阅读说明：本技术 一种黑色素瘤细胞在药物-黑色素结合能力测定中的应用 (Application of melanoma cells in determination of drug-melanin binding capacity ) 是由 王若男 李华 钱仪敏 宋硕 蔡丹凤 于 2020-12-03 设计创作，主要内容包括：本发明公开了一种黑色素瘤细胞在药物-黑色素结合能力测定中的应用。所述黑色素瘤细胞为小鼠黑色素瘤细胞B16-F10。本发明验证了将小鼠黑色素瘤细胞用于药物-黑色素结合能力的测定,其测定结果的准确性和稳定性优于现有的原代RPE细胞测定方法及游离黑色素测定的方法；并且小鼠黑色素瘤细胞(例如B16 F10)对于药物的耐受性高于原代RPE细胞,其在用于测定药物-黑色素结合能力中可以耐受更强的药物副作用即适用于药物浓度更高的测定场景。(The invention discloses an application of melanoma cells in determination of drug-melanin binding capacity. The melanoma cell is mouse melanoma cell B16-F10. The invention verifies that the melanoma cells of the mice are used for determining the binding capacity of the medicament-melanin, and the accuracy and the stability of the determination result are superior to those of the prior primary RPE cell determination method and the free melanin determination method; and the tolerance of mouse melanoma cells (such as B16F 10) to drugs is higher than that of primary RPE cells, and the mouse melanoma cells can tolerate stronger drug side effects in the determination of drug-melanin binding capacity, namely the mouse melanoma cells are suitable for determination scenes with higher drug concentration.)

1. Use of a melanoma cell in an assay for drug-melanin binding ability, wherein the melanoma cell is mouse melanoma cell B16.

2. The use of claim 1, wherein the melanoma cell is mouse melanoma cell B16-F10.

3. Use according to claim 1 or 2, wherein said determination comprises the steps of: and mixing the melanoma cells with the medicament and then culturing.

4. The use according to claim 3, wherein the incubation time is 15-25 h; preferably, the time of the cultivation is 20 h.

5. The use of claim 4, wherein the melanoma cells have a density of 1 x 10⁵2X 10 per mL⁵Per mL; preferably, the density of melanoma cells is 1.25 × 10⁵one/mL.

6. A method for detecting a drug-melanin binding ability, wherein the method comprises the steps of:

(1) mixing mouse melanoma cell B16 with the medicine and culturing;

(2) detecting the binding of said drug to said melanin in said mouse melanoma cells.

7. The assay of claim 6, wherein the incubation time in step (1) is 15-25h, preferably 20 h;

and/or the mouse melanoma cell B16 is B16-F10.

8. The detection method according to claim 6, wherein the density of the melanoma cells is 1X 10 when the culture is performed in step (1)⁵2X 10 per mL⁵one/mL.

9. The assay of claim 8, wherein the melanoma cells are at a density of 1.25 x 10⁵one/mL.

10. The assay of any one of claims 6 to 9 wherein the binding in step (2) is expressed by the concentration of the drug in the culture supernatant after cell culture.

Technical Field

The invention belongs to the field of drug detection, and particularly relates to application of melanoma cells in determination of drug-melanin binding capacity.

Background

Melanin is an anionic polymer and is found widely in the eye, skin, brain and inner ear. Melanin is distributed in the eye in the uveal layer (including iris, ciliary body and choroid) and the Retinal Pigment Epithelium (RPE). Many drugs bind to melanin, accumulate in ocular tissues, affect their pharmacokinetics in the eye, interfere with normal retinal function, and have unpredictable effects on vision. Not only ophthalmic drugs, but also many non-ophthalmic drugs administered systemically are combined with ocular melanin, for example, hydroxychloroquine, which is the most widely used drug for treating systemic lupus erythematosus, and the most clinically significant adverse reaction is ocular toxicity such as macular degeneration and retinal atrophy. It has been found that chloroquine and hydroxychloroquine have affinity for pigmented tissues, particularly with the RPE in the eye, and thus, ocular toxicity resulting from administration of hydroxychloroquine may be associated with melanin binding; the antipsychotic thioridazine also has an affinity for pigmented tissues, accumulates in the RPE and causes destruction of retinal pigment at high doses. Fundus examination of patients taking thioridazine for a prolonged period of time revealed that the RPE of the patient formed a pigmented large plaque with a loss of choroidal capillaries, which could ultimately lead to progressive macular degeneration and loss of visual function if rigorous visual function monitoring was not performed during dosing. In addition, chlorpromazine, tamoxifen, memantine and other drugs all show high eye melanin binding capacity, although not all drugs with high affinity with melanin will certainly cause eye toxicity, the phenomenon of drug-melanin binding should be paid attention to. On the one hand, drug-melanin binding can affect the pharmacokinetics and pharmacodynamics of the drug itself; on the other hand, RPE plays an important role in maintaining the integrity and physiological functions of retinal photoreceptor cells, and toxic effects on RPE will eventually affect the optic nerve, resulting in loss of visual function.

Currently, there are two main methods for in vitro study of the binding ability of melanin, namely: one is in vitro incubation of free melanin with drugs, the melanin is artificially synthesized or extracted from animal eyes; the other type is in vitro incubation of drugs and melanin-containing cells, a common cell model is primary RPE cells of pigs or cows, the freshly extracted primary RPE cells are rich in melanin, but the extraction process is complex and is easily polluted by other types of cells in tissues, the primary RPE cells have no melanin synthesis capacity, and the content of melanin in the cells is continuously reduced along with the increase of the number of passages.

Disclosure of Invention

The invention aims to solve the technical problems that in the prior art, the difference between the drug-melanin binding numerical value and in-vivo metabolism of the free melanin determination is large, and the melanin content in primary RPE cells is reduced along with the increase of the passage number, and provides the application of melanoma cells in the drug-melanin binding capacity determination.

The inventor of the invention who uses melanoma cells does not perform experiments in the field, so that the feasibility of the mouse melanoma cell B16 in determination of drug-melanin binding capacity is verified, and the effects of simplifying experimental procedures and stabilizing test results can be achieved by unexpectedly finding that melanoma cells, particularly B16-F10, have excellent effects.

In order to solve the above technical problems, one of the technical solutions provided by the present invention is: use of a melanoma cell in an assay for drug-melanin binding ability, wherein the melanoma cell is mouse melanoma cell B16.

The mouse melanoma cell B16 can be conventional in the art, and is preferably a mouse melanoma cell B16-F10.

Preferably, the assay comprises the steps of: and mixing the melanoma cells with the medicament and then culturing. The culture time is preferably 15-25 h; more preferably, the time of the cultivation is 20 hours.

The density of the melanoma cells during the culturing process is preferably 1 × 10⁵2X 10 per mL⁵one/mL, more preferably 1.25X 10⁵one/mL.

In order to solve the above technical problems, the second technical solution provided by the present invention is: a method for detecting a drug-melanin binding ability, wherein the method comprises the steps of:

(1) mixing mouse melanoma cell B16 with the medicine and culturing;

(2) detecting the binding of said drug to said melanin in said mouse melanoma cells.

The preferable definition in the above steps is one of the technical solutions as described above, specifically as follows.

Preferably, the culturing time in the step (1) is 15-25h, preferably 20 h;

wherein the mouse melanoma cell is B16, preferably B16-F10; for example, B16-F10 available from Shanghai Rich Biotech, Inc.

Preferably, the density of the melanoma cells is 1X 10 when the culturing is performed in step (1)⁵2X 10 per mL⁵one/mL, preferably 1.25X 10⁵one/mL.

Preferably, the binding in step (2) is expressed by the concentration of the drug in the culture supernatant after cell culture.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

the invention adopts mouse melanoma cells (preferably B16F 10), which are rich in melanin and have rich and easily-obtained sources, and compared with extracted melanin, the invention has complete barrier and transport mechanism, can regulate the absorption and elimination of drugs from the cells, and is more close to the real biological environment in vivo. The invention verifies that the melanoma cells of the mice are used for determining the binding capacity of the medicament-melanin, and the accuracy and the stability of the determination result are superior to those of the prior primary RPE cell determination method and the free melanin determination method; and the tolerance of mouse melanoma cells (such as B16F 10) to drugs is higher than that of primary RPE cells, and the mouse melanoma cells can tolerate stronger drug side effects in the determination of drug-melanin binding capacity, namely the mouse melanoma cells are suitable for determination scenes with higher drug concentration.

Drawings

FIG. 1 is a standard curve of the relationship between absorbance and melanin concentration measured by an ultraviolet spectrophotometer.

FIG. 2 shows the lower limit of LC-MS/MS quantitation: an analyte.

FIG. 3 shows the lower limit of LC-MS/MS quantitation: an internal standard.

FIG. 4 shows the upper limit of LC-MS/MS quantitation: an analyte.

FIG. 5 shows the upper limit of LC-MS/MS quantitation: an internal standard.

FIG. 6 is a standard curve diagram of LC-MS/MS determination peak area and hydroxychloroquine sulfate drug concentration.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Experimental materials, reagents, instrument information used in the examples:

1. cell line

Mouse B16F 10 cells, cat no: FH0361, Shanghai Rich and well-balanced Biotech Co., Ltd

2. Reagent

Chloroquine bisulfate:

batch number: 190804, Chongqing Kangle pharmaceuticals, Inc., expiration date: 2021.05.05.

synthesizing melanin:

batch number: BCBZ9117, source: Sigma-Aldrich, effective date: 2029.12.18.

1640 medium:

batch number: 2174114, source: gibco, expiration date: 2020.11.30.

fetal bovine serum:

batch number: 1928700, source: gibco, expiration date: 2024.07.31.

trypsin:

batch number: NM01, source: abrin, effective period: 2021.04.05.

3. materials and instruments

6, a pore plate:

the model is as follows: 3516, brand: costar

Uv-vis spectrophotometer:

the brand model is as follows: agilent Carry60 UV-VIs

Example 1B16 cell culture

Mouse melanoma B16 cells were plated in a cell culture medium containing 10% fetal calf serum and streptomycin (100 kU. L)^－1) 1640 medium (pH 7.2) at 37 ℃ with 5% CO₂And relative saturation humidity, cells in logarithmic growth phase were taken for subsequent experiments.

EXAMPLE 2 preparation of Hydroxychloroquine sulfate

Adding a certain amount of hydroxychloroquine sulfate into PBS (pH7.4) with a specific volume to make the concentration of the hydroxychloroquine sulfate be 1mM, filtering and sterilizing the hydroxychloroquine sulfate by using a 0.22 mu m microporous filter membrane, diluting the mixture to a corresponding concentration by using a 1640 culture medium, and marking the mixture for later use.

Example 3 Standard Curve for melanin content and determination of drug binding ability to free melanin

10mg of synthetic melanin was weighed, 10mL of PBS was added, and sonication was carried out for 15min to obtain a melanin suspension of 1 mg/mL. Diluted with PBS to 100. mu.g/mL, 50. mu.g/mL, 20. mu.g/mL, 10. mu.g/mL, and 2. mu.g/mL. The absorbance at 475nm was measured. And (3) performing linear regression by taking the absorbance value as an abscissa and the concentration as an ordinate to obtain a standard curve of melanin content: 67.402x-0.5928, R²0.9998. See fig. 1.

The results of the determination of the binding capacity of the drug hydroxychloroquine sulfate to free melanin are shown in table 1.

TABLE 1 binding Capacity of Hydroxychloroquine sulfate to synthetic melanin

Example 4 determination of drug-melanin binding Capacity

B16 cells were sampled at 1.25X 10⁵Each mL^－1Inoculation in 6-well plates2mL per well, 37 ℃ and 5% CO₂The culture was carried out overnight. Adding 2mL of hydroxychloroquine sulfate with the concentration of 0.31, 1, 5, 10 and 20 mu M, setting 3 compound holes, incubating in a constant-temperature incubator at 37 ℃ for 20h, collecting supernatant, and placing in a refrigerator at 4 ℃ for testing.

After collecting the supernatant, the cells were washed 2 times with PBS, digested and collected in a centrifuge tube (total 2mL, 20. mu.L of which was diluted by a certain factor for counting), centrifuged at 1000r/min for 5min, the supernatant was discarded, 1mL of 1mol/L NaOH solution containing 10% DMSO was added, resuspended, incubated in a 80 ℃ water bath for 45min, and the absorbance value at 475nm was determined. Bring the absorbance values into the standard curve (y 67.402x-0.5928, R)²0.9998), the corresponding melanin content was determined.

In triplicate, the concentration of hydroxychloroquine sulfate in the cell culture medium after 20h incubation was determined using LC-MS/MS.

LC-MS/MS related parameters and experimental methods:

instrument

·AB SCIEX TRIPLE QUAD^TM4500 LC-MS instrument

Centrifugal machine

Microanalysis balance, to an accuracy of 0.001mg

Vortex mixer

Pure water meter (resistivity is more than or equal to 18.2M omega cm)

Analyst data acquisition software (Version 1.7.0)

Column chromatography: xbridge BEH C18,2.5 μm, 2.1X 50mm, Waters

Pipet (20. mu.L, 100. mu.L, 200. mu.L, 300. mu.L, 1000. mu.L)

Electric pipette

Second, instrument parameters

1. Liquid phase conditions:

sample introduction amount: 5 μ L

A chromatographic column: xbridge BEH C18,2.5 μm, 2.1X 50mm, Waters

Column temperature: 40.0 deg.C

Sample injector temperature: 4 deg.C

Mobile phase A: 0.1% FA in H₂O

Mobile phase B: 0.1% FA in ACN

Operating time: 5.0min

Gradient of mobile phase:

needle washing solvent: strong wash (MeOH: ACN: IPA: DMSO ═ 1:1:1:1, v/v/v/v)

Weak wash (MeOH: H)₂O＝1:1，v/v)

Needle washing procedure: rinse Type: External only;

Rinse mode:Before and after aspiration,Dip time:1s；

Rinse pump method:Rinse pump,then Port,Time:2s；

Rinse settings:Rinsing speed:35μL/s；

Rinse volume:500μL；

Measuring line purge volume:100μL；

retention time:

2. conditions of Mass Spectrometry

The instrument model is as follows: AB SCIEX TRIPLE QUADTM 4500

An ion source: ESI

Ionization mode: positive ion

MRM ion pair:

the instrument parameters are as follows:

parameter(s)	Chloroquine bisulfate	Labetalol
			IS(v)	5500	5500
TEM(℃)	500	500
			CAD	9	9
CUR(psi)	30	30
			Gas 1(psi)	25	25
Gas 2(psi)	40	40
			DP(v)	115	65
EP(v)	10	10
			CE(v)	30	20
CXP(v)	14	11

Preparation of reagent

1. Mobile phase a (mpa): 0.1% FA in H₂O

Take 1000mL of H₂And adding 1mL of FA into the O solution in a solvent bottle, and mixing uniformly. The shelf life is 1 month at room temperature.

2. Mobile phase b (mpb): 0.1% FA in ACN

1000mL of ACN was added to 1mL of FA in a solvent bottle and mixed well. The shelf life is 1 month at room temperature.

3. Strong wash Solution (SNW): MeOH ACN IPA DMSO 1:1:1:1, v/v/v 500mL MeOH, 500mL ACN, 500mL IPA, 500mL DMSO was mixed well.

The shelf life is 3 months at room temperature.

4. Weak wash solution (WNW): ACN H₂O＝1:1，v/v

1000mL of ACN and 1000mL of H were taken₂And O, and mixing uniformly. The shelf life is 3 months at room temperature.

5. Working solution diluent&The solvent (Diluent) is MeOH H₂O＝1:1，v/v

500mL of MeOH and 500mL of H were added₂And O, and mixing uniformly. The shelf life is 3 months at room temperature.

6. Precipitant (Precipitant) MeOH

500mL of MeOH was taken. Shelf life at room temperature of 3 months

7. Stock dilutions (dilution 1): MeOH

Take 30mL MeOH. The shelf life is 3 months at room temperature.

8. Labetalol stock dilution (dilution 2): DMSO (dimethylsulfoxide)

30mL of DMSO was taken. The shelf life is 3 months at room temperature.

Preparation of standard curve and quality control

The stock solution and the working solution are both stored in an ultra-low temperature refrigerator (70 ℃ below zero to 90 ℃ below zero).

1. Preparation of stock solution of standard yeast

Precisely weighing a proper amount of hydroxychloroquine sulfate in a sample bottle, adding a proper amount of methanol, ultrasonically dissolving, shaking up, and preparing into a standard yeast stock solution with the concentration of 0.500 mg/mL. (the mass conversion factor is 0.773.)

2. Standard song&Preparation of SST working solution (working solution diluent: MeOH: H)₂O＝1:1)

Standard koji & SST working liquids were obtained using standard koji stock solutions, formulated according to the following table:

preparation of hydroxychloroquine sulfate standard curve & SST working solution

3. Preparation of Standard Curve & SST samples

Standard curve & SST working solutions were used, formulated according to the following table, to obtain standard curve & SST samples:

preparation of hydroxychloroquine sulfate standard curve & SST sample

The standard curve samples were stored in an ultra-low temperature freezer (-90 to-70 ℃).

4. Preparation of quality control stock solution

Precisely weighing a proper amount of hydroxychloroquine sulfate in a sample bottle, adding a proper amount of methanol, ultrasonically dissolving, shaking uniformly, and preparing into a quality control stock solution with the concentration of 0.500 mg/mL. (the mass conversion factor of hydroxychloroquine sulfate is 0.773.)

5. Preparation of quality control working solution (working solution diluent: MeOH: H)₂O(ml:ml)＝1:1)

Using the quality control stock solution, preparing according to the following table to obtain a quality control working solution:

preparation of hydroxychloroquine sulfate quality control working solution

6. Preparation of quality control sample

Using a quality control working solution, preparing according to the following table to obtain a quality control sample:

preparation of hydroxychloroquine sulfate quality control sample

The quality control samples were stored in an ultra-low temperature freezer (-70 to-90 ℃).

7. Preparation of internal standard stock solution

Accurately weighing a proper amount of labetalol hydrochloride into a brown sample bottle, adding a proper amount of labetalol hydrochloride stock solution diluent, dissolving and shaking up to prepare an internal standard stock solution with the concentration of 1.000 mg/mL. (the mass conversion factor of labetalol hydrochloride is 0.998.)

Preparation of internal standard working solution (working solution diluent: MeOH: H)₂O＝1:1)

Internal standard working solutions were prepared according to the following table using internal standard stock solutions:

preparation of labetalol hydrochloride internal standard working solution

Fifthly, sample treatment step

The LC-MS/MS results are shown in FIGS. 2-5.

The standard curve of LC-MS/MS determination peak area and hydroxychloroquine sulfate drug concentration is shown in FIG. 6, and the standard curve is that y is 0.00569x-0.000292, R²＝0.9958。

And (3) performing linear least square regression calculation on the theoretical concentration of the analyte in the standard curve by comparing the peak area of the analyte with the peak area of the internal standard, and calculating the actually measured concentration of the analyte in the sample by using the obtained regression equation. The concentration of hydroxychloroquine sulfate initially added is [ L ]₀]And the concentration of hydroxychloroquine sulfate in the supernatant measured after 20h incubation was [ L]Calculating the ratio of the hydroxychloroquine sulfate at different initial concentrations to the concentration of hydroxychloroquine sulfate in the culture medium according to the formula (1):

V_wvolume of culture Medium per well, V_cellCell volume (3. mu.L/10)⁶Individual cell), m is the mass of melanin in the cell.

The results of the binding capacity assay of hydroxychloroquine sulfate to mouse B16-F10 cells are shown in Table 2.

TABLE 2 binding Capacity of Hydroxychloroquine sulfate to B16 cells

From the above table, it can be seen that: the use of melanoma cells allows for accurate and stable determination of free melanin.