JP4595257B2 - Skin sensitization test method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for assaying skin sensitization of a test substance.
[0002]
[Prior art]
Since allergic reactions may occur when chemical substances come into contact with the skin, for example, when developing products containing chemical substances such as medicine, agricultural chemicals, and cosmetics, testing the skin sensitization of the content substances Is required.
Conventionally, as a method for testing the skin sensitization property of a test substance, a test substance is transdermally administered to a laboratory animal, and the skin reaction at the administration site is observed (Maximization test, Buehler test, etc.). A method of examining the proliferation of lymphocytes by administering a test substance transdermally (such as Local Lymph Node Assay) is known. Such an assay method requires laborious work such as administration of a test substance to a laboratory animal, skin observation, blood test, etc. in addition to breeding of a laboratory animal, and a period for that.
[0003]
[Problems to be solved by the invention]
In the development of medicines, agricultural chemicals, cosmetics, and the like, since strict toxicity tests are required, finally, it is also necessary to test for the presence or absence of skin sensitization by animal experiments as described above. However, when such an animal experiment is carried out for all of the development candidate compounds in the early stage of development, a large number of experimental animals, a large amount of test substance, and a lot of time are required. There is a strong demand for the development of a simpler and quicker assay method for these test substances.
An object of the present invention is to provide a method for easily and rapidly assaying the skin sensitization of a test substance without conducting such complicated animal experiments.
[0004]
[Means for Solving the Problems]
As a result of intensive studies under such circumstances, the present inventors have found that the cysteine-containing peptide having a molecular weight of about 50,000 or less as a monomer and a test substance are mixed and reacted when the peptide and the test substance are bound. By measuring the presence or absence, it was found that the test substance could be tested for skin sensitization, and the present invention was achieved.
That is, the present invention
A test method for skin sensitization of a test substance, comprising mixing a cysteine-containing peptide having a molecular weight of 50,000 or less as a monomer and a test substance, and then measuring the presence or absence of a conjugate of the peptide and the test substance Is to provide.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the assay method of the present invention will be described.
The peptide that can be used in the assay method of the present invention is a peptide containing one or more cysteine residues and having a molecular weight of about 50,000 or less as a monomer. Specific examples include glutathione. An aqueous buffer solution containing such a peptide, for example, an inorganic acid salt such as an alkali metal phosphate such as sodium phosphate or potassium phosphate, or an organic acid salt such as an acetate such as sodium acetate, potassium acetate or ammonium acetate. Alternatively, it is dissolved in water or a mixed solvent of these with an organic solvent so as to have a concentration of, for example, about 0.01 μM to about 1 M, usually about 10 μM to about 100 mM.
The test substance is dissolved in, for example, an organic solvent such as methanol, ethanol, acetonitrile, acetone, or a mixed solvent thereof to have a concentration of about 0.01 μM to about 1 M, usually about 1 mM to about 500 mM. .
Next, the peptide solution and the test substance solution are mixed and reacted so that the molar concentration ratio of the peptide and the test substance is, for example, 1: 100 to 20: 1. In the reaction, the mixed solution of the peptide solution and the test substance solution is usually allowed to stand for about 10 minutes to about 2 days while keeping the temperature in a temperature range of about 4 ° C to about 60 ° C.
[0006]
The presence / absence of a conjugate of the test substance and peptide generated by such a reaction is measured by analyzing a mixed solution of the peptide solution and the test substance solution as follows.
Examples of the method for analyzing the mixed liquid include high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography (TLC), and mass spectrometry (MS). Also, an analytical method (LC-MS, GC-MS, TLC-MS) in which any one of HPLC, GC, or TLC and MS can be used. According to this method, even if a sample contains a plurality of components, they can be separated individually and subjected to mass spectrometry for each.
Examples of chromatographic techniques that can be used for the above HPLC, GC, or TLC include reverse phase, normal phase, and ion exchange. Commercially available columns and TLCs that can be used for such chromatographic methods include, for example, SUMIPAX ODS A-212 (manufactured by Sumika Chemical Analysis Center), L-column ODS (manufactured by Chemicals Research Association), and Cosmo as LC columns. Examples of the seal include 5C18-AR-II (manufactured by Nacalai Tesque), and examples of the TLC plate include silica gel 60F254 (manufactured by Merck) and Silica Gel 60 Plate (manufactured by Nacalai Tesque).
Examples of ionization methods that can be used in mass spectrometry include matrix-assisted laser ionization (MALDI), electrospray ionization (ESI), atmospheric pressure ionization (API), electron impact ionization (EI), Fast atom bombardment ionization (FAB) method. Examples of commercially available ion sources that can be used for such ionization methods include ion sources such as Shimadzu / KRATOS, PE Biosystems, Micromass, and Bruker in the case of MALDI. In the case of the ESI method, ion sources such as those from Finnigan Mat, Micromass, Sciex, Hewlett-Packard, and PE Biosystems may be mentioned. Examples of the mass spectrometer include a magnetic field type, a quadrupole type, an ion trap type, a Fourier transform-ion cyclotron resonance type, and a time-of-flight type mass spectrometer.
The analysis result of the mixed solution obtained as described above is compared with, for example, the analysis results of the peptide solution and the test substance solution before mixing, is not detected from the peptide solution and the test substance solution before mixing, and By examining the presence / absence of a component detected only from the mixed solution after the incubation, the presence / absence of a conjugate of the peptide and the test substance can be measured. When mass spectrometry such as MS, LC-MS, GC-MS, and TLC-MS is used for such analysis, the mass, structure, etc. are analyzed for each component based on information obtained from the mass spectrum, and the composition and By confirming the structure, a conjugate of the peptide and the test substance can also be specified.
[0007]
【Example】
EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.
[0008]
Example 1
0.1 ml of 1-chloro-2,4-dinitrobenzene (hereinafter referred to as DNCB; positive skin sensitization, mass 202) dissolved in acetone to a concentration of 2 mg / ml (10 mM), and glutathione described below 1 ml of any of solutions 1 to 3 was mixed and incubated at 37 ° C. for 1 hour, and then 20 μl thereof was subjected to HPLC under the conditions described below. The obtained chromatogram was compared with the chromatogram obtained by analyzing each of the above-mentioned DNCB solution and glutathione solution in the same manner. As a result, when any of the glutathione solutions 1 to 3 was used, a peak (hereinafter referred to as peak 1) that was not recognized in the chromatograms of the DNCB solution and the glutathione solution was detected.
[0009]
[Glutathione solution]
1; Glutathione dissolved in 0.2M NaH ₂ PO ₄ -Na ₂ HPO ₄ pH 6 to 3.0mg / ml (9.8mM) 2; 0.2M NaH ₂ PO in glutathione to 3.0mg / ml (9.8mM) ₄ -Na ₂ HPO ₄ dissolved in pH 8 3; glutathione dissolved in 0.2 M Na ₂ CO ₃ -NaHCO ₃ pH 10 to 3.0 mg / ml (9.8 mM)
[HPLC measurement conditions]
Shimadzu LC-10AD
Column: SUMPAX ODS A-212 (6 mm x 150 mm)
Column temperature: 25 ° C
Flow rate: 1 ml / min.
Detection wavelength: 220nm
Eluent A: Solution containing 0.1% TFA in distilled water Eluent B: Solution containing 0.08% TFA in acetonitrile Elution conditions: Elution mixed with 95% eluent B and 5% eluent B After injecting the sample into the column equilibrated with the solution, the mixture of eluent A and eluent B was allowed to flow while increasing the proportion of eluent B from 5% to 80% over 30 minutes.
[0011]
0.4 ml of a solution in which DNCB was dissolved in acetone to 20 mg / ml (100 mM) and 2 ml of a solution in which 0.2 mg ammonium acetate was dissolved in glutathione to 6.5 mM were mixed and incubated at 37 ° C. for 1 hour. Lyophilized. The lyophilizate was dissolved in methanol and subjected to MS. As a result, the mass number of the compound corresponding to the above peak 1 was measured to be 473. The mass number corresponds to the mass of the compound formed by detaching chlorine from DNCB and binding to glutathione (mass 307).
[0012]
[MS measurement conditions]
PE Biosystems Voyager DE-STR mass spectrometer ionization; MALDI method
Example 2
0.1 ml of a solution of sodium trinitrobenzenesulfonate (hereinafter referred to as TNBS, positive skin sensitization, mass 315) dissolved in acetone to 3.5 mg / ml (10 mM), and glutathione solution 2 described in Example 1 1 ml of the mixture was mixed and incubated at 37 ° C. for 1 hour, and then 20 μl thereof was subjected to HPLC under the conditions described in Example 1. The obtained chromatogram (FIG. 3) was compared with the chromatogram (FIGS. 1 and 2) obtained by analyzing the glutathione solution and the TNBS solution in the same manner. As a result, peaks that were not observed in the chromatograms of the TNBS solution and the glutathione solution (FIG. 3; peaks (1) and (2)) were detected.
The above mixture of TNBS and glutathione was subjected to LC-MS under the conditions described below. From the MS spectra of the peaks corresponding to the above peaks (1) and (2) (FIGS. 4 and 5), the mass numbers of the compounds corresponding to these peaks are 518 for (1) and 518 for (2). And measured. The mass number corresponds to the mass of the compound formed by detaching the —SO ₃ Na group from TNBS and binding to glutathione.
Moreover, it replaced with the glutathione solution 2 and analyzed similarly using the glutathione solution 1. Results similar to those described above using glutathione solution 2 were obtained.
[0014]
[LC-MS measurement conditions]
LC: Hitachi L-6200 (UV is L-4000)
Column: SUMPAX ODS A-212 (6 mm x 150 mm)
Column temperature: 25 ° C
Flow rate: 1 ml / min.
Detection wavelength: 254nm
Eluent A: Solution containing 0.1% TFA in distilled water Eluent B: Solution containing 0.08% TFA in acetonitrile Elution conditions: Elution mixed with 95% eluent B and 5% eluent B After injecting the sample into the column equilibrated with the solution, the mixture of eluent A and eluent B was allowed to flow while increasing the proportion of eluent B from 5% to 80% over 30 minutes.
[0015]
MS: Finnigan Mat TSQ700 mass spectrometer Ionization: ESI spray voltage: 4.5kV
Capillary temperature: 285 ° C
Sheath gas: 80psi
Auxiliary gas: 10Unit
[0016]
Example 3
0.1 ml of 1,3-dicyclohexylcarbodiimide (hereinafter referred to as DCC, positive skin sensitization, mass 206) dissolved in methanol to a concentration of 21 mg / ml (100 mM), and glutathione solution 1 described in Example 1 1 ml of any of ˜3 was mixed and incubated at 37 ° C. for 1 hour, and then 20 μl thereof was subjected to LC-MS under the conditions described in Example 2. The chromatogram obtained by LC was compared with the chromatogram obtained by analyzing each of the glutathione solution and the DCC solution in the same manner. As a result, when any one of the glutathione solutions 1 to 3 was used, peaks that were not recognized in the chromatograms of the DCC solution and the glutathione solution were detected. From the MS spectrum of this peak, the mass number of the compound contained in the peak was measured to be 513. The mass number corresponds to the mass of the combination of DCC (mass 206) and glutathione (mass 307).
[0017]
Example 4
0.1 ml of a solution of 1,2-epoxyoctane (hereinafter referred to as 1,2EO; positive skin sensitization, mass 128) dissolved in methanol to 12 mg / ml (100 mM), and glutathione described in Example 1 Any 1 ml of the solutions 1 to 3 were mixed and incubated at 37 ° C. for 1 hour, and then 20 μl thereof was subjected to LC-MS under the conditions described in Example 2. The chromatogram obtained by LC was compared with the chromatogram obtained by analyzing each of the glutathione solution and the 1,2EO solution in the same manner. As a result, when any of the glutathione solutions 1 to 3 was used, peaks that were not observed in the chromatograms of the 1,2EO solution and the glutathione solution were detected. From the MS spectrum of this peak, the mass number of the compound contained in the peak was measured to be 435. The mass number corresponds to the mass of the combination of 1,2EO (mass 128) and glutathione (mass 307).
[0018]
Example 5
0.1 ml of a solution prepared by dissolving 1-methyl-3-nitro-1-nitrosoguanidine (hereinafter referred to as MNNG; positive skin sensitization, mass 147) in methanol to a concentration of 14 mg / ml (100 mM); The glutathione solution 1 or 2 described in 1) was mixed with 1 ml and incubated at 37 ° C. for 1 hour, and then 20 μl thereof was subjected to LC-MS under the conditions described in Example 2. The chromatogram obtained by LC was compared with the chromatogram obtained by analyzing each of the glutathione solution and the MNNG solution in the same manner. As a result, when either 1 or 2 glutathione solutions were used, peaks that were not observed in the chromatograms of the MNNG solution and the glutathione solution were detected. From the MS spectrum of this peak, the mass number of the compound contained in the peak was measured to be 394. The mass number coincides with the mass of the compound formed by elimination of the —N (NO) CH ₃ group from MNNG and binding to glutathione.
[0019]
Example 6
0.1 ml of a solution of formaldehyde (37% aqueous solution) (hereinafter referred to as FA, positive skin sensitization, mass 30) in methanol to give 3 mg / ml (37 mM), and glutathione solution 1 described in Example 1 Alternatively, either 1 ml of 2 was mixed and incubated at 37 ° C. for 1 hour, and then 20 μl thereof was subjected to LC-MS under the conditions described in Example 2. The chromatogram obtained by LC was compared with the chromatogram obtained by analyzing each of the glutathione solution and the FA solution in the same manner. As a result, when either 1 or 2 glutathione solutions were used, peaks that were not observed in the chromatograms of the FA solution and the glutathione solution were detected. From the MS spectrum of this peak, the mass number of the compound contained in the peak was measured to be 337. The mass number corresponds to the mass of the conjugate of FA and glutathione.
[0020]
Example 7
0.1 ml of a solution of glutaraldehyde (25% aqueous solution) (hereinafter referred to as GA, positive skin sensitization, mass 100) dissolved in methanol to give 52 mg / ml (130 mM), and the glutathione solution described in Example 1 1 ml of either 1 or 2 was mixed and incubated at 37 ° C. for 1 hour, and then 20 μl thereof was subjected to LC-MS under the conditions described in Example 2. The chromatogram obtained by LC was compared with the chromatogram obtained by analyzing each of the glutathione solution and the GA solution in the same manner. As a result, when any one of the glutathione solutions 1 or 2 was used, two peaks that were not observed in the chromatograms of the GA solution and the glutathione solution were detected. From the MS spectra of these peaks, the mass numbers of the compounds contained in these peaks were measured as 407 and 489, respectively. The mass number 407 corresponds to the mass of the conjugate of GA and glutathione. The mass number 489 corresponds to the mass of a compound formed by combining two molecules of GA and one molecule of glutathione with the elimination of two atoms of hydrogen and one atom of oxygen.
[0021]
Example 8
0.1 ml of a solution prepared by dissolving methyl salicylate (hereinafter referred to as MSC; negative skin sensitization, mass 152) in methanol so as to be 15 mg / ml (100 mM), and either glutathione solution 1 or 2 described in Example 1 1 ml was mixed and incubated at 37 ° C. for 1 hour, and then 20 μl thereof was subjected to LC-MS under the conditions described in Example 2. The chromatogram obtained by LC was compared with the chromatogram obtained by analyzing each of the glutathione solution and the MSC solution in the same manner. As a result, no peak was detected in the chromatograms of the MSC solution and glutathione solution when either 1 or 2 glutathione solution was used.
[0022]
Example 9
0.1 ml of a solution of phenol (hereinafter referred to as PH, negative skin sensitization, mass 94) dissolved in methanol to 9 mg / ml (100 mM), and either of the glutathione solutions 1 or 2 described in Example 1 1 ml was mixed and incubated at 37 ° C. for 1 hour, and then 20 μl thereof was subjected to LC-MS under the conditions described in Example 2. The chromatogram obtained by LC was compared with the chromatogram obtained by analyzing each of the glutathione solution and the PH solution in the same manner. As a result, no peak was detected in the chromatograms of the PH solution and the glutathione solution when either 1 or 2 glutathione solution was used.
[0023]
Example 10
0.4 ml of a solution of ethylsulfonylbenzene (hereinafter referred to as PVS, positive skin sensitization, mass 168) dissolved in acetone so as to be 16 mg / ml (100 mM), and glutathione 2 mg / ml (6.5 mM) 2 ml of a solution dissolved in 0.2M ammonium acetate was mixed, kept at 37 ° C. for 1 hour, and then lyophilized. The lyophilized product was dissolved in methanol and subjected to MS under the conditions described in Example 1. As a result, it was found that the sample contained a compound having a mass number of 475 and a compound having a mass number of 643. The mass number 475 corresponds to the mass of a compound formed by binding one molecule of PVS and one molecule of glutathione. The mass number 643 coincides with the mass of a compound formed by binding two molecules of PVS and one molecule of glutathione.
[0024]
Example 11
0.4 ml of a solution in which maleic anhydride (hereinafter referred to as MA, positive skin sensitization, mass 98) is dissolved in acetone to be 9.8 mg / ml (100 mM), and glutathione is 2 mg / ml (6.5 mM). The solution was mixed with 2 ml of a solution dissolved in 0.2 M ammonium acetate, kept at 37 ° C. for 1 hour, and then lyophilized. The lyophilized product was dissolved in methanol and subjected to MS under the conditions described in Example 1. As a result, it was found that the sample contained a compound having a mass number of 423 and a compound having a mass number of 521. The mass number 423 corresponds to the mass of the NH ₄ ⁺ salt of a compound formed by binding one molecule of MA and one molecule of glutathione. The mass number 521 corresponds to the mass of the NH ₄ ⁺ salt of a compound formed by binding two molecules of MA and one molecule of glutathione.
[0025]
Example 12
0.4 ml of a solution of methyl methanesulfonate (hereinafter referred to as MMS, positive skin sensitization, mass 110) in acetone so as to be 10 mg / ml (100 mM), and glutathione 2 mg / ml (6.5 mM) 2 ml of a solution dissolved in 0.2M ammonium acetate was mixed, kept at 37 ° C. for 1 hour, and then lyophilized. The lyophilized product was dissolved in methanol and subjected to MS under the conditions described in Example 1. As a result, it was found that the sample contained a compound having a mass number of 497. The mass number corresponds to the mass of a compound formed by detaching a methyl group from two molecules of MMS and binding to one molecule of glutathione.
[0026]
Example 13
0.4 ml of a solution of fluorescein isothiocyanate (hereinafter referred to as FITC, positive skin sensitization, mass number 389) dissolved in acetone to 40 mg / ml (100 mM) and glutathione 2 mg / ml (6.5 mM) The solution was mixed with 2 ml of a solution dissolved in 0.2 M ammonium acetate, kept at 37 ° C. for 1 hour, and then lyophilized. The lyophilized product was dissolved in methanol and subjected to MS under the conditions described in Example 1. As a result, it was found that the sample contained a compound having a mass number of 696 (FIG. 7). The mass number corresponds to the mass of the conjugate of FITC and glutathione.
[0027]
【The invention's effect】
In the early stages of development of medicines, agricultural chemicals, cosmetics, etc., quickly and easily test which of a number of candidate compounds expresses skin sensitization with a small amount of test compound without conducting animal experiments. It becomes possible to do.
[Brief description of the drawings]
FIG. 1 is a liquid chromatogram of glutathione solution 2 (pH 8). GSH indicates a glutathione peak.
FIG. 2 is a liquid chromatogram of a sodium trinitrobenzene sulfonate solution.
TNBS shows the peak of sodium trinitrobenzene sulfonate.
FIG. 3 is a liquid chromatogram of a solution obtained by mixing and reacting a sodium trinitrobenzene sulfonate solution and a glutathione solution 2 (pH 8). GSH indicates a glutathione peak, and TNBS indicates a sodium trinitrobenzene sulfonate peak. The TNBS / GSH reaction product (1) and the TNBS / GSH reaction product (2) show peaks that were not detected in the liquid chromatograms of the glutathione solution 2 (pH 8) and the sodium trinitrobenzene sulfonate solution, respectively.
FIG. 4 is an ESI-MS spectrum of TNBS · GSH reactant (1).
FIG. 5 is an ESI-MS spectrum of TNBS · GSH reaction product (2).
FIG. 6 is a MALDI-MS spectrum of fluorescein isothiocyanate (FITC).
FIG. 7 is a MALDI-MS spectrum of a reaction product of FITC and glutathione.

Claims (4)

Glutathione and test substance are mixed in the absence of oxidase and oxygen donor, and then the presence or absence of a binding substance between the glutathione and test substance is measured. Method.   The method according to claim 1, wherein the presence or absence of the bound substance is measured using a mass spectrometer. The method according to claim 1 or 2, wherein the mixing conditions of glutathione and the test substance are 37 ° C to 60 ° C and 1 hour to 2 days. The concentration of the test substance is 1 mM to 500 mM, the concentration of glutathione is 10 μM to 100 mM, and the molar concentration ratio of glutathione to the test substance is 1: 100 to 20: 1. Method.

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