JP2007114091A - Screening method of drugs - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To construct a system capable of developing a method for measuring the drug transport of a drug transporter with high sensitivity, without having to use using a radiation label body to apply the same to the screening or the like of the lead compound of a pharmaceutical enterprise. <P>SOLUTION: The screening method of the drugs transported by the drug transporter is constituted of a process for preparing a cell membrane vehicle from cells which develops the drug transporter; a process for bringing the obtained cell membrane vehicle into contact with a drug to be inspected in the presence of ATP (adenosine triphosphate); a process for trapping the cell membrane vehicle having come into contact with the drug to be inspected; a process for solubilizing the trapped cell membrane vehicle, to obtain a liquid extract having extracted the drug to be inspected taken in the cell membrane vehicle; a process for obtaining a measuring sample, by removing components other than the drug to be inspected from the liquid extract; and a process for quantifying the amount of the drug to be inspected in the measuring sample by a liquid chromatograph and tandem mass analysis (LC/MS/MS). <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for screening a drug transported by a drug transporter.

  The transporter forms a pathway necessary for substances to pass through the cell membrane, and is intended to absorb, metabolize, or excrete substances that are essential for maintaining biological functions such as nutrients or metabolites in the body. Plays an important role. And since such a transporter also recognizes a drug having a structure similar to these substances or in-vivo metabolites, it is called a drug transporter particularly when a drug is considered as a transport substrate. There are many types of substrates recognized by one drug transporter, and a drug having a structure not similar to the substrate recognition site may be transported, and the mechanism of drug selection is unclear.

  As such drug transporters, ABC (ATP binding site) transporters, organic ion transporters, peptide transporters, and the like are known. For example, ABC transporters include about 50 types of ABC transporters from human genome analysis. The presence of a gene is predicted.

  ABC transporters, such as P-glycoprotein (ABCB1), cMOAT / MRP2 (ABCC2), and BCRP (ABCG2), transport a wide variety of drugs. Recent studies have shown that it has a profound effect on However, a system for screening many kinds of compounds has not been fully established yet. Even if there is an analogy, as will be described later, it is a transport experiment using a radiolabel and a method for measuring ATPase activity. Even if a pharmaceutical company wants to introduce a screening system for transporters when searching for new drugs, it is too expensive and impractical to radiolabel all lead compounds.

  Currently, the dropout of a large number of development candidate compounds in the preclinical and clinical development stages of drug discovery is a serious and serious problem. Approximately 50% of discontinued cases are due to pharmacokinetic defects (ie, low absorption rates, low blood levels, fast clearance, etc.) and toxicity. Many pharmaceutical companies are beginning to seriously re-examine new drug discovery and development strategies as new drug development costs increase. To speed up drug discovery, pharmacokinetic screening must be incorporated from the initial stage of exploratory research.

The following four methods are currently reported as methods for evaluating the drug transport ability of ABC transporters, but each has potential problems when screening a large amount of compounds.
(1) Measurement of drug transport or inhibition at the cellular level: Since cultured cells are used, it is unsuitable for screening large amounts of compounds, and the experimental values vary, making dynamic analysis difficult.
(2) Measurement of ATPase activity using cell membrane fraction: ATPase activity of ABC transporter accompanying transport of substrate is measured, but other ATPase activities inherent in the cell membrane fraction are high and signal / noise ratio is high. (For example, the present inventor has constructed a screening system for measuring ATPase activity, but the method is currently applicable only to P-glycoprotein (ABCB1) and BCRP (ABCG2)).
(3) Measurement by photoaffinity labeling of [ ³² P] -8-azido-ATP: In cell-free system using cell membrane fraction, ABC transporter recognizes substrate and binds ATP to ATP binding site. Using properties, [ ³² P] -8-azido-ATP is photoaffinity labeled. Since proteins are separated by gel electrophoresis, they are unsuitable for screening large quantities of compounds.
(4) Using a cell membrane vesicle of a cell expressing ABC transporter to measure the incorporation of the radiolabeled compound into the membrane vesicle using a liquid scintillation counter: this method is the most sensitive and transportable. While having the advantage of measuring the process itself, radiolabeled compounds must be used. Therefore, radioactivity handling facilities and radiolabeling are necessary.

  An object of the present invention is to develop a method for measuring drug transport of a drug transporter with high sensitivity without using a radiolabel, and to construct a system that can be applied to screening of lead compounds of pharmaceutical companies. . For this purpose, the cell membrane vesicle transport system of cells expressing ABC transporter is combined with LC / MS / MS to realize high-sensitivity measurement, and by using, for example, a 96-well or 384-well filter plate The purpose is to shorten the sample processing time and realize high-speed screening.

The present invention provides the following inventions in order to solve the above problems.
(1) preparing a cell membrane vesicle from cells expressing a drug transporter; contacting the obtained cell membrane vesicle with a test drug in the presence of ATP (adenosine triphosphate); contacting with the test drug Trap the cell membrane vesicles; solubilize the trapped cell membrane vesicles to obtain an extraction solution in which the test drug incorporated in the cell membrane vesicles is extracted; remove components other than the test drug from the extraction solution Obtaining a measurement sample; and quantifying the amount of a test drug in the measurement sample by liquid chromatography and tandem mass spectrometry (LC / MS / MS), and a drug transported by a drug transporter Screening method of
(2) The drug screening method according to (1), wherein the drug transporter is an ABC transporter;
(3) The drug screening method according to (1) or (2), wherein cell membrane vesicles are trapped with a well filter plate;
(4) The method for screening a drug according to any one of (1) to (3), wherein the trapped cell membrane vesicle is washed and then solubilized; and (5) components other than the test drug are membrane proteins, phospholipids and A method for screening a drug according to any one of (1) to (4), which comprises a solubilizing agent;
It is in.

  According to the screening method of the present invention, it is not necessary to radiolabel the lead compound, and highly sensitive measurement can be performed by LC / MS / MS. Moreover, by using a 96-well or 384-well filter plate, the sample processing time can be shortened and high-speed screening can be realized. Therefore, by introducing the screening method, the probability that a new drug will drop out in the development stage due to pharmacokinetic problems is greatly reduced, and pharmaceutical companies are expected to be able to reduce the amount of research and development costs. .

  The drug screening method of the present invention comprises preparing a cell membrane vesicle from a cell expressing a drug transporter; contacting the obtained cell membrane vesicle with a test drug in the presence of ATP (adenosine triphosphate); Trapping cell membrane vesicles that have come into contact with the test drug; solubilizing the trapped cell membrane vesicles to obtain an extraction solution in which the test drug incorporated into the cell membrane vesicles is extracted; And obtaining a measurement sample; and quantifying the amount of a test drug in the measurement sample by liquid chromatography and tandem mass spectrometry (LC / MS / MS).

  Drug transporters are not particularly limited, and include ABC transporters, organic ion transporters, peptide transporters, etc., and ABCB1 (P-glycoprotein, MDR1) and ABCG2 (BCRP) are suitable as human ABC transporters Used for.

  When preparing a cell membrane vesicle from a cell expressing a drug transporter, a conventional method can be used.

  For example, a cDNA encoding ABC protein is incorporated into an antibiotic resistance expression vector, transfected into a cultured cell, this cell is cultured in the presence of an antibiotic, and a cell expressing the ABC protein expression vector is selected and used. Mass culture. Subsequently, the ABC protein-expressing cells are collected, crushed and centrifuged to obtain a target cell membrane vesicle.

  Next, in the method of the present invention, the obtained cell membrane vesicle is brought into contact with a test drug in the presence of ATP. For example, cell membrane vesicles are prepared from cells expressing ABC transporter, and incubated with a test drug in the presence of ATP. Drugs that serve as substrates for ABC transporters are incorporated into membrane vesicles. On the other hand, drugs that do not become substrates are not taken up.

  Next, the cell membrane vesicle that has come into contact with the test drug is trapped on a well filter plate, and the cell membrane vesicle is further solubilized to obtain an extraction solution in which the test drug taken into the cell membrane vesicle is extracted. Cell membrane vesicles can be trapped by various methods, but a well filter plate such as 96-well or 384-well is preferably used. For example, membrane vesicles are trapped with a 96-well filter plate (well diameter 0.2-0.4 μm) and washed with buffer. Next, the membrane vesicle is solubilized to extract the drug accumulated inside. As the solubilizer, a solvent such as NaOH solution, DMSO (dimethyl sulfoxide), various surfactants, and the like can be used. For example, membrane vesicles can be easily solubilized with good recovery by using a 10 mM NaOH solution.

  Next, components other than the test drug are removed from the extracted solution to obtain a measurement sample. Components other than the test drug include membrane proteins, phospholipids, and solubilizers. For this purpose, the well filter plate is preferably used.

  In the present invention, the step of solubilizing the membrane vesicle, completely extracting the test drug accumulated therein, and subjecting it to desalting / deproteinization is particularly important. Most preferably, the recovery rate is substantially 100%. Further, for example, a 96-well filter plate or the like can be used to simplify the operation and realize high-speed screening.

  Further, in the present invention, the amount of the test drug in the measurement sample is quantified with a liquid chromatograph and a tandem mass spectrometer (LC / MS / MS). Preferably, the test drug is measured, for example, by LC / MS / MS equipped with an autosampler compatible with a 96-well filter plate and an appropriate column and eluent. By comparing with the standard sample, the amount of the test drug taken into the membrane vesicle can be quantitatively calculated. In the LC / MS / MS analysis, first, a measurement sample is separated by high performance liquid chromatography (HPLC), an eluted component is introduced into an ionization chamber, and ionized to obtain an MS spectrum. That is, by selecting ions in the first MS, colliding with an inert gas in the collision chamber to dissociate the ions, and selecting and analyzing specific ions in the second MS, it is possible to analyze with high selectivity and high sensitivity. A value can be obtained.

  The method of the present invention does not need to be radiolabeled with a test compound, and can be applied to a high-speed automated screening apparatus by using, for example, a 96-well filter plate. This method of applying LC / MS / MS makes it possible to screen many kinds of lead compounds from the viewpoint of drug transport in drug discovery, and drugs with high bioavailability (absorption in the small intestine) It has the advantage that it can be applied to exploration and development of drugs that act selectively in the nervous system or new anticancer agents. Furthermore, it can be applied as a screening method for searching for unknown substrates in the functional analysis of the novel ABC transporter obtained by genome analysis.

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
(A) Preparation of insect cell membrane expressing ABCG2 Human ABC transporter was expressed in insect cells, and functional analysis was performed using the cell membrane. First, ABCG2 cDNA was incorporated into a vector (pFastBac1). After the expression bacmid was prepared from the vector, insect cells were infected with the genetically modified virus to express the transporter protein. The method is as follows.

  The pFastBac1 vector carrying ABCG2 cDNA was transformed into DH10Bac competent cells, and ABCG2 cDNA was recombined into bacmid. Thereafter, the recombinant bacmid was introduced into Sf9 insect cells using Cellfectin reagent (Invitrogen Co.) to obtain a genetically modified virus having ABCG2 cDNA in the genome. Sf9 insect cells were infected with this genetically modified virus to express ABCG2 protein.

In order to confirm the expression of ABCG2 protein, Western blotting using ABCG2-specific monoclonal antibody BXP-21 (SIGNET) was performed. The protein-transferred nitrocellulose membrane was blocked with p0.5% (w / v) skim milk-containing TTBS (0.05% Tween 20-containing TBS) and then diluted 500-fold with TTBS containing 0.5% (w / v) skim milk. -21 was soaked at room temperature for 1.5 hours for antigen-antibody reaction. The nitrocellulose membrane was thoroughly washed with TTBS, and a secondary antibody reaction was performed by immersing in an anti-mouse IgG antibody diluted 3000 times with TTBS containing 0.5% (w / v) skim milk at room temperature for 1 hour. The nitrocellulose membrane was thoroughly washed with TTBS, then immersed in Western Lightning Chemiluminescence Reagent Plus, and the chemiluminescence was detected with Lumino Imaging Analyzer FAS-1000 (TOYOBO Co., Ltd.).
(B) Screening of transport activity using membrane vesicles and sample preparation
Plasma membranes were prepared from insect cells expressing ABCG2, and transport activity was screened using the membrane vesicles. In the high-speed screening method, a 96-well filter plate (Nihon Millipore “MultiScreen”) and an automated system (Biotech “EDR384S”) were used to increase the speed. Transport activity was examined using methotrexate (MTX), a substrate of ABCG2.
The standard assay system for MTX transport measurement is as follows.

53 μl 250 mM sucrose / 10 mM Tris / HEPES (pH7.4) solution
30 μl 3.33 mM ATP, 33.3 mM creatine phosphate, 33.3 mM MgCl ₂
(Or 33.3 mM creatine phosphate / 33.3 mM MgCl ₂ )
5 μl 2 mg / ml creatine kinase
2 μl 10 mM MTX (final concentration 200 μM)
10 μl Sf9 cell membrane sample (total 50 μg protein)
100 μl total
Incubations were performed at 37 ° C. 0, 10 and 20 minutes after the start of the reaction, 1 ml of a 250 mM sucrose / 2 mM EDTA / 10 mM Tris / HEPES (pH 7.4) solution cooled in ice was rapidly added to the reaction mixture to stop the reaction. . 270 μl of the solution was poured into the Millipore “MultiScreen ^” well and aspirated. Each well was washed four times with 200 μl of an ice-cooled 250 mM sucrose / 10 mM Tris / HEPES (pH 7.4) solution. In order to quantify MTX incorporated into cell membrane vesicles, 200 μl of 10 mM NaOH solution was added to each well and allowed to stand at room temperature for 10 minutes. By this treatment, the membrane vesicles were broken, and the MTX trapped in the vesicles was dissolved in the NaOH solution (recovery rate 99% or more). The NaOH solution was then transferred to a new 96-well plate through the bottom filter by aspiration.

Subsequently, in order to deproteinize, 100 μl of acetonitrile was added to 50 μl of the sample solution and centrifuged. 10 μl of the supernatant was collected and mixed with 190 μl of milli-Q water to obtain a measurement sample.
(C) Quantitative analysis by LC / MS / MS The above samples were quantitatively analyzed using LC / MS / MS. The equipment used is as follows.

LC (Liquid Chromatography): “Waters 2695 Separation Module”
MS / MS (mass spectrometry): “Waters Quattro micro API”
First, the standard MTX sample was subjected to LC (liquid chromatography), and the elution time was measured (A in FIG. 1). The column used was an Atlantis dC ₁₈ 3 mm, 2.1 x 150 mm reverse phase column kept at 40 ° C. The mobile phase of LC consists of three phases A, B, and C, and the following gradient was applied. The flow rate was 0.2 ml / min. Under these conditions, the elution time of MTX was 7.61 minutes.

Time (min) A (%) B (%) C (%)
0 80 10 10
10 0 90 10
12 80 10 10
In order to ionize MTX, measurement was performed by general ESI (Electron Spray ionization) by an atmospheric pressure ionization method. In MS1 scan mode, the optimum cone voltage (pull-in voltage) of MTX is examined, and the mass spectrum of MTX at each cone voltage is shown in Fig. 1B. The optimum cone voltage was 30V, and MTX was observed as a monovalent protonated molecule (m / z = 455).

  Next, in order to optimize the energy applied to the collision cell between MS1 and MS2, mass spectra of standard MTX samples at various collision energies were measured and shown in FIG. The higher the collision energy, the more the molecular weight related ions fragmented. Since the sensitivity at m / z = 308 at the collision energy of 20 eV was the highest, this was used as the monitor ion.

  Finally, quantitative and reproducibility in LC / MS / MS measurement was investigated. Different concentrations of MTX solution samples were used and analyzed under the LC / MS / MS conditions described above. Three injections were repeated for each concentration point. As a result, the calibration curve shown in FIG. 3 was obtained. When LC / MS / MS analysis was performed under these conditions, it was found that reproducibility was good and even a small amount of MTX could be measured with high reliability.

  Under the above basic conditions, LC / MS / MS analysis of MTX confined inside the membrane vesicle was performed. The result is shown in FIG. The S / N ratio was also good, and an MTX signal of m / z = 308 at a collision energy of 20 eV was observed.

  Based on the LC / MS / MS analysis conditions thus obtained, MTX transport into the membrane vesicle by ABCG2 was measured. The time course is shown in FIG. 5 (average + standard deviation (N = 3)). Even without using radiolabeled MTX, the transport function of ABCG2 could be measured accurately and rapidly by combining the high-speed vesicle screening method and the LC / MS / MS analysis method as in this example. This method is basically applicable to other ABC transporters or SLC (solute carrier) transporters as long as membrane vesicle transport experiments are possible.

  By using the method of the present invention for measuring drug transport of a drug transporter with high sensitivity without using a radiolabel, a system that can be applied to screening of lead compounds of pharmaceutical companies can be provided.

LC and mass spectrum of standard methotrexate (MTX). Mass spectrum of standard MTX at various collision energies. Standard MTX calibration curve by LC / MS / MS analysis. LC / MS / MS analysis results of MTX transported inside the membrane vesicle. Transport of MTX into membrane vesicles by ABCG2.

Claims (5)

Preparing a cell membrane vesicle from a cell expressing a drug transporter;
Contacting the obtained cell membrane vesicle with a test drug in the presence of ATP;
Trapping cell membrane vesicles in contact with the test drug;
Solubilizing the trapped cell membrane vesicle to obtain an extraction solution from which the test drug incorporated in the cell membrane vesicle is extracted;
Remove components other than the test drug from the extracted solution to obtain a measurement sample; and quantify the amount of the test drug in the measurement sample by liquid chromatography and tandem mass spectrometry (LC / MS / MS) thing,
A method for screening a drug transported by a drug transporter characterized by the above.   The drug screening method according to claim 1, wherein the drug transporter is an ABC transporter.   The drug screening method according to claim 1 or 2, wherein the cell membrane vesicle is trapped by a well filter plate.   The method for screening a drug according to any one of claims 1 to 3, wherein the trapped cell membrane vesicle is washed and then solubilized.   The drug screening method according to claim 1, wherein components other than the test drug include a membrane protein, a phospholipid, and a solubilizer.

Images