JPH11299478A - Analysis of metabolism of medicine and instrument therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for analyzing the metabolism of a medicine in a tissue by culturing plural tissue pieces in a culture liquid containing the prescribed medicine, subsequently collecting samples from the plural tissues, respectively, at plural time points and quantifying the medicine or a metabolite in the tissues, respectively. SOLUTION: This method for analyzing the metabolism of a medicine comprises culturing a tissue piece in a culture liquid containing the prescribed medicine, collecting samples from at least one of the tissue piece and the culture liquid at two or more time points, quantitatively analyzing at least one of the non-changed medicine or the metabolites remaining or produced in the sample with the passage of time by the absorption, metabolism and excretion of the medicine in the tissue, and subsequently tracing and organizing the changes in the quantities of the non-changed medicine or metabolite with the passage of time. Therein, a plurality of the tissue pieces are separately or simultaneously cultured in a plurality of culture vessels 1 fixed to the upper surface of a substrate 2 in a mutually connected state. Fine amounts of samples are simultaneously collected from the plural tissue pieces and/or the culture liquid at two or more time points, and then analyzed to determine the medicine-metabolizing function of the tissue such as liver.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for analyzing drug metabolism and a device therefor, and more particularly to a method for analyzing drug metabolism and a device therefor which can directly evaluate the function of a tissue such as a liver with respect to drug metabolism using a small amount of a biological sample.

[0002]

2. Description of the Related Art During the development of new drugs, experimental animals have been widely used as human substitutes. However, there is a great difference in pharmacokinetics between humans and experimental animals, and serious side effects frequently occur in humans even with drugs that are safe as a result of animal experiments.

[0003] The side effects of a drug are closely related to metabolism in the liver, and knowing the absorption rate when passing through the liver, the metabolic rate in the liver, and the excretion rate from the liver include the side effects of the drug. Is extremely important in assessing safety.
In order to grasp these factors, it is necessary to track changes in the residual amount or the production amount of the drug itself (so-called unchanged form) and metabolite over time. As an experimental method for this, a part of the liver (for example, liver lobule)
Is cultured in a culture solution containing a target substance, and a method of tracking a temporal change in the amount of an unchanged substance and a metabolite is useful.

[0004] In such a culture test, when performing cultivation with different concentrations of the target substance and the composition of the culture solution, in order to ensure the accuracy of the experiment, reactions in a plurality of culture solutions are allowed to proceed at the same time. A sample for analysis from the culture solution,
It is desirable to collect the samples at multiple points in time and analyze those samples.

[0005]

However, although it is easy to simultaneously advance the reactions of a plurality of culture solutions, it is necessary to simultaneously sample a plurality of samples from each solution at a number of times and to immediately stop the reactions. Required extremely complex and therefore expensive equipment.

Further, when the culturing reaction is carried out in a desired atmosphere, for example, under a constant partial pressure of oxygen or carbon dioxide gas, even if the reactions can proceed simultaneously, it is possible to collect a large number of samples through the atmosphere at the same time. Was extremely difficult.

Therefore, an object of the present invention is to culture a plurality of tissue pieces in a plurality of culture solutions to which a predetermined substance has been added, under a predetermined atmosphere, and to metabolize the substance by the tissue during culture. It is to provide a method for analyzing the.

[0008] Another object of the present invention is to simultaneously culture a plurality of tissue pieces in a plurality of culture solutions to which a predetermined substance has been added under a predetermined atmosphere at the same time. Samples can be taken at the same time from the solution, the reaction can be stopped immediately, and those samples can be used for analysis,
It is an object of the present invention to provide an improved method so that simultaneous sampling of a plurality of samples and stopping of the reaction can be performed with an inexpensive apparatus.

[0009] Another object (2) of the present invention is to simultaneously culture a plurality of tissue pieces in a plurality of culture solutions to which a predetermined substance is added under a predetermined atmosphere, and to simultaneously culture the two or more pieces. It is an object of the present invention to provide a simple and inexpensive apparatus that can simultaneously collect samples from respective liquids at a point in time, immediately stop the reaction, and provide the samples for analysis.

[0010]

SUMMARY OF THE INVENTION The object of the present invention is to cultivate a tissue piece in a culture medium to which a predetermined drug has been added, and to simultaneously obtain a sample from at least one of the tissue piece and the culture medium at two or more time points. As a result of the absorption, metabolism, and excretion of the drug, at least one of the unchanged substance and the metabolite remaining or generated in the sample over time is quantitatively analyzed, and the time of the amount of the unchanged substance and the metabolite is determined. By tracking changes,
In a method of analyzing drug metabolism by a tissue, a plurality of tissue pieces are individually but simultaneously cultured in a culture solution in a plurality of connected and fixed culture vessels, respectively.
At each of the above-mentioned time points, it was achieved by a drug metabolism analysis method, wherein a sample is simultaneously collected from at least one of a plurality of tissue pieces and a culture solution.

[0011] The object of the present invention is particularly to provide a container or container in which culture vessels connected to each other are housed in a sealable bag or box, and a pipe for introducing a predetermined gas is connected to one end of the culture vessel and the other end is connected to the other end. Is provided with an exhaust hole whose size is such that the gas composition in the bag is maintained at a predetermined composition, and a predetermined gas is introduced from the gas introduction pipe so as to maintain the gas composition in the bag at a predetermined composition. The above-described drug metabolism analysis method in which culture was performed while introducing gas was achieved more effectively.

[0012] The object (2) of the present invention is to store culture vessels connected to each other in a sealable bag or box (including a cylindrical one) and to introduce a predetermined gas into one end thereof. A pipe is connected, and the other end is provided with an exhaust hole large enough to keep the gas composition in the bag or the box at a predetermined composition,
This was achieved by a culture instrument adapted to carry out culturing while introducing a predetermined gas from a gas inlet tube so as to maintain the gas composition in the bag or the box at a predetermined composition.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The drug is not limited to a synthetic product, but also includes a natural product and a substance existing in an organism. The number of culture vessels and the number of tissue pieces are not always the same, and two or more tissue pieces may be accommodated in one culture vessel. A sample may be collected from either a tissue piece or a culture solution, or a sample may be collected from both the tissue piece and the culture solution. In the latter case, samples are taken simultaneously from both the tissue piece and the culture.

Although the shape of the culture vessel is not related to the effects of the present invention, a dish-shaped dish with a flat bottom (a Petri dish) is practically convenient. In order to prevent the culture solution from spilling out, the culture vessel preferably has a volume that is at least three times the volume of the culture solution.

A plurality of culture vessels having the same reaction time under the same atmosphere and the same temperature are connected and fixed to each other. However, if necessary, it may be divided into two or more connected groups (when fixed to a substrate, it may be divided into two or more sheets). The term “fixed” includes detachable. The arrangement of the connected culture vessels may be one row, two or more rows, or circumferentially, and is arbitrary. When the culture vessels are fixed to one or more substrates and connected to each other, it is preferable that the substrates are chemically stable and do not deform or deteriorate at the reaction temperature.

Culture is performed in a culture solution in a culture vessel. If necessary, the culture solution is placed in a desired atmosphere with the culture vessel to perform the culture. In this case, in order to keep the gas composition of the atmosphere constant, the culture solution container is accommodated in a bag or box (hereinafter, referred to as a bag or the like) having an appropriate shape, and a gas having a predetermined composition is appropriately applied from one end of the bag or the like. It is possible to adopt a method in which the gas is introduced at a flow rate and the gas is discharged to the outside through a hole of an appropriate size provided at the other end. Instead of providing a hole at the other end of the bag or the like, a porous bag (for example, a paper bag) or the like may be used. In the vicinity of the liquid surface of the culture solution, since the concentration of water such as water, carbon dioxide, ammonia, and nitric oxide generated as a result of metabolism and excretion from the tissue pieces during culture tends to increase, the flow of gas is promoted, It is necessary to distribute a gas having a predetermined composition through a bag or the like. If a closed container or a bag having only one gas inlet / outlet is used, it is difficult to keep the gas composition on the culture solution constant. The bag or the like may be opaque or translucent, but a transparent bag is convenient because the culture solution can be observed. If translucent or transparent, bags etc. are colored (wavelength selectivity)
Alternatively, it may be impermeable to ultraviolet light. In order to sterilize the inside of the culture instrument, it is preferable to be transparent to ultraviolet rays.

The culture solution must contain sufficient nutrients required by the tissue. The volume of the culture solution must be at least 10 times the volume of the tissue piece, and is preferably 15 times or more.

The thickness of the tissue piece is preferably 1 mm or more and 3 mm or less. The preparation of the tissue piece is desirably performed at 3 ° C. or lower, and a temperature close to 0 ° C. is preferable. However, the temperature is higher than the temperature at which the culture solution freezes. The preparation of the tissue piece is preferably completed within 20 minutes. It is preferable that the time from the preparation of the tissue piece to the start of culture does not exceed 10 minutes.

It is preferable that the liver tissue piece is a unit of liver lobule and does not include a plurality of liver lobules.

Separation of components extracted from a tissue slice under each culture condition and a sample obtained from a culture solution can be performed simultaneously and simultaneously as simultaneously as possible, for example, by performing chromatographic development on one thin layer chromatographic plate at the same time. It is preferred to do so.

FIG. 1 shows a connected body of culture vessels used in the present invention. The culture dish 1 containing a culture solution (not shown)
It has a flat bottom and is integrally connected by being fixed to a flat substrate 2.

FIG. 2 shows a culture device used in the present invention, which is provided with a bag for placing a culture solution under a constant atmosphere. The culture dish 1 is fixed in a line to a flat substrate 2 and is housed in a cylindrical bag 3. The bag 3 has a gas introduction pipe 4 at one end and a small hole 5 and an exhaust pipe 6 at the upper end of the other end. A gas having a desired composition for culturing is supplied to the bag 3 from the gas introduction pipe 4. Part of the gas in the bag 3 is gradually released from the hole 5 through the exhaust pipe 6 to the outside of the bag.

FIG. 3 shows a culture apparatus used in the present invention.
The bag 3 containing the culture dish 1 fixed to the substrate 2 is
The substrate 2 is placed on a support base 8 so as to be horizontal, placed in a thermostat 7 at a desired temperature, and a culture reaction is performed for a required time while shaking the whole by a shaking means (not shown).

[0024]

The present invention will be described more specifically with reference to the following examples. [Example 1] Prepare Waymouth medium containing nafamostat mesilate 5% xylitol and 10 ^{@ 8} mol / L (including physiologically buffered saline, essential amino acids, vitamins),
The temperature was set to 0 ° C. (hereinafter referred to as culture solution A).

A part of the liver (liver lobule) collected from a human at the time of surgery is perfused with a perfusion solution having the following composition to remove blood in the tissue, and then transported at a temperature of about 0 ° C. . The transit time was about 50 hours.

Immediately after arrival, the liver tissue was sliced into slices having a thickness of 2 mm and a weight of about 100 mg in slice solution B, and suspended in the solution. Weigh the tissue pieces and measure 1 per 100 mg
0 pg of Diazepam (including ^2-14 C for 3.7 kBq), was added to the slice liquid B was suspended tissue pieces, placed in a culture dish 1, as shown in FIG. 1, it detachably on the substrate 2 Fixed.

[0027]

As shown in FIG. 2, the substrate 2 was wrapped in a cylindrical bag 3 made of polyethylene, and oxygen gas was supplied from the gas introduction pipe 4 at a liquid volume of 100 ml / min. As shown in FIG. 3, the culture reaction was started in a thermostat at a temperature of 28 ° C. During the reaction time, the culture dish 1 on the substrate 2 was shaken by a shaking device (not shown), and the culture solution was stirred.

After 15 minutes, 1, 2, 4 and 24 hours from the start of the reaction, the culture containers on the substrate corresponding to the respective reaction times are taken out, and 2 ml of methanol is added to the culture solution to stop the reaction. I let it. Remove the tissue piece from the culture,
Extraction was performed in 0.5 ml of methanol at a temperature of −25 ° C. for 16 hours, the extraction was repeated 5 times, the extracts were combined, the solvent was removed under reduced pressure, redissolved in 20 μl of methanol, and concentrated. The extract was spotted at a position 10 mm from the lower end of a silica gel thin layer chromatography plate (Merck No. 60F254) (hereinafter, TLC plate). The culture was centrifuged, and the supernatant was spotted on a TLC plate.

The thin-layer chromatograph plate is developed with a developing solvent having the following composition, dried, brought into close contact with an imaging plate (Fuji Photo Film Co., Tokyo), and an image analyzer BAS is used.
Analyzed at 2000 (Fuji Photo Film, Tokyo). The contact time was set in three stages of 24 hours, 48 hours, and 72 hours, and the optimal value for the required image was used. [Composition of developing solvent] Chloroform 95 parts Methanol 5 parts Ammonia water 0.1 part

The relative radioactivity concentration of the fraction on each TLC plate obtained by the image analysis was expressed as a relative value with the radioactivity concentration in the culture solution at the start of the culture as 100. The results were as shown in Tables 1 and 2.

Table 1 Radioactivity in culture solution Culture time (hr) 0.25 1 2 4 24 Unchanged 84.82 0.00 0.00 0.00 0.00 Metabolite M1 0.00 0.00 0.00 0.00 0.00 M2 0.00 0.00 0.00 0.00 0.00 M3 0.25 94.7 91.0 91.4 83.8

Table 2 Radioactivity in tissue pieces Culture time (hr) 0.25 1 2 4 24 Unchanged substance U 14.75 0.13 0.20 0.08 0.21 Metabolite M1 0.07 0.00 0.02 0.01 0.01 M2 0.07 0.005 0.007 0.01 0.04 M3 0.05 5.17 8.95 7.98 13.90 Total metabolites 0.19 5.17 8.98 8.00 13.95 unchanged + metabolite T 14.94 5.30 9.18 8.08 14.16 100 U / T 98.7 2.45 2.18 0.99 1.48

As can be seen in Tables 1 and 2, the reaction time
The drug incorporated into the liver tissue in 15 minutes is about 15% (14.94 + 0.25%) of the drug added to the culture, and 14.75% unchanged in the tissue. At this point, about 99% of the drug absorbed into the liver remains unchanged (see 100 U / T column), and only a small portion of the drug has been metabolized.

After one hour, all of the added drug was taken into the liver tissue. Among them, a small amount remains in the tissue as unchanged, and most of the metabolite M3 is excreted in the culture solution, but a part of the metabolite M3 remains in the tissue.
The same applies after 2 hours. Other metabolites M1 and M2 are
It is hardly found in the culture solution. After 1 hour, the metabolite M3 in the tissue increases, but M1 and M2 decrease.

[0036] [Example 2] ^[2-14 C] of Example 1 Diazep
The same experiment and analysis were performed using ring- ^14C- labeled 7-ethoxycoumarin instead of am. N-
A mixed solution of 6 parts of hexane and 4 parts of ethyl acetate was used. TL
The relative radioactivity concentration of each fraction obtained by image analysis of radioactivity on the C plate was calculated as 10
Tables 3 and 4 show the results expressed as relative values of 0. The three metabolites were designated as M11, M12 and M13.

Table 3 Radioactivity in culture solution Culture time (hr) 0.25 1 24 24 unchanged 85.2 78.84 57.15 26.25 0.77 Metabolite M11 0.58 1.21 3.34 5.60 1.89 M12 0.00 0.00 0.00 0.00 0.00 M13 1.79 4.21 11.27 29.22 80.45

Table 4 Radioactivity in tissue pieces Culture time (hr) 0.25 1 24 24 unchanged 11.35 16.83 13.63 15.16 0.91 metabolite M11 0.098 0.034 0.61 0.62 0.24 M12 0.073 0.315 0.82 1.08 0.53 M13 0.915 4.77 13.29 21.70 14.56 Metabolism Total 1.086 5.12 14.72 23.40 15.33 unchanged + metabolite 12.44 21.95 28.35 38.56 16.24

Table 3 and Table 4 show the uptake and metabolism of ring- ^14C- labeled 7-ethoxycoumarin in the tissues. As the unchanged form was incorporated into the tissue, the unchanged form in the culture solution decreased, and the unconverted form in the tissue decreased. Variants increase once but decrease significantly after 24 hours. Metabolites generated in tissues are mainly M13. Metabolites in tissues increase up to 4 hours for each component. On the other hand, M13 in the culture medium was reduced to 24
It can be seen that the time increases significantly.

[0040]

According to the present invention, a plurality of tissue pieces are connected to each other,
Individually but simultaneously in culture in a plurality of fixed culture vessels, at two or more time points,
By simultaneously collecting a sample from at least one of the plurality of tissue pieces and the culture solution, the plurality of tissue pieces are cultured under a predetermined atmosphere in a plurality of culture solutions to which a predetermined substance is added. It has now become possible to analyze the metabolism of that substance by tissues in culture.

In particular, the culture vessels connected to each other are housed in a sealable bag or box, and a tube for introducing a predetermined gas is connected to one end thereof, and the bag (or box) is connected to the other end. )
An exhaust hole having a size enough to keep the gas composition in the predetermined composition is provided, and a predetermined gas is supplied from the gas introduction pipe so as to maintain the gas composition in the bag (or the box) at the predetermined composition. By using a culture instrument adapted to perform culture while being introduced, a plurality of tissue pieces are cultured under a predetermined atmosphere in a plurality of culture solutions to which a predetermined substance is added, respectively. Analyzing the metabolism of substances has become simple and inexpensive.

[Brief description of the drawings]

FIG. 1 is a sectional view of a connected body of a culture vessel used in the present invention.

FIG. 2 is a cross-sectional view of the culture device used in the present invention.

FIG. 3 is an explanatory view of a culture device used in the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Culture Petri dish 2 Substrate 3 Bag 4 Gas introduction pipe 5 Hole 6 Exhaust pipe 7 Constant temperature bath 8 Support stand

Claims (13)

[Claims] 1. A method for culturing a tissue piece in a culture solution to which a predetermined drug has been added, collecting a sample from at least one of the tissue piece and the culture solution at two or more time points, absorbing the drug in tissue, As a result of metabolism and excretion, at least one of an unchanged substance and a metabolite remaining or generated in the sample over time is quantitatively analyzed to track a temporal change in the amount of the unchanged substance and the metabolite. Thus, in a method of analyzing drug metabolism by tissue, a plurality of tissue pieces are individually but simultaneously cultured in the culture solution in a plurality of connected and fixed culture vessels, respectively, at two or more time points, A method for analyzing drug metabolism, comprising simultaneously collecting a sample from at least one of the plurality of tissue pieces and the culture solution. 2. The drug metabolism analysis method according to claim 1, wherein samples are simultaneously collected from both the plurality of tissue pieces and the culture solution. 3. The method according to claim 1, wherein the culture vessel has a flat bottom and is fixed on a flat substrate. 4. The method according to claim 1, wherein the drug is a radiolabeled drug. 5. The culture vessel is housed in a sealable bag or box, and a tube for introducing a predetermined gas is connected to one end of the culture vessel, and the bag or box has a gas composition therein. Has an exhaust hole of a size that can be maintained at a predetermined composition,
The drug metabolism analysis method according to any one of claims 1 to 4, wherein the culture is performed while introducing a predetermined gas from the tube so as to maintain a gas composition in the bag or the box at a predetermined composition. 6. The plurality of culture vessels are divided into groups having the same culture time, and the culture vessels of each group are connected to each other,
6. The drug metabolism analysis method according to claim 5, wherein the cells are fixed and stored in the bag or box for each group, and when a predetermined culture time has elapsed, the culture is terminated by separating from the other groups. 7. The method for analyzing drug metabolism according to claim 5, wherein a plurality of groups of the culture vessels respectively housed in the bag or the box are shaken and cultured by a common instrument in the same thermostat. 8. The drug metabolism analysis method according to claim 6, wherein the culture vessels of each group are arranged in a line on the same substrate. 9. The method according to claim 1, wherein the tissue is a liver. 10. The drug metabolism analysis method according to claim 9, wherein the tissue piece is obtained from a liver slice. 11. A plurality of culture vessels connected to each other,
It comprises a sealable bag or box housing this, a pipe connected to one end thereof for introducing a predetermined gas, and an exhaust hole provided at the other end of the bag or box. Is a size that can maintain the gas composition in the bag or the box at a predetermined composition, and a predetermined gas is supplied from the gas introduction pipe so as to maintain the gas composition in the bag or the box at the predetermined composition. A culture instrument for culturing while introducing the cultivation. 12. The culture instrument according to claim 11, wherein said culture vessels are arranged on the same substrate. 13. A plurality of culture vessels connected to each other,
A bag or box that can be sealed, a pipe for introducing a predetermined gas connected to one end thereof, and the bag or box provided at the other end of the bag or box. A culture device having an exhaust hole large enough to maintain the gas composition at a predetermined composition, a thermostatic bath for maintaining the culture device at a constant temperature, and a rocking means for rocking the culture container. A culture device.