Evaluation method of ABC transporter substrate

Abstract

The invention provides an evaluation method of an ABC transporter substrate, and belongs to the technical field of biomedicine. According to the invention, MDCK II cells of overexpression human multidrug resistance protein 1 and breast cancer drug resistance protein are adopted as a model, and a transport buffer solution added with bovine serum albumin is adopted, so that in an ABC transporter substrate experiment for evaluating the adsorptive compound, the recovery rate of the adsorptive compound in the ABC transporter substrate experiment is remarkably improved, and the recovery rate of the adsorptive compound in the ABC transporter substrate experiment is remarkably increased. The characteristics of the substrate are accurately judged, and a more accurate preclinical basis is provided for drug interaction.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an evaluation method for an ABC transporter substrate. Background Art

[0002] Transporters are a class of functional proteins expressed on the cell membrane that participate in the absorption, distribution, and excretion of drugs. The loss or downregulation of their functions will affect the body's disposal of drugs. In addition, transporter-mediated drug interactions (DDIs) can lead to significant changes in the clinical disposal of drugs in the body, which can directly or indirectly affect drug efficacy and even induce toxicity in severe cases. Drug regulatory authorities in the United States, Europe, and China have successively issued guidelines that recommend in vitro evaluation of potential interactions between major transporters and test drugs to provide a reference for in vivo studies.

[0003] Multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) are members of the ATP-binding cassette (ABC) membrane transporter superfamily associated with tumor resistance and are considered to be one of the important causes of tumor resistance. Literature reports show that a certain Protac (Proteolysis Targeting Chimera) drug interacts with rosuvastatin. Subjects who took the cholesterol-lowering drug rosuvastatin and the Protac drug at the same time had elevated AST (aspartate aminotransferase) / ALT (alanine aminotransferase). The symptoms disappeared after rosuvastatin was discontinued and treatment could be continued. The reason is that the Protac drug can inhibit the BCRP transporter, and rosuvastatin is a known BCRP transporter substrate. The combination of rosuvastatin and BCRP inhibitors may inhibit its efflux in the intestine to increase oral absorption, and inhibit its bile excretion in the liver, thereby increasing the blood concentration of rosuvastatin and causing corresponding adverse reactions.

[0004] At present, the in vitro evaluation models of DDI caused by ABC transporters mainly include membrane vesicle models, polarized monolayer cell models, and "sandwich" cultured hepatocyte models. Among them, the polarized monolayer cell model refers to a model that uses a cell line that can spontaneously polarize to form a complete cell monolayer for transporter DDI research. Caco-2 cells are a commonly used cell model. In addition, epithelial cell lines with high expression of specific transporters, such as LLC-PK1 (Lewis lung cancer pig kidney cells 1) and MDCK, can also be used for related research. Transwell chambers are used to assist in the study of drugs crossing cell membranes. Cells are inoculated on Transwell chambers and cultured to form a complete polarized cell monolayer. Bidirectional permeability experiments (apical-basolateral, AB and BA directions) are carried out, and the bidirectional apparent permeability coefficient (apical permeability coefficient, P app ) and the calculation of efflux ratio (ER) to evaluate whether the compound is a substrate for the ABC transporter. In addition, by measuring the net flow of the probe substrate in the BA direction or the change in ER value, the inhibitory effect of the compound on a specific transporter can also be evaluated.

[0005] However, when using the Transwell model to conduct bidirectional permeability experiments to study interactions with ABC transporters, special attention should be paid to the following three points: (1) Recovery rate: If the test compound has strong nonspecific adsorption or is easily retained in the cell, the recovery rate may be insufficient (usually less than 70.0%); in addition, the incubation time of the model cells is long (usually more than 60 minutes), and factors such as intracellular metabolic enzymes need to be considered. (2) The transmembrane ability of the compound itself: The substrate binding site of the efflux transporter is located in the cell. The test compound needs to cross the membrane to reach the cell and interact with the substrate binding site. If the passive diffusion of the test compound is poor and it is difficult to enter the cell through the mediation of the transporter, it will affect the DDI evaluation. (3) Test concentration: The bidirectional transport of the test compound in the Transwell model is the result of the superposition of the dual mechanisms of passive transport and active transport. Its passive diffusion conforms to Fick's diffusion law. If the concentration of the test compound is too high, the amount of compound passively transported may be much higher than the amount of active transport by the transporter, which masks the role of active transport by the transporter. On the other hand, based on the Michaelis-Menten model, high concentrations of the test compound will saturate the binding sites of the transporter, resulting in a false negative result.

[0006] The strong adsorption of the compound causes it to adhere more to the cell surface and test consumables, resulting in insufficient recovery in the test. At the same time, compounds with strong adsorption usually also have poor water solubility (such as Protac drugs), which further affects the test recovery. The low recovery rate has a significant impact on the accuracy of ABC transporter substrate judgment, which in turn misleads the risk of transporter drug interaction DDI assessment.

[0007] Therefore, it is of great significance to develop a method that can improve the accuracy of ABC transporter substrate judgment. Summary of the invention

[0008] In view of the problems existing in the prior art, the object of the present invention is to provide a method for evaluating an ABC transporter substrate.

[0009] The present invention provides a method for evaluating an ABC transporter substrate, the method comprising the following steps:

[0010] (1) dissolving an adsorbent compound in an organic solvent to prepare an adsorbent compound solution;

[0011] (2) preparing a transport buffer containing bovine serum albumin;

[0012] (3) preparing a drug resistance protein inhibitor solution;

[0013] (4) A bidirectional permeability experiment was carried out using a Transwell model using an adsorbed compound solution, a transport buffer solution, and a drug-resistant protein inhibitor solution. After administration, the cell layer in the Transwell model was cleaned, and samples from the administration side and the receiving side were collected. The efflux ratio A of the adsorbed compound in the absence of the drug-resistant protein inhibitor and the efflux ratio B of the adsorbed compound in the presence of the drug-resistant protein inhibitor were calculated. When the following conditions were met: A was greater than 2.0, and the reduction rate of B compared with A was greater than 50%, the adsorbed compound was judged to be an ABC transporter substrate.

[0014] The present invention provides a method for improving the recovery rate of an adsorbable compound in an ABC transporter substrate experiment, the method comprising the following steps:

[0015] (1) dissolving an adsorbent compound in an organic solvent to prepare an adsorbent compound solution;

[0016] (2) preparing a transport buffer containing bovine serum albumin;

[0017] (3) preparing a drug resistance protein inhibitor solution;

[0018] (4) using the adsorbed compound solution, transport buffer and drug resistance protein inhibitor solution, a bidirectional permeability experiment was carried out using the Transwell model. After the administration, the cell layer in the Transwell model was washed, and the samples on the administration side and the receiving side were collected to calculate the recovery rate of the adsorbed compound;

[0019] Recovery rate of adsorbed compound = (total amount of compound on the administration side at the end of the experiment + total amount of compound on the receiving side at the end of the experiment) / (total amount of compound on the administration side at the beginning of the experiment) × 100%.

[0020] Furthermore, the Transwell model is a model that meets the following criteria: using an ABC transporter positive substrate solution, a transport buffer and a drug-resistant protein inhibitor solution, a bidirectional permeability experiment is carried out using the Transwell model, after the administration is completed, the cell layer in the Transwell model is cleaned, and samples from the administration side and the receiving side are collected, and the efflux ratio C of the ABC transporter positive substrate in the absence of the drug-resistant protein inhibitor and the efflux ratio D of the ABC transporter positive substrate in the presence of the drug-resistant protein inhibitor are calculated, and the following criteria are met: C is greater than 2.0, and the reduction rate of D compared with C is more than 50%;

[0021] The ABC transporter positive substrate solution is prepared according to the following method: dissolving the ABC transporter positive substrate with an organic solvent to prepare the ABC transporter positive substrate solution.

[0022] Furthermore, in step (1), the adsorptive compound is a Protac compound.

[0023] Furthermore, in step (2), the transport buffer contains 0.1-1% bovine serum albumin, preferably 0.5% bovine serum albumin.

[0024] Furthermore, in step (3), the drug-resistant protein is multidrug resistance protein 1 or breast cancer resistance protein.

[0025] Furthermore, in step (3), the multidrug resistance protein 1 inhibitor is oxaquinoxaline; and the breast cancer resistance protein inhibitor is Ko143.

[0026] Furthermore, the multidrug resistance protein 1 substrate is digoxin; and the breast cancer resistance protein substrate is teriflunomide.

[0027] Furthermore, in step (1), the organic solvent is dimethyl sulfoxide;

[0028] In step (3), the solvent for preparing the drug resistance protein inhibitor solution is an organic solvent, preferably dimethyl sulfoxide.

[0029] Further, the solvent in the dosing side solution or the receiving side solution is a transport buffer containing bovine serum albumin;

[0030] The concentration of dimethyl sulfoxide in the dosing side solution or the receiving side solution is 0.1-0.3%, preferably 0.2%.

[0031] Furthermore, the concentration of the multidrug resistance protein 1 inhibitor in the dosing side solution or the receiving side solution is 3-7 μM, preferably 5 μM; the concentration of the breast cancer resistance protein inhibitor in the dosing side solution or the receiving side solution is 0.1-2 μM, preferably 1 μM.

[0032] Furthermore, the cells used in the Transwell model are MDCKⅡ cells; MDCKⅡ cells are obtained by culturing MDCKⅡ cells with drug resistance protein gene knocked out.

[0033] Furthermore, the culture conditions are culturing in a DMEM medium containing 10% fetal bovine serum, 1% non-essential amino acids and 1% penicillin-streptomycin at 37° C., 5% CO 2 and relative saturated humidity.

[0034] Furthermore, the conditions of the bidirectional permeability experiment are: incubation at 37° C. for 90 to 120 minutes.

[0035] The present invention has achieved the following beneficial effects:

[0036] The present invention has experimentally found that for adsorbent Protac drugs, the use of transport buffers with added bovine serum albumin (BSA), while processing and collecting cell layer samples, can greatly improve the recovery rate of Protac molecules in ABC transporter substrate experiments, and accurately determine the substrate characteristics. In addition to the Protac drugs used in the specific embodiments of the present invention, other Protac drugs are also applicable to the method of the present invention.

[0037] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0038] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. DETAILED DESCRIPTION

[0039] The raw materials used in the present invention are synthesized standard reference compounds, and the equipment used are all known products.

[0040] 1. Source of raw materials of the present invention

[0041] (1) Test compound:

[0042] Palbociclib: non-adsorbed control compound, molecular weight 447.5;

[0043]

[0044] BSJ-03-123 (a CDK6 protein degrader): Protac compound, molecular weight 937.01.

[0045]

[0046] (2) Sources of main reagents are shown in Table 1:

[0047] Table 1. Main reagents

[0048]

[0049]

[0050] (3) Cell source:

[0051] The cells used in the present invention were purchased from Sigma, and they are canine MDR1 knockout human MDR1 knockout MDCKⅡ cells (Cat. No.: MTOX1303) and canine MDR1 knockout human BCRP knockout MDCKⅡ cells (Cat. No.: MTOX1304), and are represented by MDCKⅡ-MDR1 and MDCKⅡ-BCRP cells in the specification, respectively.

[0052] 2. Data processing method

[0053] (1) Fluorescent yellow penetration:

[0054] Apparent permeability coefficient (P app (LY) = (acceptor side volume / (membrane area × culture time)) × ((acceptor side fluorescence value at the end of culture – blank solution fluorescence value) / (administration side fluorescence value at the beginning of culture – blank solution fluorescence value))

[0055] (2) Permeability of test compounds:

[0056] Apparent permeability coefficient (P app ) = (receiving side volume / (membrane area × culture time)) × (receiving side drug concentration at the end of culture) / (drug concentration on the administration side at the beginning of culture × dilution multiple);

[0057] This experiment uses Millipore cell culture plates: membrane area = 0.11 cm 2, receiving side volume = 0.25 mL (AB) or 0.075 mL (BA);

[0058] 1) When the test drug is Palbociclib, the incubation time = 90 minutes. In the test, the samples of the positive group and the Palbociclib 10 μM group were diluted 10 times and then injected for testing.

[0059] 2) When the test drug was BSJ-03-123, incubation time = 120 minutes;

[0060] (3) Drug excretion ratio:

[0061] Efflux ratio (ER) = P app (BA) / P app (AB)

[0062] (4) ER reduction percentage after using inhibitors (%):

[0063] ER reduction percentage (%) = (ER without inhibitor - ER after adding inhibitor / without inhibitor

[0064] ER)×100%

[0065] (5) Recovery rate of compound solution:

[0066] Recovery rate (%) = (total amount of compound on the administration side after incubation time + total amount of compound on the receiving side after incubation time) / (total amount of compound on the administration side at 0 minutes) × 100%

[0067] 3. Evaluation criteria

[0068] When the specific inhibitor is absent, the efflux ratio of the positive substrate is greater than 2.0, and compared with the absence of the specific inhibitor, the ER value reduction rate in the presence of the specific inhibitor exceeds 50%, indicating that the test system is qualified.

[0069] According to the technical guidelines for drug interaction studies issued by the China National Medical Products Administration (NMPA) (Technical Guidelines for Drug Interaction Studies (Trial) (National Medical Products Administration Review Center, January 2021) and the United States Food and Drug Administration (USFDA) (InVitro Drug Interaction Studies-Cytochrome P450 Enzyme-and Transporter-Mediated Drug Interactions (Guidance for Industry). FDA, January 2020): When the ER value of the test substance is greater than or equal to 2 in the absence of the specific inhibitor, and the ER value reduction rate in the presence of the specific inhibitor is more than 50% compared with the absence of the specific inhibitor, the test substance is judged to be a substrate of the transporter.

[0070] Example 1. Evaluation of the transport effect of ABC transporters on Palbociclib

[0071] 1. Cell culture

[0072] MDCKⅡ-MDR1 and MDCKⅡ-BCRP cells were routinely cultured in DMEM medium containing fetal bovine serum (10%), non-essential amino acids (1%) and penicillin-streptomycin (1%) at 37°C, 5% CO2 and relative saturated humidity. When the cell confluence reached more than 80%, 1.35×10 5 cells / mL (MDCKⅡ-MDR1) and 1.26×10 5 The cells were inoculated at a density of 10 cells / mL (MDCKⅡ-BCRP) in Transwell-96 well cell culture plates, and the medium was changed on the 1st and 4th days after plating. The cells were taken out on the 5th day and used for transport experiments.

[0073] 2. Preparation of dosing solution

[0074] (1) Preparation of compound stock solutions

[0075] Test compound: Palbociclib stock solution was prepared at a concentration of 10 mM using DMSO as solvent;

[0076] Positive inhibitors: Prepare a 5 mM Zosuquidar stock solution in DMSO and a 1 mM Ko143 stock solution in DMSO;

[0077] Positive substrate: Prepare a 10 mM digoxin stock solution in DMSO and a 5 mM teriflunomide stock solution in DMSO.

[0078] (2) Preparation of transport buffer

[0079] 1) Dissolve 48.04 g of hydroxyethylpiperazineethanesulfonic acid (HEPES) and 1.720 g of sodium chloride (NaCl) in 200 mL of H2O to prepare a 1 M HEPES-0.85% NaCl solution;

[0080] 2) Take 51.2 mL of 1 M HEPES-0.85% NaCl solution and mix it with 2000 mL of HBSS (1×), and adjust the pH to 7.21 with 2 M NaOH to obtain a blank transport buffer.

[0081] Use blank transport buffer to prepare the dosing solution as follows (end A is the upper chamber, end B is the lower chamber):

[0082] Table 2. Dosage formulation

[0083]

[0084] Note: The concentrations listed in the table are all final concentrations. The total concentration of organic solvent (DMSO) in the A-end and B-end drug solutions of all groups in the experiment was 0.2%.

[0085] 3. Transport experiment

[0086] After 5 days of culture, MDCKⅡ-MDR1 and MDCKⅡ-BCRP cells were taken out for drug transport test. The culture medium was removed and the cells were rinsed twice with preheated blank transport buffer. Then, the drug solution was added to both sides of the cell plate as listed in the table below, and samples were collected after the incubation (37℃, 90min):

[0087] Table 3. Dosing methods for transport experiments

[0088]

[0089] 4. Sample processing

[0090] (1) Palbociclib sample processing:

[0091] Table 4. Palbociclib Sample Processing

[0092]

[0093] (2) Digoxin sample processing:

[0094] Table 5. Digoxin sample processing

[0095]

[0096]

[0097] (3) Teriflunomide sample processing:

[0098] Table 6. Teriflunomide Sample Processing

[0099]

[0100] Note: "Taking the supernatant for sampling" means taking the supernatant into a 96-well plate for LC-MS / MS to detect drug concentration.

[0101] 5. Cell membrane integrity test (fluorescent yellow permeability test)

[0102] Before incubation (0 minutes), take 100 μL of sample from each well of the cell plate A and place it in a 96-well black fluorescence measurement plate; after 90 minutes of incubation, take 100 μL of sample from each well of the cell plate B and place it in a 96-well black fluorescence measurement plate (for specific sample collection methods, see the transport experiment in step 3). The sample is read with an ELISA reader to detect the fluorescence intensity (excitation wavelength 485 nm, emission wavelength 535 nm).

[0103] 6. Chromatographic and mass spectrometry conditions

[0104] (1) Digoxin detection conditions

[0105] Chromatographic column: ACQUITY UPLC-HSS T3, 1.8 μm, 2.1 × 50 mm, Waters

[0106] Column temperature: 40°C

[0107] Ion source: electrospray ionization (ESI)

[0108] Mobile phase A: pure water

[0109] Mobile phase B: acetonitrile

[0110] Internal Standard (IS): Tolbutamide

[0111] Gradient elution:

[0112] Table 7. Gradient elution

[0113]

[0114] Retention time: t R(DGX) ≈0.89min; t R(IS) ≈0.93min

[0115] Capillary voltage: 2kV

[0116] Ion source temperature: 150°C

[0117] Desolvation temperature: 350°C

[0118] Desolvation gas flow rate: 650L / h

[0119] Cone gas flow rate: 150L / h

[0120] Atomizing gas pressure: 6 bar

[0121] Detection method: Multiple reaction monitoring (MRM) in negative ion mode

[0122] Ion pairs: m / z 779.54→m / z 84.98 (DGX), m / z 268.91→m / z 170.06 (IS) Cone voltage: 55 V (DGX) and 46 V (IS)

[0123] Impact voltage: 50eV (DGX) and 16eV (IS)

[0124] (2) Teriflunomide detection conditions

[0125] Chromatographic column: ACQUITY UPLC-HSS T3, 1.8 μm, 2.1 × 50 mm, Waters

[0126] Column temperature: 40°C

[0127] Ion source: electrospray ionization (ESI)

[0128] Mobile phase A: pure water

[0129] Mobile phase B: acetonitrile Internal standard (IS): teriflunomide-D4

[0130] Gradient elution:

[0131] Table 8. Gradient elution

[0132]

[0133] Retention time: t R(TLF) ≈1.22min; t R(IS) ≈1.22min

[0134] Capillary voltage: 2kV

[0135] Ion source temperature: 150°C

[0136] Desolvation temperature: 550°C

[0137] Desolvation gas flow rate: 800L / h

[0138] Cone gas flow rate: 150L / h

[0139] Atomizing gas pressure: 6 bar

[0140] Detection method: Multiple reaction monitoring (MRM) in negative ion mode

[0141] Ion pairs: m / z 269.07→m / z 159.98 (TLF), m / z 273.03→m / z 164.04 (IS) Cone voltage: 45 V (TLF) and 49 V (IS)

[0142] Impact voltage: 26eV (TLF) and 28eV (IS)

[0143] (3) Palbociclib testing conditions

[0144] Chromatographic column: ACQUITY UPLC-BEH C18, 1.7 μm, 2.1 × 50 mm, Waters

[0145] Column temperature: 40°C

[0146] Ion source: electrospray ionization (ESI)

[0147] Mobile phase A: 0.1% formic acid-5 mM ammonium acetate water

[0148] Mobile phase B: 0.1% formic acid-acetonitrile

[0149] Internal Standard (IS): Palbociclib-D6

[0150] Gradient elution:

[0151] Table 9. Gradient elution

[0152]

[0153] Retention time: t R(Palbociclib) ≈0.63min; t R(IS) ≈0.63min

[0154] Capillary voltage: 3kV

[0155] Ion source temperature: 150°C

[0156] Desolvation temperature: 550°C

[0157] Desolvation gas flow rate: 650L / h

[0158] Cone gas flow rate: 150L / h

[0159] Atomizing gas pressure: 7 bar

[0160] Detection method: Positive ion mode multiple reaction monitoring (MRM)

[0161] Ion pairs: m / z 439.61→m / z 324.95 (Palbociclib), m / z 38.32→m / z 322.55 (IS)

[0162] Cone voltage: 30V (Palbociclib) and 30V (IS)

[0163] Collision voltage: 30eV (Palbociclib) and 30eV (IS)

[0164] 7. Test results

[0165] (1) Cell membrane integrity (permeability of the cell monolayer to fluorescent yellow)

[0166] The permeability coefficients of MDCKⅡ-MDR1 and MDCKⅡ-BCRP cells to fluorescein in each well of the test cell plate were all lower than 5.0×10 -6 cm / s, indicating that the cell layer has good integrity.

[0167] Table 10. Apparent permeability coefficient of fluorescent yellow in MDCKⅡ-MDR1 cells

[0168]

[0169]

[0170] Table 11. Apparent permeability coefficient of fluorescent yellow in MDCKⅡ-BCRP cells

[0171]

[0172] (2) Transport behavior of Palbociclib in the MDCKⅡ-MDR1 cell system

[0173] In the MDCKⅡ-MDR1 cell experimental system, the efflux ratio of 10μM digoxin was 21.78 when no inhibitor was added, and the efflux ratio was 1.00 under the action of 5μM positive inhibitor Zosuquidar, and the ER reduction percentage was 95.39%, indicating that the test system is normal and suitable for the present invention. At the concentration levels of 1μM and 10μM, the efflux transport ratios of Palbociclib on MDCKⅡ-MDR1 cells were 3.68 and 3.49, respectively. After using MDR1 inhibitors, the efflux ratios were 0.91 and 0.76, and the ER reduction percentages were 75.27% and 78.22%, respectively (Table 12).

[0174] Table 12. Effect of MDR1 on the transport of Palbociclib and digoxin

[0175]

[0176] Note: The apparent permeability coefficient (P app ) and recovery rate (%) are the average values ​​of three groups of samples.

[0177] (3) Transport behavior of Palbociclib in the MDCKⅡ-BCRP cell system

[0178] In the MDCKⅡ-BCRP cell experimental system, the efflux ratio of 5μM teriflunomide was 67.78 without the addition of inhibitors, and the efflux ratio was 1.22 under the action of 1μM positive inhibitor Ko143, and the ER reduction percentage was 98.20%, indicating that the test system is normal and suitable for this study. At the concentration levels of 1μM and 10μM, the efflux transport ratios of Palbociclib on MDCKⅡ-BCRP cells were 65.33 and 38.37, respectively. After the use of BCRP inhibitors, the efflux ratios were 1.06 and 0.96, and the ER reduction percentages were 98.34% and 97.50%, respectively. (Table 13).

[0179] Table 13. Effect of BCRP on the transport of Palbociclib and Teriflunomide

[0180]

[0181] In summary, the efflux ratio (ER) of Palbociclib in MDCKⅡ-MDR1 cells at 1 and 10 μM concentrations was 3.68 and 3.49, respectively. After using the MDR1 inhibitor Zosuquidar, the efflux ratios were 0.91 and 0.76, respectively, and the ER reduction percentages were 75.27% and 78.22%, respectively. At the same test concentration, the efflux ratios of Palbociclib on MDCKⅡ-BCRP cells were 65.33 and 38.37, respectively. After using the BCRP inhibitor Ko143, the efflux ratios were 1.06 and 0.96, respectively, and the ER reduction percentages were 98.34% and 97.50%, respectively. The above results show that Palbociclib exhibits substrate characteristics of efflux transporters MDR1 and BCRP at 1 μM and 10 μM concentrations.

[0182] Example 2: Evaluation of the transport effect of ABC transporters on BSJ-03-123

[0183] 1. Cell culture

[0184] MDCKⅡ-MDR1 and MDCKⅡ-BCRP cells were routinely cultured in DMEM medium containing fetal bovine serum (10%), non-essential amino acids (1%), and penicillin-streptomycin (1%) at 37°C, 5% CO2, and relative saturated humidity. When the cell confluence reached about 90%, 0.95×105 cells / mL (MDCKⅡ-MDR1) and 1.15×10 5 The cells were inoculated at a density of 10 cells / mL (MDCKⅡ-BCRP) in a Transwell-96 well cell culture plate and used for transport experiments after 6 days of culture. During this period, the medium was changed every 2 to 3 days.

[0185] 2. Preparation of dosing solution

[0186] (1) Preparation of compound stock solutions

[0187] Test compound: 20 mM BSJ-03-123 stock solution was prepared using DMSO as solvent;

[0188] Positive inhibitors: prepare a 5 mM Zosuquidar stock solution in DMSO and a 1 mM Ko143 stock solution in DMSO;

[0189] Positive substrate: Prepare a 10 mM digoxin stock solution in DMSO and a 5 mM teriflunomide stock solution in DMSO.

[0190] (2) Preparation of transport buffer:

[0191] 1) 47.87 g of hydroxyethylpiperazineethanesulfonic acid (HEPES) and 1.7088 g of sodium chloride (NaCl) were dissolved in 200 mL of H2O to prepare a 1 M HEPES-0.85% NaCl solution;

[0192] 2) Mix 48.45 mL of 1 M HEPES-0.85% NaCl solution and 1938 mL of HBSS, add 3.875 g of bovine serum albumin (BSA) to a final concentration of 0.2%, and adjust the pH to 7.24 with 2 M NaOH to obtain a transport buffer.

[0193] The compound group and the compound + inhibitor group were administered according to the method of Example 1, except that the "1, 10 μM Palbociclib solution" was replaced with "2, 10, 50 μM BSJ-03-123 solution".

[0194] 3. Transport experiment

[0195] After 6 days of culture, MDCKⅡ-MDR1 and MDCKⅡ-BCRP cells were taken out for drug transport test. The culture medium was removed and the cells were rinsed twice with preheated blank transport buffer. Then, the drug solution was added to both sides of the cell plate as listed in the table below, and samples were collected after the incubation (37℃, 120min):

[0196] Table 14. Dosing methods for transport experiments

[0197]

[0198]

[0199] The specific steps for collecting cell lysate are as follows:

[0200] Place the washed Transwell plate on a new 96-well low-adsorption plate, add 100 μL of 4°C precooled acetonitrile solution containing the internal standard into the Transwell well, shake it on a shaker for 5 minutes, and then use a pipette to repeatedly blow and extract to collect the cell lysate.

[0201] 4. Sample processing

[0202] (1) BSJ-03-123 sample processing:

[0203] Table 15. BSJ-03-123 sample processing

[0204]

[0205] (2) Digoxin sample processing: as described in Example 1.

[0206] (3) Teriflunomide sample treatment: as described in Example 1.

[0207] 5. Cell membrane integrity test (fluorescent yellow permeability test)

[0208] Before incubation, take out 100 μL of sample from each well of the cell plate A and place it in a 96-well black fluorescence measurement plate; after 120 minutes of incubation, take out 100 μL of sample from each well of the cell plate B and place it in a 96-well black fluorescence measurement plate (see the transport experiment in step 3 for the specific sample collection method). The sample is read with an ELISA reader to detect the fluorescence intensity (excitation wavelength 485 nm, emission wavelength 535 nm). The blank control solution is the blank transport buffer.

[0209] 6. Chromatographic and mass spectrometry conditions

[0210] (1) Digoxin detection conditions: as described in Example 1.

[0211] (2) Teriflunomide detection conditions: refer to the detection conditions of teriflunomide in Example 1, except that the retention time: t R(TLF) ≈1.12min;t R(IS) ≈1.12min.

[0212] (3) BSJ-03-123 test conditions:

[0213] Chromatographic column: IC-U 1.6μm 3.0mmI.D.×100mmL, DAICEL CORPORATION

[0214] Column temperature: 35°C

[0215] Ion source: electrospray ionization (ESI)

[0216] Mobile phase A: 10 mM ammonium acetate in water

[0217] Mobile phase B: 30% isopropanol-acetonitrile solution

[0218] Internal standard (IS): BSJ-03-123-D4

[0219] Gradient elution:

[0220] Table 16. Gradient elution

[0221]

[0222] Retention time: t R(BSJ-03-123) ≈1.88min; t R(IS) ≈2.9min

[0223] Capillary voltage: 2kV

[0224] Ion source temperature: 150°C

[0225] Desolvation temperature: 600°C

[0226] Desolvation gas flow rate: 700L / h

[0227] Cone gas flow rate: 150L / h

[0228] Atomizing gas pressure: 6 bar

[0229] Detection method: Positive ion mode multiple reaction monitoring (MRM)

[0230] Ion pairs: m / z 897.57→m / z 517.04 (IS), m / z 892.50→m / z 512.12 (BSJ-03-123)

[0231] Cone voltage: 5V (IS) and 23V (BSJ-03-123)

[0232] Impact voltage: 38eV (IS) and 38eV (BSJ-03-123)

[0233] 7. Total recovery rate of the test substance

[0234] Total recovery rate (%) = (total amount of compound on the administration side at 120 minutes + total amount of compound on the receiving side at 120 minutes + total amount of compound in cell lysate) / (total amount of compound on the administration side at 0 minutes) × 100%.

[0235] 8. Test results

[0236] (1) Cell membrane integrity (permeability of the cell monolayer to fluorescent yellow)

[0237] The permeability of MDCKⅡ-MDR1 and MDCKⅡ-BCRP cells to fluorescent yellow in each well of the test cell plate was less than 5.0×10 -6 cm / s, indicating that the cell layer has good integrity.

[0238] Table 17. Apparent permeability coefficient of fluorescent yellow in MDCKⅡ-MDR1 cells

[0239]

[0240]

[0241] Table 18. Apparent permeability coefficient of fluorescent yellow in MDCKⅡ-BCRP cells

[0242]

[0243] (2) Transport behavior of BSJ-03-123 in the MDCKⅡ-MDR1 cell system

[0244] Table 19. Effect of transport buffer without BSA treatment and without washing the cell layer on transport

[0245]

[0246]

[0247] Table 20. Effect of BSA treatment in transport buffer without washing the cell layer on transport

[0248]

[0249] Table 21. Effects of adding BSA to the transport buffer and washing the cell layer on transport

[0250]

[0251]

[0252] (3) Transport behavior of BSJ-03-123 in the MDCKⅡ-BCRP cell system

[0253] Table 22. Effect of transport buffer without BSA treatment and without washing the cell layer on transport

[0254]

[0255] Table 23. Effect of adding BSA to the transport buffer without washing the cell layer on transport

[0256]

[0257]

[0258] Table 24. Effects of adding BSA to the transport buffer and washing the cell layer on transport

[0259]

[0260] The above results show that when BSA was not added to the transport buffer and the cell layer was not washed, the solution recovery rate of BSJ-03-123 in the MDCKⅡ-MDR1 cell system was low, the total recovery rate was abnormally high, and the efflux ratio result could not accurately determine the substrate; when BSA was added to the transport buffer and the cell layer was not washed, the solution recovery rate increased, but the total recovery rate was still abnormally high, which could show the substrate characteristics to a certain extent; when BSA was added to the transport buffer and the cell layer was washed, the solution recovery rate was further improved, the total recovery rate tended to be normal, and the efflux ratio could determine the substrate characteristics. Therefore, BSJ-03-123 showed substrate characteristics of low permeability and efflux transporters MDR1 and BCRP at concentration levels of 2μM, 10μM and 50μM.

[0261] The above results show that compared with the case without adding BSA, the solution recovery rate was significantly improved after adding BSA to the transport buffer, and the solution recovery rate was further significantly improved by further adding the cell layer washing operation.

[0262] In summary, the present invention provides an evaluation method for ABC transporter substrates. The present invention uses MDCKⅡ cells overexpressing human multidrug resistance protein 1 and breast cancer resistance protein as a model, and uses a transport buffer added with bovine serum albumin. In the ABC transporter substrate experiment for evaluating adsorbent compounds, the recovery rate of the adsorbent compounds in the ABC transporter substrate experiment is significantly improved, and the substrate characteristics are accurately determined, providing a more accurate preclinical basis for drug interactions.

Claims

1. A method for evaluating an ABC transporter substrate, characterized in that: The method comprises the following steps: (1) dissolving an adsorbent compound in an organic solvent to prepare an adsorbent compound solution; (2) preparing a transport buffer containing bovine serum albumin; (3) preparing a drug resistance protein inhibitor solution; (4) A bidirectional permeability experiment was carried out using a Transwell model using an adsorbed compound solution, a transport buffer solution, and a drug-resistant protein inhibitor solution. After administration, the cell layer in the Transwell model was cleaned, and samples from the administration side and the receiving side were collected. The efflux ratio A of the adsorbed compound in the absence of the drug-resistant protein inhibitor and the efflux ratio B of the adsorbed compound in the presence of the drug-resistant protein inhibitor were calculated. When the following conditions were met: A was greater than 2.0, and the reduction rate of B compared with A was greater than 50%, the adsorbed compound was judged to be an ABC transporter substrate.

2. A method for improving the recovery rate of an adsorbent compound in an ABC transporter substrate experiment, characterized in that: The method comprises the following steps: (1) dissolving an adsorbent compound in an organic solvent to prepare an adsorbent compound solution; (2) preparing a transport buffer containing bovine serum albumin; (3) preparing a drug resistance protein inhibitor solution; (4) using the adsorbed compound solution, transport buffer and drug resistance protein inhibitor solution, a bidirectional permeability experiment was carried out using the Transwell model. After the administration, the cell layer in the Transwell model was washed, and the samples on the administration side and the receiving side were collected to calculate the recovery rate of the adsorbed compound; Recovery rate of adsorbed compound = (total amount of compound on the administration side at the end of the experiment + total amount of compound on the receiving side at the end of the experiment) / (total amount of compound on the administration side at the beginning of the experiment) × 100%.

3. The method according to claim 1 or 2, characterized in that: The Transwell model is a model that meets the following criteria: using an ABC transporter positive substrate solution, a transport buffer and a drug-resistant protein inhibitor solution, a bidirectional permeability experiment is carried out using the Transwell model, after the administration is completed, the cell layer in the Transwell model is cleaned, and samples on the administration side and the receiving side are collected, and the efflux ratio C of the ABC transporter positive substrate in the absence of the drug-resistant protein inhibitor and the efflux ratio D of the ABC transporter positive substrate in the presence of the drug-resistant protein inhibitor are calculated, and the following criteria are met: C is greater than 2.0, and the reduction rate of D compared with C is greater than 50%; The ABC transporter positive substrate solution is prepared according to the following method: dissolving the ABC transporter positive substrate with an organic solvent to prepare the ABC transporter positive substrate solution.

4. The method according to claim 1 or 2, characterized in that: In step (1), the adsorptive compound is a Protac compound.

5. The method according to claim 1 or 2, characterized in that: In step (2), the transport buffer contains 0.1-1% bovine serum albumin, preferably 0.5% bovine serum albumin.

6. The method according to claim 1 or 2, characterized in that: In step (3), the drug-resistant protein is multidrug resistance protein 1 or breast cancer resistance protein.

7. The method according to claim 6, characterized in that In step (3), the multidrug resistance protein 1 inhibitor is oxaquinoxaline; and the breast cancer resistance protein inhibitor is Ko143.

8. The method according to claim 1 or 2, characterized in that: The cells used in the Transwell model are MDCKⅡ cells; MDCKⅡ cells are obtained by culturing MDCKⅡ cells with drug-resistant protein genes knocked out.

9. The method according to claim 8, characterized in that The culture conditions are 37° C., 5% CO 2 and relative saturated humidity in a DMEM medium containing 10% fetal bovine serum, 1% non-essential amino acids and 1% penicillin-streptomycin.

10. The method according to claim 1 or 2, characterized in that: The conditions of the bidirectional permeability experiment are: incubation at 37° C. for 90 to 120 minutes.

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