitamin E, vitamin K, etc.), steroids (hormones such as cortisol (also known as hydrocortisone), norepinephrine, cholesterol, etc.), and fatty acids (lauric acid, myristic acid, linoleic acid, linolenic acid, etc.). The poorly water-soluble substance is preferably selected from at least one of fat-soluble vitamins, steroids, and fatty acids, and more preferably selected from at least one of fat-soluble vitamins and fatty acids.

[0039] The method for dissolving a poorly water-soluble substance in water using the solubilizing agent or solubilizing solution of the present invention is described below. Examples of such methods include a method for dissolving a poorly water-soluble substance in water by mixing a poorly water-soluble substance or a mixture containing a poorly water-soluble substance (e.g., a sample containing a poorly water-soluble substance), the solubilizing agent of the present invention, and water.

[0040] There is no particular limitation on the method for mixing the poorly soluble substance, the solubilizing agent of the present invention, and water, and examples thereof include the following methods:

[0041] (a) a method of mixing the solubilizing agent of the present invention with water to prepare a solubilized solution, and mixing the obtained solubilized solution with a poorly water-soluble substance or a mixture containing a poorly water-soluble substance (e.g., a sample containing a poorly water-soluble substance),

[0042] (b) a method of mixing a poorly water-soluble substance or a mixture containing a poorly water-soluble substance with water, and mixing the resulting mixture containing the poorly water-soluble substance and water with the solubilizing agent of the present invention or the solubilizing aqueous solution of the present invention,

[0043] (c) When the mixture containing the poorly water-soluble substance contains water (for example, when the mixture containing the poorly water-soluble substance is an aqueous dispersion of the poorly water-soluble substance), the mixture is mixed with the solubilizing agent of the present invention or the solubilizing aqueous solution of the present invention.

[0044] The temperature during stirring of the mixture containing the poorly water-soluble substance, the solubilizing agent of the present invention, and water is, for example, 4 to 37° C. In the method for dissolving a poorly water-soluble substance in water using the solubilizing agent or solubilizing solution of the present invention, the solubilizing agent (particularly compound (1)), the solubilizing solution, and the poorly water-soluble substance are as described above.

[0045] The content of compound (1) in a mixture containing a solubilizing agent, water, and a poorly water-soluble substance is preferably 0.025 to 5.0% by mass, more preferably 0.05 to 5.0% by mass, relative to the total mass of the mixture. The content of water in a mixture containing a solubilizing agent, water, and a poorly water-soluble substance is preferably 85.00 to 99.974% by mass, more preferably 90.00 to 99.94% by mass, relative to the total mass of the mixture. The content of the poorly water-soluble substance in a mixture containing a solubilizing agent, water, and a poorly water-soluble substance is preferably 0.001 to 10.0% by mass, more preferably 0.01 to 5.0% by mass, relative to the total mass of the mixture.

[0046] Next, the use of the solubilizing agent of the present invention is described. For example, the use thereof includes dissolving a poorly water-soluble substance in water or an aqueous solution (eg, a buffer solution) using the solubilizing agent of the present invention for measuring the poorly water-soluble substance.

[0047] In the measurement of the poorly water-soluble substance as described above, the content of the compound (1) in the mixture containing the measuring reagent, the poorly water-soluble substance to be measured, water and the compound (1) is preferably 0.025 to 5.0% by mass, more preferably 0.05 to 5.0% by mass, based on the entire mixture.

[0048] [Example]

[0049] The present invention will be further described in detail by the following examples and comparative examples, but the present invention is not limited thereto. In the following examples, the compounds obtained in the following Synthesis Examples 1 to 3 were used as solubilizing agents.

[0050] Synthesis of compound (1)

[0051] Synthesis example 1

[0052] 14.7635 g (0.050 mol) of 2-methacryloyloxyethyl-2-trimethylammoniumethyl phosphate (MPC) and 8.0460 g (0.055 mol) of 1-octanethiol were dissolved in 81.00 g of ethanol, and 0.2226 g (0.0022 mol) of diisopropylamine was added as a catalyst, and the mixture was reacted at room temperature for 24 hours. After completion of the reaction, the reaction solution was concentrated, and the concentrate was added to ethyl acetate. The precipitate was recovered by filtration and dried in a vacuum dryer to obtain 2-[3-(octylsulfanyl)-2-methylpropionyloxy]ethyl-2-(trimethylammonium)ethyl phosphate (a compound wherein X in formula (1) is methyl and n is 7) (12 g, yield: 50%) as a white powder.

[0053] Synthesis example 2

[0054] 2-[3-(dodecylsulfanyl)-2-methylpropionyloxy]ethyl-2-(trimethylammonio)ethyl phosphate (a compound wherein X of formula (1) is methyl and n is 11) was obtained as a white powder in the same manner as in Synthesis Example 1, except that 1-dodecylmercaptan was used instead of 1-octanethiol and the added amount was changed so that the molar ratio of the raw materials was equal to that in Synthesis Example 1.

[0055] Synthesis example 3

[0056] 2-[3-(hexadecylsulfanyl)-2-methylpropionyloxy]ethyl-2-(trimethylammonio)ethyl phosphate (a compound wherein X of formula (1) is methyl and n is 15) was obtained as a white powder in the same manner as in Synthesis Example 1, except that 1-hexadecylmercaptan was used instead of 1-octanethiol and the added amount was changed so that the molar ratio of the raw materials was equal to that in Synthesis Example 1.

[0057] Preparation of solubilization solution

[0058] Preparation Example 1-1-1

[0059] The compound obtained in Synthesis Example 1 was dissolved in phosphate-buffered saline (hereinafter referred to as "PBS") so as to have a concentration of 5.000% by mass to prepare a solubilized solution.

[0060] Preparation Example 1-1-2

[0061] The compound obtained in Synthesis Example 1 was dissolved in PBS so as to have a concentration of 0.500% by mass to prepare a solubilized solution.

[0062] Preparation Example 1-1-3

[0063] The compound obtained in Synthesis Example 1 was dissolved in PBS so as to have a concentration of 0.050% by mass to prepare a solubilized solution.

[0064] Preparation Example 1-1-4

[0065] The compound obtained in Synthesis Example 1 was dissolved in PBS so as to have a concentration of 0.025% by mass to prepare a solubilized solution.

[0066] Preparation Example 1-1-5

[0067] The compound obtained in Synthesis Example 1 was dissolved in PBS so as to have a concentration of 0.010% by mass to prepare a solubilized solution.

[0068] Preparation Example 1-2-1

[0069] The compound obtained in Synthesis Example 2 was dissolved in PBS so as to have a concentration of 5.000% by mass to prepare a solubilized solution.

[0070] Preparation Example 1-2-2

[0071] The compound obtained in Synthesis Example 2 was dissolved in PBS so as to have a concentration of 0.500% by mass to prepare a solubilized solution.

[0072] Preparation Example 1-2-3

[0073] The compound obtained in Synthesis Example 2 was dissolved in PBS so as to have a concentration of 0.050% by mass to prepare a solubilized solution.

[0074] Preparation Example 1-2-4

[0075] The compound obtained in Synthesis Example 2 was dissolved in PBS so as to have a concentration of 0.025% by mass to prepare a solubilized solution.

[0076] Preparation Example 1-2-5

[0077] The compound obtained in Synthesis Example 2 was dissolved in PBS so as to have a concentration of 0.010% by mass to prepare a solubilized solution.

[0078] Preparation Example 1-3-1

[0079] The compound obtained in Synthesis Example 3 was dissolved in PBS so as to have a concentration of 5.000% by mass to prepare a solubilized solution.

[0080] Preparation Example 1-3-2

[0081] The compound obtained in Synthesis Example 3 was dissolved in PBS so as to have a concentration of 0.500% by mass to prepare a solubilized solution.

[0082] Preparation Example 1-3-3

[0083] The compound obtained in Synthesis Example 3 was dissolved in PBS so as to have a concentration of 0.050% by mass to prepare a solubilized solution.

[0084] Preparation Example 1-3-4

[0085] The compound obtained in Synthesis Example 3 was dissolved in PBS so as to have a concentration of 0.025% by mass to prepare a solubilized solution.

[0086] Preparation Example 1-3-5

[0087] The compound obtained in Synthesis Example 3 was dissolved in PBS so as to have a concentration of 0.010% by mass to prepare a solubilized solution.

[0088] Preparation Example 1-4-1

[0089] Tween (registered trademark) 20 manufactured by Bio-Rad Laboratories was dissolved in PBS to a concentration of 5.000% by mass to prepare a solubilized solution.

[0090] Preparation Example 1-4-2

[0091] Tween (registered trademark) 20 was dissolved in PBS to a concentration of 0.500% by mass to prepare a solubilized solution.

[0092] Preparation Example 1-4-3

[0093] Tween (registered trademark) 20 was dissolved in PBS to a concentration of 0.050% by mass to prepare a solubilized solution.

[0094] Preparation Example 1-4-4

[0095] Tween (registered trademark) 20 was dissolved in PBS to a concentration of 0.025% by mass to prepare a solubilized solution.

[0096] Preparation Example 1-4-5

[0097] Tween (registered trademark) 20 was dissolved in PBS to a concentration of 0.010% by mass to prepare a solubilized solution.

[0098] Preparation Example 1-5-1

[0099] Triton (registered trademark) X-100 manufactured by Nacalai Tesque Co., Ltd. was dissolved in PBS to a concentration of 5.000% by mass to prepare a solubilized solution.

[0100] Preparation Example 1-5-2

[0101] Triton (registered trademark) X-100 was dissolved in PBS to a concentration of 0.500% by mass to prepare a solubilized solution.

[0102] Preparation Example 1-5-3

[0103] Triton (registered trademark) X-100 was dissolved in PBS to a concentration of 0.050% by mass to prepare a solubilized solution.

[0104] Preparation Example 1-5-4

[0105] Triton (registered trademark) X-100 was dissolved in PBS to a concentration of 0.025% by mass to prepare a solubilized solution.

[0106] Preparation Example 1-5-5

[0107] Triton (registered trademark) X-100 was dissolved in PBS to a concentration of 0.010% by mass to prepare a solubilized solution.

[0108] Preparation Example 1-6-1

[0109] Triton (registered trademark) X-405 manufactured by Nacalai Tesque Co., Ltd. was dissolved in PBS to a concentration of 5.000% by mass to prepare a solubilized solution.

[0110] Preparation Example 1-6-2

[0111] Triton (registered trademark) X-405 was dissolved in PBS to a concentration of 0.500% by mass to prepare a solubilized solution.

[0112] Preparation Example 1-6-3

[0113] Triton (registered trademark) X-405 was dissolved in PBS to a concentration of 0.050% by mass to prepare a solubilized solution.

[0114] Preparation Example 1-6-4

[0115] Triton (registered trademark) X-405 was dissolved in PBS to a concentration of 0.025% by mass to prepare a solubilized solution.

[0116] Preparation Example 1-6-5

[0117] Triton (registered trademark) X-405 was dissolved in PBS to a concentration of 0.010% by mass to prepare a solubilized solution.

[0118] Cholecalciferol (Vitamin D3) Solubilization Test

[0119] Example 1-1-1 and Example 1-1-2

[0120] Cholecalciferol (vitamin D3) was dissolved in ethanol to a concentration of 20 mg / mL to prepare a cholecalciferol solution. The solubilized solution obtained in Preparation Example 1-1-3 or Preparation Example 1-1-4 was dispensed into a 96-well plate in an amount of 100 μL / well. The cholecalciferol solution was dispensed in an amount of 1 μL / well. After stirring the mixture in each well for 1 minute, the absorbance of the mixture at a wavelength of 450 nm was measured. In addition, the absorbance of only PBS without cholecalciferol at a wavelength of 450 nm was measured. The difference between the absorbance of the mixture of the solubilized solution and the cholecalciferol solution at a wavelength of 450 nm and the absorbance of only PBS at a wavelength of 450 nm was taken as the "turbidity of the mixture of the solubilized solution and the cholecalciferol solution". The smaller the turbidity, the more water-soluble substances were dissolved.

[0121] The "turbidity of the mixture of PBS and cholecalciferol solution" (i.e., the difference between the absorbance of the mixture of PBS and cholecalciferol solution at 450 nm and the absorbance of PBS alone at 450 nm) was calculated in the same manner as above except that PBS was used instead of the solubilization solution.

[0122] Cholecalciferol is insoluble in PBS. Therefore, the following formula is used:

[0123] Turbidity ratio relative to PBS (%) = 100 × (turbidity of the mixture of the solubilized solution and the cholecalciferol solution) / (turbidity of the mixture of the PBS and the cholecalciferol solution)

[0124] The turbidity ratio relative to PBS was calculated as an indicator for evaluating whether cholecalciferol was dissolved. When the turbidity ratio relative to PBS was less than 10%, cholecalciferol was evaluated as dissolved.

[0125] Example 1-2-1 and Example 1-2-2

[0126] A solubilization test was performed in the same manner as in Examples 1-1-1 and 1-1-2, except that the solubilization solution obtained in Preparation Example 1-2-3 or Preparation Example 1-2-4 was used instead of the solubilization solution obtained in Preparation Example 1-1-3 or Preparation Example 1-1-4.

[0127] Example 1-3-1 and Example 1-3-2

[0128] A solubilization test was performed in the same manner as in Examples 1-1-1 and 1-1-2, except that the solubilization solution obtained in Preparation Example 1-3-3 or Preparation Example 1-3-4 was used instead of the solubilization solution obtained in Preparation Example 1-1-3 or Preparation Example 1-1-4.

[0129] Comparative Example 1-1-1

[0130] A solubilization test was performed in the same manner as in Example 1-1-1 and Example 1-1-2, except that the solubilization solution obtained in Preparation Example 1-4-3 was used instead of the solubilization solution obtained in Preparation Example 1-1-3 or Preparation Example 1-1-4.

[0131] Comparative Example 1-2-1

[0132] A solubilization test was performed in the same manner as in Examples 1-1-1 and 1-1-2, except that the solubilization solution obtained in Preparation Example 1-5-3 was used instead of the solubilization solution obtained in Preparation Example 1-1-3 or Preparation Example 1-1-4.

[0133] Comparative Example 1-3-1

[0134] A solubilization test was performed in the same manner as in Examples 1-1-1 and 1-1-2, except that the solubilization solution obtained in Preparation Example 1-6-3 was used instead of the solubilization solution obtained in Preparation Example 1-1-3 or Preparation Example 1-1-4.

[0135] Table 1 shows the solubilizing solution used, the solubilizing agent used, the concentration of the solubilizing agent in the solubilizing solution (referred to as "solubilizing agent concentration" in Table 1), the turbidity of the mixture of the solubilizing solution and the cholecalciferol solution (referred to as "turbidity" in Table 1), the turbidity of the mixture of PBS and the cholecalciferol solution (referred to as "PBS turbidity" in Table 1), and the turbidity ratio relative to PBS. In addition, "-m" in Table 1 indicates the end of each example and each comparative example. For example, the row "-1" in "Example 1-1" indicates "Example 1-1-1". In addition, the preparation example of preparing the solubilizing solution is described in the solubilizing solution column. In addition, the synthesis example of the synthetic solubilizing agent or the trade name of the surfactant is described in the solubilizing agent column.

[0136]

Table 1

[0137]

[0138] As shown in Table 1, in Examples 1-1-1 to 1-3-2, the turbidity ratio relative to PBS was less than 10%, indicating that cholecalciferol, a poorly water-soluble substance, was dissolved. On the other hand, in Comparative Examples 1-1 to 1-3, the turbidity ratio relative to PBS was 10% or greater. These results demonstrate that the solubilizing agents and solubilizing solutions used in Examples 1-1-1 to 1-3-2 exhibit superior solubilization performance for cholecalciferol, a poorly water-soluble substance, compared to the solubilizing agents and solubilizing solutions used in Comparative Examples 1-1 to 1-3.

[0139] Solubilization Test of DL-alpha-Tocopherol (Vitamin E)

[0140] Example 2-1-1 and Example 2-1-2

[0141] DL-α-tocopherol (vitamin E) was dissolved in ethanol to a concentration of 20 mg / mL to prepare a DL-α-tocopherol solution. This solution was used instead of the cholecalciferol solution. The solubilization test was performed in the same manner as in Examples 1-1-1 and 1-1-2, and the "turbidity of the mixture of the solubilized solution and the DL-α-tocopherol solution" (i.e., the difference between the absorbance of the mixture of the solubilized solution and the DL-α-tocopherol solution at a wavelength of 450 nm and the absorbance of PBS alone at a wavelength of 450 nm) was calculated.

[0142] The "turbidity of the mixture of PBS and DL-α-tocopherol solution" (i.e., the difference between the absorbance of the mixture of PBS and DL-α-tocopherol solution at 450 nm and the absorbance of PBS alone at 450 nm) was calculated in the same manner as above except that PBS was used instead of the solubilization solution.

[0143] DL-α-tocopherol is insoluble in PBS. Therefore, it is prepared using the following formula:

[0144] Turbidity ratio relative to PBS (%) = 100 × (turbidity of the mixture of the solubilized solution and the DL-α-tocopherol solution) / (turbidity of the mixture of the PBS and the DL-α-tocopherol solution)

[0145] The turbidity ratio relative to PBS was calculated as an indicator for evaluating whether DL-α-tocopherol was dissolved. When the turbidity ratio relative to PBS was less than 10%, DL-α-tocopherol was evaluated as dissolved.

[0146] Example 2-2-1

[0147] A solubilization test was performed in the same manner as in Example 2-1-1 and Example 2-1-2, except that the solubilization solution obtained in Preparation Example 1-2-3 was used instead of the solubilization solution obtained in Preparation Example 1-1-3 or Preparation Example 1-1-4.

[0148] Comparative Example 2-2-1

[0149] A solubilization test was performed in the same manner as in Example 2-1-1 and Example 2-1-2, except that the solubilization solution obtained in Preparation Example 1-5-3 was used instead of the solubilization solution obtained in Preparation Example 1-1-3 or Preparation Example 1-1-4.

[0150] Shown in the table 2 are the turbidity (being called " turbidity " in the table 2) of the concentration of the solubilizing solution of use, the solubilizing solution of use, the solubilizing solution, the turbidity (being called " turbidity " in the table 2) of the mixture of solubilizing solution and DL-α-tocopherol solution, the turbidity (being called " turbidity " in the table 2) of the mixture of PBS and DL-α-tocopherol solution, the turbidity ratio with respect to PBS.In addition, " m " that table 2 is recorded represents the end of each embodiment and each comparative example.In addition, in the row of solubilizing solution, put down in writing the preparation example of preparation solubilizing solution.In addition, in the row of solubilizing solution, put down in writing the synthesis example of synthetic solubilizing agent or the trade name of surfactant.

[0151]

Table 2

[0152]

[0153] As shown in Table 2, in Examples 2-1-1 to 2-2-1, the turbidity ratio relative to PBS was less than 10%, indicating that DL-α-tocopherol, a poorly water-soluble substance, was dissolved. On the other hand, in Comparative Example 2-2-1, the turbidity ratio relative to PBS was 10% or greater. These results demonstrate that the solubilizing agents and solubilizing solutions used in Examples 2-1-1 to 2-2-1 exhibit superior solubilization performance for DL-α-tocopherol, a poorly water-soluble substance, compared to the solubilizing agent and solubilizing solution used in Comparative Example 2-2-1.

[0154] Solubilization test of hydrocortisone

[0155] Example 3-3

[0156] Hydrocortisone was dissolved in ethanol to a concentration of 20 mg / mL to prepare a hydrocortisone-soluble solution, which was used instead of the cholecalciferol solution. The hydrocortisone solution was dispensed at 125 μL / well. The solubilization solution obtained in Preparation Example 1-3-3 was used instead of the solubilization solution obtained in Preparation Example 1-1-3 or Preparation Example 1-1-4. Otherwise, the solubilization test was performed in the same manner as in Example 1-1-1 and Example 1-1-2, and the "turbidity of the mixture of the solubilized solution and the hydrocortisone solution" (i.e., the difference between the absorbance of the mixture of the solubilized solution and the hydrocortisone solution at a wavelength of 450 nm and the absorbance of PBS alone at a wavelength of 450 nm) was calculated.

[0157] The "turbidity of the mixture of PBS and hydrocortisone solution" (i.e., the difference between the absorbance of the mixture of PBS and hydrocortisone solution at 450 nm and the absorbance of PBS alone at 450 nm) was calculated in the same manner as above except that PBS was used instead of the solubilizing solution.

[0158] Hydrocortisone is insoluble in PBS. Therefore, the following formula is used:

[0159] Turbidity ratio relative to PBS (%) = 100 × (turbidity of the mixture of the solubilized solution and the hydrocortisone solution) / (turbidity of the mixture of the PBS and the hydrocortisone solution)

[0160] The turbidity ratio relative to PBS was calculated as an indicator for evaluating whether hydrocortisone was dissolved. When the turbidity ratio relative to PBS was less than 10%, hydrocortisone was evaluated as dissolved.

[0161] Comparative Example 3-1

[0162] A solubilization test was performed in the same manner as in Example 3-3, except that the solubilization solution obtained in Preparation Example 1-4-3 was used instead of the solubilization solution obtained in Preparation Example 1-3-3.

[0163] Comparative Example 3-2

[0164] A solubilization test was performed in the same manner as in Example 3-3, except that the solubilization solution obtained in Preparation Example 1-5-3 was used instead of the solubilization solution obtained in Preparation Example 1-3-3.

[0165] Comparative Example 3-3

[0166] A solubilization test was performed in the same manner as in Example 3-3, except that the solubilization solution obtained in Preparation Example 1-6-3 was used instead of the solubilization solution obtained in Preparation Example 1-3-3.

[0167] Shown among the table 3 is the turbidity (being called " turbidity " in the table 3) of the concentration of the solubilizing solution of use, the solubilizing solution of use, the solubilizing solution, the turbidity (being called " turbidity " in the table 3) of the mixture of solubilizing solution and hydrocortisone solution, PBS and the turbidity ratio of hydrocortisone solution, with respect to PBS.In addition, in the row of solubilizing solution, have put down in writing the preparation example of preparation solubilizing solution.In addition, in the row of solubilizing solution, have put down in writing the synthesis example of synthetic solubilizing agent or the trade name of surfactant.

[0168]

Table 3

[0169]

[0170] As shown in Table 3, in Example 3-3, the turbidity ratio relative to PBS was less than 10%, indicating that hydrocortisone, a poorly water-soluble substance, was dissolved. On the other hand, in Comparative Examples 3-1 to 3-3, the turbidity ratio relative to PBS was 10% or greater. These results demonstrate that the solubilizing agent and solubilizing solution used in Example 3-3 exhibit superior solubilization performance for hydrocortisone, a poorly water-soluble substance, compared to the solubilizing agents and solubilizing solutions used in Comparative Examples 3-1 to 3-3.

[0171] Solubilization test of lauric acid

[0172] Example 4-1

[0173] Lauric acid was dissolved in ethanol to a concentration of 100 mg / mL to prepare a lauric acid solution, which was used instead of the cholecalciferol solution. The lauric acid solution was dispensed at 3 μL / well, and the solubilization solution obtained in Preparation Example 1-1-1 was used instead of the solubilization solution obtained in Preparation Example 1-1-3 or Preparation Example 1-1-4. The solubilization test was performed in the same manner as in Examples 1-1-1 and 1-1-2, and the "turbidity of the mixture of the solubilized solution and the lauric acid solution" (i.e., the difference between the absorbance of the mixture of the solubilized solution and the lauric acid solution at a wavelength of 450 nm and the absorbance of PBS alone at a wavelength of 450 nm) was calculated.

[0174] The same procedure as above was repeated except that PBS was used instead of the solubilization solution, and the "turbidity of the mixture of PBS and lauric acid solution" (i.e., the difference between the absorbance of the mixture of PBS and lauric acid solution at 450 nm and the absorbance of PBS alone at 450 nm) was calculated.

[0175] Lauric acid is insoluble in PBS. Therefore, the following formula is used:

[0176] Turbidity ratio relative to PBS (%) = 100 × (turbidity of the mixture of the solubilized solution and the lauric acid solution) / (turbidity of the mixture of the PBS and the lauric acid solution)

[0177] The turbidity ratio relative to PBS was calculated as an indicator for evaluating whether lauric acid was dissolved. When the turbidity ratio relative to PBS was less than 10%, it was evaluated that lauric acid was dissolved.

[0178] Comparative Example 4-1

[0179] A solubilization test was performed in the same manner as in Example 4-1, except that the solubilization solution obtained in Preparation Example 1-4-1 was used instead of the solubilization solution obtained in Preparation Example 1-1-1.

[0180] Comparative Example 4-2

[0181] A solubilization test was performed in the same manner as in Example 4-1, except that the solubilization solution obtained in Preparation Example 1-5-1 was used instead of the solubilization solution obtained in Preparation Example 1-1-1.

[0182] Comparative Example 4-3

[0183] A solubilization test was performed in the same manner as in Example 4-1, except that the solubilization solution obtained in Preparation Example 1-6-1 was used instead of the solubilization solution obtained in Preparation Example 1-1-1.

[0184] Table 4 shows the solubilizing solution used, the solubilizing agent used, the concentration of the solubilizing agent in the solubilizing solution (referred to as "concentration of solubilizing agent" in Table 4), the turbidity of the mixture of the solubilizing solution and the lauric acid solution (referred to as "turbidity" in Table 4), the turbidity of the mixture of PBS and the lauric acid solution (referred to as "turbidity of PBS" in Table 4), and the turbidity ratio relative to PBS. In addition, the preparation example of the solubilizing solution is described in writing in the column of the solubilizing solution. In addition, the synthesis example of the synthetic solubilizing agent or the trade name of the surfactant is described in the column of the solubilizing agent.

[0185]

Table 4

[0186]

[0187] As shown in Table 4, in Example 4-1, the turbidity ratio relative to PBS is less than 10%, so it can be seen that lauric acid, which is a poorly water-soluble substance, is dissolved. On the other hand, in Comparative Example 4-3, the turbidity ratio relative to PBS is more than 10%. From these results, it can be seen that the solubilizing agent and solubilizing solution used in Example 4-1 are superior in solubilizing lauric acid, which is a poorly water-soluble substance, compared to the solubilizing agent and solubilizing solution used in Comparative Example 4-3. In addition, the turbidity relative to PBS of Example 4-1 is equal to or less than the turbidity of Comparative Example 4-1 and Comparative Example 4-2. From these results, it can be seen that the performance of the solubilizing agent and solubilizing solution used in Example 4-1 in solubilizing lauric acid is equal to or greater than the solubilizing agent (i.e., commercially available Tween (registered trademark) 20 or Triton (registered trademark) X-100) and solubilizing solution used in Comparative Example 4-1 or Comparative Example 4-2.

[0188] Solubilization test of myristic acid

[0189] Example 5-1

[0190] Myristic acid was dissolved in ethanol to a concentration of 100 mg / mL to prepare a myristic acid-soluble solution, which was used instead of the cholecalciferol solution. The myristic acid solution was dispensed at 3 μL / well. The solubilization test was performed in the same manner as in Examples 1-1-1 and 1-1-2, except that the solubilized solution obtained in Preparation Example 1-1-1 was used instead of the solubilized solution obtained in Preparation Example 1-1-3 or Preparation Example 1-1-4. The "turbidity of the mixture of the solubilized solution and the myristic acid solution" (i.e., the difference between the absorbance of the mixture of the solubilized solution and the myristic acid solution at a wavelength of 450 nm and the absorbance of PBS alone at a wavelength of 450 nm) was calculated.

[0191] The same procedure as above was followed except that PBS was used instead of the solubilization solution, and the "turbidity of the mixture of PBS and myristic acid solution" (i.e., the difference between the absorbance of the mixture of PBS and myristic acid solution at 450 nm and the absorbance of PBS alone at 450 nm) was calculated.

[0192] Myristic acid is insoluble in PBS. Therefore, the following formula is used:

[0193] Turbidity ratio relative to PBS (%) = 100 × (turbidity of the mixture of the solubilized solution and the myristic acid solution) / (turbidity of the mixture of the PBS and the myristic acid solution)

[0194] The turbidity ratio relative to PBS was calculated as an indicator for evaluating whether myristic acid was dissolved. When the turbidity ratio relative to PBS was less than 10%, myristic acid was evaluated as dissolved.

[0195] Comparative Example 5-1

[0196] A solubilization test was performed in the same manner as in Example 5-1, except that the solubilization solution obtained in Preparation Example 1-4-1 was used instead of the solubilization solution obtained in Preparation Example 1-1-1.

[0197] Comparative Example 5-2

[0198] A solubilization test was performed in the same manner as in Example 5-1, except that the solubilization solution obtained in Preparation Example 1-5-1 was used instead of the solubilization solution obtained in Preparation Example 1-1-1.

[0199] Comparative Example 5-3

[0200] A solubilization test was performed in the same manner as in Example 5-1, except that the solubilization solution obtained in Preparation Example 1-6-1 was used instead of the solubilization solution obtained in Preparation Example 1-1-1.

[0201] Table 5 shows the solubilizing solution used, the solubilizing agent used, the concentration of the solubilizing agent in the solubilizing solution (referred to as "concentration of solubilizing agent" in Table 5), the turbidity of the mixture of the solubilizing solution and the myristic acid solution (referred to as "turbidity" in Table 5), the turbidity of the mixture of PBS and the myristic acid solution (referred to as "turbidity of PBS" in Table 5), and the turbidity ratio relative to PBS. In addition, the preparation example of preparing the solubilizing solution is recorded in the column of the solubilizing solution. In addition, the synthesis example of the synthetic solubilizing agent or the trade name of the surfactant is recorded in the column of the solubilizing agent.

[0202]

Table 5

[0203]

[0204] As shown in Table 5, in Example 5-1, the turbidity ratio relative to PBS was less than 10%, indicating that myristic acid, a poorly water-soluble substance, was dissolved. On the other hand, in Comparative Examples 5-1 to 5-3, the turbidity ratio relative to PBS was 10% or greater. These results demonstrate that the solubilizing agent and solubilizing solution used in Example 5-1 exhibit superior solubilization performance for myristic acid, a poorly water-soluble substance, compared to the solubilizing agents and solubilizing solutions used in Comparative Examples 5-1 to 5-3.

[0205] The above results show that poorly water-soluble substances can be solubilized by using the solubilizing agent of the present invention.

[0206] Industrial Applicability

[0207] The solubilizing agent of the present invention can be used, for example, for measuring poorly water-soluble substances.

[0208] This application is based on Japanese Patent Application No. 2020-074983 filed in Japan, the entire contents of which are incorporated herein by reference.

Claims

1. Use of a solubilizing agent comprising a compound represented by formula (1) for solubilizing a poorly water-soluble substance. Formula (1): ； In the formula, X represents a hydrogen atom or a methyl group, and n represents an integer of 5 to 15; in, The poorly water-soluble substance is at least one selected from fat-soluble vitamins, steroids and fatty acids.

2. The use according to claim 1, wherein The compound represented by formula (1) is a component of a solubilization solution, and the solubilization solution comprises the compound represented by formula (1) and water.

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