JP2000139462A - System for high speed screening of medicine absorbability by measurement of ph change in cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an evaluation method capable of quickly evaluating the intestinal absorption of a medicine in vivo. SOLUTION: This method for evaluating the intestinal absorption of a medicine comprises (1) a process for forming a cell layer, (2) a process for introducing a fluorescent pigment into the cell layer, (3) a process for bringing the fluorescent pigment-introduced cell layer into contact with a buffer solution containing the medicine to allow the cell layer to absorb the medicine, and (4) a process for subjecting the pH change in the cells of the cell layer obtained in the process (3) to a fluorescent light measurement to evaluate the amount of the medicine contained in the cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for evaluating the absorption of a drug for oral administration. In particular, the present invention relates to a method for rapid in vitro evaluation (screening) of intestinal absorption of orally administered drugs.

[0002]

2. Description of the Related Art Drugs administered orally are absorbed into the body mainly in the intestinal tract and exert their effects. Therefore, whether or not the drug is absorbed in the intestinal tract is very important as a criterion for determining whether or not the drug can be orally administered. In the drug discovery stage, especially in the lead compound search stage,
It is extremely important to establish a method that can test the intestinal absorption of a drug in a short time on a large number of samples.

[0003] Intestinal absorption of a drug is generally performed using the intestine of a rat. That is, a buffer solution containing a drug is injected into the intestine of a rat, and it is tested whether or not the drug penetrates into a dominant blood vessel of the intestine. However, this method is becoming difficult to perform because of the use of large amounts of rats, especially in the United States, in view of animal abuse. Further, one of the problems is a method of quantifying a drug which consumes cost and time as described later.

[0004] As an alternative to the method using rat intestinal tract, a method using Caco-2 cells derived from human colon cancer is becoming mainstream in Europe and the United States. This method
It measures the amount of drug that passes through a Caco-2 monolayer formed on a porous membrane. As a specific method, for example, the bottom is formed of a porous film, and C is formed on the film.
Using a container formed with a monolayer of aco-2, a buffer solution (A) containing a drug is poured into the container, and the container is immersed in a buffer solution (B) containing no drug, and after a certain period of time, Caco
-2 is a method for quantifying the drug that passes through the monolayer and is present in the buffer solution (B). As a method of measuring the passing amount,
High performance liquid chromatography (HPLC) or liquid chromatography / mass spectrometry (LC / MS) is used.

When a drug is absorbed in the intestinal tract,
It is believed that certain drugs are absorbed once by cells in the intestinal epithelial cells via transporters on the surface of the epithelial cells and subsequently released into the blood vessels of the intestinal tract.
When a drug is absorbed into a cell by a transporter, it is known that some transporters simultaneously absorb hydrogen ions in a solution together with the drug into the cell. Therefore, in the art, the intracellular pH changes along with the absorption of the drug, and a change in the intracellular pH due to the transport of the drug (that is, including the absorption as the first step) is reported. Few, slightly Wenzel et al.'S The Journal of Pha
rmacology and Experimental Therapeutics, 277, 831-
839 (1996) using Caco-2 cells and frog oocytes expressing the transporter.

[0006] Further, the pH change in the liposome due to the permeation of an acidic drug such as salicylic acid into the liposome is described in
urnal of Pharmacology and Experimental Therapeutic
s, 285 ( 3), 1175-1180 (1998). Ca
The present inventors have reported a change in intracellular pH due to permeation of salicylic acid when using co-2 cells at the annual meeting of the Pharmaceutical Society of Japan in March 1998. Tsuji and others reported similar results at the same year's meeting.

[0007] The methods of measuring intracellular pH change reported in the above report all involve filling a cell with a pH-sensitive fluorescent dye, and measuring the intracellular pH based on the change in the fluorescence intensity.

[0008]

At present, a method for detecting a drug in a drug permeability test using Caco-2 is a high performance liquid chromatography (HPLC) method or a liquid chromatography / mass spectrometry (LC / MS) method. Law.

However, in the HPLC method, generally, 1
At present, the analysis time per sample is 1-2 hours, and a large amount of sample is rapidly supplied by a method such as combinatorial chemistry. The problem is that even so much time is required. On the other hand, LC /
In the MS method, although the analysis time is reduced to about several tens of minutes, there is a problem in that the handling of the test agent such as sampling from a test device is complicated.

[0010] On the other hand, many drugs have either acidic or basic properties, and when these drugs are absorbed into cells, the intracellular pH may naturally change. . Therefore, if it is possible to measure this change in pH quickly and quantitatively, it can be considered that this can be used as an evaluation standard for intracellular absorption of a drug and further for intestinal absorption of a drug.

[0011]

According to one embodiment of the present invention, there are provided: (1) a step of forming a cell layer; (2) a step of introducing a fluorescent dye into the cell layer; Bringing the cell layer into which the fluorescent dye is introduced into contact with a buffer solution containing a drug to absorb the drug into the cell layer; and (4) detecting the pH change in the cell of the cell layer obtained in (3) by fluorescence. Evaluating the abundance of the drug in the cells by analyzing the method (screening system)
It is.

[0012] In another embodiment of the present invention, Caco-2 cells derived from human colon cancer or HT2
The above-mentioned evaluation method using 9 cells or MDCK cells derived from kidney tubules.

Further, as another embodiment of the present invention,
The above evaluation method uses a cell line in which a transporter is expressed at a high density by genetic manipulation as a cell.

Further, as another embodiment of the present invention,
The above evaluation method uses a buffer having a pH in the range of 5.0 to 8.0.

Another embodiment of the present invention is the above-mentioned evaluation method, wherein the buffer is an isotonic buffer.

Further, as another embodiment of the present invention,
The above evaluation method using a buffer having a drug concentration of 0.01 mM to 100 mM.

As another embodiment of the present invention, (3)
The above evaluation method is performed at a temperature within a range that does not affect cells.

Further, another embodiment of the present invention is the above-mentioned evaluation method in which fluorescence analysis is performed using a fluorescence reader.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, compounds (drugs) whose intestinal absorption can be tested include weakly acidic compounds, weakly basic compounds, and peptide-like compounds having a possibility of becoming a substrate for a peptide transporter. . For example, examples of the weakly acidic compound include salicylic acid, m-hydroxybenzoic acid, o-anisic acid, succinic acid, benzenesulfonic acid, acetic acid, sulfanilic acid, nicotinic acid, and benzoic acid. Examples of the weakly basic compound include propranolol, atheronol, imipramine and the like. Peptide analogs that have the potential to be substrates for peptide transporters include cefdinir, benzylpenicillin, cefazolin, dicloxacillin, captopril, oxacillin, ceftizoxime, cefixime, carbenicillin, ceftibbutene,
Salbenicillin, lisinopril and the like can be mentioned.

The cell types that can be used in the test method of the present invention are generally cultured epithelial cells having the same morphology as gastrointestinal epithelial cells.
Co-2 cells and HT29 cells; MDCK cells derived from kidney tubules; and cell lines and the like in which transporters are expressed at high density by gene transfer.
Expressing the transporter at a high density by this gene transfer is effective for increasing the intracellular absorption of the drug, increasing the intracellular pH change, and thus improving the sensitivity of the method of the present invention. is there.

As is well known in the art, the culture of various cells used in the present invention is desirably performed under culture conditions most suitable for the cells used. Furthermore, various short-term culture methods can be adopted for the purpose of speeding up the evaluation.

The cells cultured by the above method may form a monolayer or a multi-layer. It is preferable that the cell layer forms a uniform layer at least in the fluorescence measurement section. The cells forming the cell layer are preferably in a tightly connected state.

The concentration of the drug used in the measurement is 0.01 mM
It is preferably in the range of １００100 mM, and more preferably in the range of 0.1 mM to 10 mM. This concentration can be determined in view of the intracellular pH change caused by the drug to be evaluated.

As the composition of the solution used for the measurement, it is desirable to use a buffer solution isotonic with the cells to be used. Examples of such buffers include HBSS, KHBB
Etc. can be used.

The pH of the above buffer may be between 5.0 and 8.0. Preferably, it can be prepared based on the actual pH in the digestive tract (small intestine). The pH in the digestive tract is usually in the range of 5.0 to 7.5, and it is preferable to perform the measurement of the present invention within that range.

The temperature at which the drug absorption into the cell layer is tested should be within a range that does not affect the cells used.
Preferably it is in the range of 0 ° C to 50 ° C, more preferably in the range of 4 ° C to 42 ° C, most preferably in the range of 4 ° C to 42 ° C.
It is in the range of ℃ to 38 ℃.

In order to measure intracellular pH change, p
A fluorescent reagent showing a change in the fluorescence intensity depending on H can be introduced into cells and used. Such reagents are commercially available, for example, BCECF (Dojindo Laboratories), BCECF-AM (Dojindo Laboratories), 5 (and 6) -carboxy-2 ', 7'-dichlorofluorescein, 5 (and 6) -carboxy-
2 ', 7'-dichlorofluorescein diacetate, 5
(And 6) -carboxy-2 ', 7'-dichlorofluorescein diacetate succimidyl ester, 5
(And 6) -carboxy-2 ', 7'-dichlorofluorescein succimidyl ester, 5 (and 6)-
Carboxy-fluorescein and the like can be used. It is preferable to use BCECF or BCECF-AM.

The pH change in the cells into which the above-mentioned fluorescent reagent has been introduced can be measured using a usual device for measuring fluorescence intensity, and for example, a device such as a fluorescence reader can be used. In order to improve the measurement accuracy, it is preferable to use a method for removing background fluorescence. As an example of such a method, FLIP
R (Fluorescence Imaging Plate Reader (FLuorometric
Imaging Plate Reader)) There is a system. The system integrates a powerful argon laser irradiation system, CCD image camera, cell layer separation optics, 96-well pipettor, performs fluorescence analysis on all 96 wells on a microplate, and records dynamic data in seconds. Can be analyzed. In other words, a fluorescent reagent suitable for the specific change to be measured is excited using an argon laser to generate fluorescence, background fluorescence generated outside the cell is removed by a cell layer separation optical system, and a cooled CCD camera is integrated and detected. Fluorescence intensity is measured using the instrument.

[0029]

EXAMPLES (1) Intracellular pH using Caco-2 cells
Determination of sulfanilic acid (SU) using the following method
A), m-hydroxybenzoic acid (m-HBA), benzoic acid (BA), nicotinic acid (NA), salicylic acid (S
A), Cac using benzenesulfonic acid (BSA)
The fluorescence intensity corresponding to the pH change in o-2 cells was measured. The fluorescence intensity of the well into which HBBS containing no drug was injected as a control well was measured.

Caco-2 cells were cultured on a 96-well black / clear well plate coated with collagen. The culture method includes a 3-day culture method (3-day BIOCOAT (registered trademark) HT) developed by Becton Dickinson.
The S Caco-2 Assay System) was modified using a 4-day culture method. After completion of the culture, the fluorescent substance BCECF is added to each well.
And Caco- at 37 ° C. for 1 hour.
Transfected into 2 cells. Thereafter, it is washed four times with Hank's saline buffer (HBSS), and finally, H is added to each well.
90 μL of BSS was left. The well plate in this state was attached to the FLIPR system, and 45 μL of an HBSS solution (pH 6.0) containing each test agent (3 mM or 15 mM) was simultaneously added to each well using a 96-well pipettor. At this time, the drug concentration in each well is 1 mM or 3 mM, respectively. The fluorescence intensity of each well was measured at intervals of 2 seconds for 15 minutes immediately after injection of the drug. The difference between the fluorescence intensity of each well and the fluorescence intensity of the control well was calculated, and the value was used as an index of intracellular pH change due to the drug.

FIG. 1 shows the changes over time in the fluorescence intensity of salicylic acid and of the control wells.

(2) Test of rat small intestinal mucosal permeability Using the following method, sulfanilic acid (SU) was used as a drug.
A), m-hydroxybenzoic acid (m-HBA), benzoic acid (BA), nicotinic acid (NA), salicylic acid (S
A) The permeability of rat small intestinal mucosa was measured using benzenesulfonic acid (BSA).

Wister male rats (230-280 g)
After anesthesia with pentobarbital, the abdomen was opened, and a polyethylene tube was cannulated above and below about 15 cm to about 15 cm from the upper part of the jejunum to form a small intestinal loop. Flow rate 0.2-0.4 mL from the top of the loop
Per minute containing the drug at a rate of
The perfusate flowing out from the lower part of the loop was collected every 10 minutes for 0 minutes. The intestinal permeability of a drug is determined from the difference between the drug concentration in the perfusate before and after perfusion (corresponding to the amount of membrane permeation).
It was calculated according to the mpletely radial mixing model. As the perfusion solution, NaCl 140 mM, KCl 5 mM,
MES / HEPES 10 mM and FITC-Dex
Using an isotonic solution (pH 6.5) containing 1 mM of tran, bubbling was always performed with a mixed gas of O ₂ : CO ₂ = 95: 5. Here, FITC-Dextran was added to correct the absorption of water from the small intestine that occurred during the perfusion, and the concentration of the drug was corrected by the absorption of water from the concentration ratio before and after the perfusion.

(3) Permeability test using Caco-2 monolayer membrane Sulfanilic acid (SU) was used as a drug by the following method.
A), m-hydroxybenzoic acid (m-HBA), benzoic acid (BA), nicotinic acid (NA), salicylic acid (S
A), Cac using benzenesulfonic acid (BSA)
The permeability using the o-2 single layer film was measured.

A transport medium (TM) having the following composition was prepared.

NaCl 136.89 mM Glucose 19.45 mM KCl 5.36 mM CaCl ₂ 1.26 mM Na ₂ HPO ₄ 0.34 mM MgCl ₂ .6H ₂ O 0.49 mM KH ₂ PO ₄ 0.44 mM NaHCO ₃ 4.17 mM MgSO ₄ .7H ₂ O 0.41 mM Hepes 10.00 mM T. M was heated to 37 ° C. before the experiment, and adjusted to pH 7.4 and pH 6.0, respectively. Further, each drug was added to T.M. M, adjusted to pH 7.4 and pH 6.0 again,
It was used as a drug sample solution.

2.6 on the serosal side of the well with insert
mL of T.I. M (pH 7.4), T.M. M
(PH 6.0) was introduced and pre-incubated at 37 ° C. for 20 minutes. Next, T.S. M (pH 7.
4) Transfer the cup to the well containing 2.6 mL,
After introducing drug sample solution (1.5 mL) to mucosal side, 10
Every minute, sampling was performed for 0.1 minute from the serosal side for 60 minutes. Immediately after sampling, T.P. Add 0.1 mL of M (pH 7.4). The drug concentration in the obtained sample was quantified, and the value was subtracted from the initial concentration to calculate the concentration of the drug remaining on the mucosal side at each time. Then, from the logarithm of the initial concentration,
From the value obtained by subtracting the logarithm of the side concentration and the slope of the time, the first-order absorption rate constant Ka is obtained, multiplied by the volume added to the mucosal side, and divided by the effective area of the cup (4.71 cm ² ) to obtain the membrane permeation. The coefficient (cm / min) was determined.

(4) Summary of Results FIG. 2 is a graph comparing the change in the pH of Caco-2 cells in (1) with the result of the permeability of the Caco-2 cell monolayer membrane obtained in (3). Indicated. In addition, FIG. 3 shows a graph comparing the change in intracellular pH of Caco-2 due to the absorption of the drug obtained in (1) with the result of the permeability of the rat small intestine mucosa obtained in (2). In each graph, the intracellular p
As the change in H, a value 10 seconds after the addition of the drug was used.

As shown in FIG. 2 and FIG.
The results of the co-2 intracellular pH change and the rat intestinal mucosal permeability showed good correlation with the Caco-2 cell monolayer permeability (correlation coefficients R were 0.99 and 0.98, respectively). .

[0040]

According to the method of the present invention, the change in Caco-2 intracellular pH can be monitored without quantifying the drug itself, which requires a lot of time and requires complicated handling of the test drug. By measuring using the intensity of the fluorescence generated by the sensitive fluorescent reagent, it became clear that the membrane permeability of the drug can be evaluated for a large number of samples at once in a short time. Therefore, the method of the present invention is extremely useful as a rapid drug absorption evaluation method at the drug discovery stage.

[Brief description of the drawings]

FIG. 1 is a graph showing the time change of the fluorescence intensity and the fluorescence intensity of a control well due to a pH change in Caco-2 cells when salicylic acid is used.

FIG. 2 is a graph showing a comparison between a change in fluorescence intensity due to a pH change in Caco-2 cells and a permeability of a Caco-2 cell monolayer.

FIG. 3 is a graph comparing the change in fluorescence intensity due to the pH change in Caco-2 cells with the permeability of rat jejunum.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12Q 1/02 C12R 1:91) (71) Applicant 595117091 1 BECTION DRIVE, FRA NKLIN LAKES, NEW JERSEY 07417-1880, UNITED STATES OF AMERICA (72) Inventor Yoko Taki 1-3-4 Minami Kyouyou, Hirakata-shi, Osaka F-term (reference) 2G045 AA40 BB52 CB01 CB02 DB03 FA11 FB12 GC15 4B024 AA11 BA80 DA02 GA11 QB4A Q QQ08 QQ61 QQ91 QQ98 QR41 QR51 QR57 QR66 QR69 QR77 QS24 QS36 QX02

Claims (8)

[Claims] (1) a step of forming a cell layer; (2)
A step of introducing a fluorescent dye into the cell layer; and (3) a step of bringing the cell layer into which the fluorescent dye is introduced into contact with a buffer solution containing a drug, and absorbing the drug into the cell layer;
(4) analyzing the pH change in the cells of the cell layer obtained in (3) by fluorescence analysis to evaluate the abundance of the drug in the cells, the intestinal absorption of the drug. Evaluation method. 2. The method according to claim 1, wherein the cell is Cac derived from human colon cancer.
The evaluation method according to claim 1, wherein the method is selected from the group consisting of o-2 cells, HT29 cells derived from human colorectal cancer, and MDCK cells derived from renal tubules. 3. The method according to claim 1, wherein the cell is a cell line in which a transporter is expressed at a high density by genetic manipulation. 4. The evaluation method according to claim 1, wherein the pH of the buffer is in the range of 5.0 to 8.0. 5. The evaluation method according to claim 1, wherein the buffer is an isotonic buffer. 6. The evaluation method according to claim 1, wherein the concentration of the drug in the buffer solution containing the drug is 0.01 mM to 100 mM. 7. The method according to claim 1, wherein the step (3) is performed at a temperature within a range that does not affect the cells. 8. The evaluation method according to claim 1, wherein the fluorescence analysis is performed using a fluorescence reader.