JP3929910B2 - Hepatocytes and liver microsomes in which cytochrome P450 expression was induced - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a hepatocyte in which cytochrome P450 expression is induced, obtained by culturing hepatocytes fractionated from a mammal and subcultured in a medium containing a cytochrome P450 inducer, and the hepatocytes prepared from the hepatocytes. Related to liver microsomes.
[0002]
[Prior art]
Culture of hepatocytes is very important for the study of various liver functions, pharmacokinetic properties (absorption, distribution, metabolism, excretion), toxicological properties, and the interaction of two or more drugs. In the early stages of drug development, in vitro metabolic tests using liver microsomes and liver cells are used to evaluate drug candidate compounds. However, primary cultured hepatocytes have large variations in drug metabolic ability due to race differences and sex differences (Non-patent Documents 1 and 2), are difficult to obtain, and are accompanied by ethical problems. It is known that the content of drug metabolism-related enzymes later decreases to 50% or less (Non-patent Document 3), and there is a problem that a long-term metabolic test maintaining a uniform and stable character is difficult. On the other hand, in the case of the subcultured cell line, the activity of drug metabolism-related enzymes, particularly cytochrome P450, is almost lost, so it was not useful for the in vitro metabolism test described above. There is also a report on a method for establishing a transformant that expresses cytochrome P450 using a liver cancer-derived cell line (Patent Document 1), but only the molecular species encoded by the introduced gene is synthesized, which improves operability and stability. There was also a problem.
[0003]
[Patent Document 1]
JP 2001-8861 [Patent Document 2]
Patent No. 2744418 [Non-Patent Document 1]
Guillouzo et al .; Biochem. Pharmacol. 1985; 34: 2991-5
[Non-Patent Document 2]
Coudouris et al .; Xenobiotica 1993; 23: 1399-409
[Non-Patent Document 3]
Maurel et al .; Adv. Drug Deliver. Rev. 1996; 22: 105-132
[Non-Patent Document 4]
Kobayashi et al .; Science 2000; 287: 1258-1262
[Non-Patent Document 5]
Hasumura et al .; Human Cell 1988; 1: 238-44
[Non-Patent Document 6]
Hasumura et al .; Human Cell 1988; 1: 98-100
[Non-Patent Document 7]
Fujise et al .; Hepato-Gastroenterol. 1990; 37: 457-460
[Non-Patent Document 8]
Homma et al .: Human Cell 1990; 3: 152-7
[0004]
[Problems to be solved by the invention]
Regarding the induction of drug-metabolizing enzymes, it is clear that there are species differences between animals, so it is difficult to extrapolate the results of animal experiments directly to humans. There is an urgent need to provide a maintained human cultured hepatocyte cell line.
That is, the present invention is useful for in vitro drug metabolism tests, and an object thereof is to provide subcultured hepatocytes and liver microsomes that can be stably supplied.
[0005]
[Means for Solving the Problems]
As a result of intensive studies in view of the above problems, the inventors of the present invention have succeeded in subculturing hepatocytes at a very high density and adding a cytochrome P450 inducer to the medium, so that the amount of cytochrome P450 mRNA expression is increased. It was highly induced, and it was found that the induced cytochrome P450 maintained the substrate metabolic capacity in vivo even in vitro, and the present invention was completed. That is, the present invention
(1) A hepatocyte in which cytochrome P450 expression is induced, obtained by three-dimensional high-density culture of hepatocytes and further culturing in a medium containing a cytochrome P450 inducer after the culture;
(2) The hepatocyte according to (1), wherein the density of the hepatocytes cultured in three-dimensional high density is 10 ⁶ cells / ml or more;
(3) The hepatocyte according to (1) above, wherein the density of the hepatocytes cultured in three-dimensional high density is 10 ⁸ cells / ml or more;
(4) The cytochrome P450 induced in the hepatocytes is CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 or CYP3A4;
(5) The hepatocyte according to (1) above, wherein the cytochrome P450 inducer is at least one of phenytoin, carbamazepine, rifampicin, dexamethasone, sulfinpyrazone or barbituric acid drugs and salts thereof;
(6) A hepatocyte in which cytochrome P450 expression is induced, obtained by three-dimensional high-density culture of human hepatocytes and further culturing in a medium containing rifampicin after the culture;
(7) Liver microsomes prepared from the hepatocytes according to any one of (1) to (6) above;
(8) Expression of cytochrome P450, comprising (a) three-dimensional high-density culture of hepatocytes; and (b) culturing the hepatocytes after the three-dimensional high-density culture in a medium containing a cytochrome P450 inducer. A method for producing hepatocytes in which is induced;
(9) (a) Three-dimensional high density culture of human hepatocytes using a holofiber bioreactor or a radial flow bioreactor; and (b) Rifampicin is added to the human hepatocytes after the three-dimensional high density culture. A method for producing human hepatocytes in which expression of cytochrome P450 is induced, comprising culturing in a medium containing the same;
(10) (a) by cytochrome P450, which comprises contacting the hepatocyte according to any one of (1) to (6) above or the liver microsome according to (7) above with a test compound. A method for assessing the sensitivity of a test compound to metabolism;
(11) (a) Incubating the hepatocyte according to any one of (1) to (6) above or the liver microsome according to (7) above and a test compound; (b) (E) extracting the reaction product obtained in (a); (c) the evaluation method according to (10), further comprising analyzing the reaction product obtained in (b);
(12) The evaluation method according to (11), wherein at least two compounds are used as the test compound.
(13) The evaluation method according to (12), which is a drug interaction evaluation method.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail by showing the meanings of symbols, terms, and the like described in the present specification, embodiments of the present invention, and the like.
[0007]
The hepatocytes used in the present invention can be obtained by sorting from a mammalian liver by a known method, but those collected from a mammalian liver cancer are preferred. Liver cancer-derived cells can be subcultured and are relatively easy to establish as cell lines. Liver cancer refers to a malignant tumor that has developed in the liver, and includes hepatocellular carcinoma, hepatoblastoma and the like, but is not particularly limited as long as the sorted cells can be subcultured. Examples thereof include HepG2 as a human hepatoblastoma-derived cell line, and B16 as a human hepatocellular carcinoma-derived cell line.
In addition, even in normal hepatocytes, the SV40T gene is expressed in the cell to become immortal, subcultured and propagated, and then the so-called "" obtained by removing the SV40T gene using the Cre-loxP system. “Primary” hepatocytes (Non-patent Document 4) can also be used in the present invention.
By using hepatocytes subcultured in this way, it is possible to stably supply cells having no variation in properties.
[0008]
In the present specification, “three-dimensional culture” refers to a culture method excluding a single-layer culture method among cell culture methods, and “high density culture” refers to a cell density of at least 10 ⁶ cells / ml, The culture is preferably 10 ⁸ cells / ml or more. Here, the monolayer culture method refers to a culture method in which cells are two-dimensionally grown on the bottom surface of a culture vessel to form a single cell layer.
“Culture methods other than monolayer culture” include suspension culture, embedded culture using soft agar, methylcellulose, collagen gel as a substrate, holofiber bioreactor and radial flow bioreactor ( Examples include a culture method using Patent Document 2). Among them, a culture method using a holofiber bioreactor or a radial flow bioreactor capable of realizing high-density culture is preferable. In particular, a radial flow type bioreactor can be cultured for a long period of 100 days or longer, and can be used for enzyme induction experiments and inhibition experiments. During the culture, the pH is preferably about 6.5 to 7.5, and the temperature is 0 to 40 ° C., preferably about 37 ° C.
[0009]
The cytochrome P450 inducer used in the present invention is added to the culture medium of the subcultured hepatocytes according to the present invention, thereby inducing the expression of cytochrome P450 in the hepatocytes. Cytochrome P450 is a drug-metabolizing enzyme that exists in the liver, intestine, and the like, and oxidizes the drug oxidatively in the presence of NADPH. There are 6 substrate recognition sites in the primary structure of cytochrome P450, and because of this difference, there are a number of molecular species. Therefore, each molecular species is followed by “CYP” (representing cytochrome P450), and the family by number. The subfamily is expressed in alphabets and the unique name is appended with a number. The CYP molecular species induced in hepatocytes according to the present invention are CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A1, CYP, which are particularly important in drug metabolism. Of particular importance is CYP3A4, which is about 50% metabolized by drugs on the market. The cytochrome P450 inducer used in the present invention is not particularly limited, but is preferably a CYP3A4 inducer, and examples thereof include barbituric acid drugs such as phenytoin, carbamazepine, rifampicin, dexamethasone, sulfinpyrazone, and phenobarbital.
[0010]
The liver microsome according to the present invention is prepared from hepatocytes in which the expression of cytochrome P450 according to the present invention is induced. A normal method can be used for the preparation. For example, a supernatant obtained by precipitating nuclei, mitochondria, and lysosomes from the homogenate of hepatocytes according to the present invention by fractional centrifugation is further added at 100,000 × g for about 1 hour. The ultracentrifugation can be performed by resuspending the fraction obtained as a precipitate. Liver microsomes contain all cytochrome P450 molecular species contained in the cells, as well as enzymes related to conjugation reactions, so they are particularly useful for in vitro drug metabolism studies in the early stages of drug development. It can also be used to analyze the interaction of two or more drugs. In addition, since it is easier to store and transport than cells, it is possible to compare the results of metabolic tests using microsomes derived from multiple cell lines. This property is particularly desirable because it has been shown that there are race, gender and species differences in the activity of liver metabolic enzymes. In conventional subcultured cells, many enzymes were inactivated before the treatment for preparing the microsomal fraction, and it was difficult to obtain a microsomal fraction that sufficiently maintained the enzyme activity. According to this method, it is possible to prepare a large amount of a uniform microsomal fraction from cells in a state where cytochrome P450 activity is maintained at a very high level.
[0011]
The hepatocytes and liver microsomes according to the present invention are most preferably derived from cells sorted from human liver. In particular, cells derived from Functional Liver Cell (hereinafter referred to as “FLC”) (Non-Patent Document 5-8), which is a human-derived human hepatocellular carcinoma established by the present inventors, have liver functions such as albumin production. Since it is relatively maintained, it is preferable as a hepatocyte used in the present invention, and among them, the FLC-5 cell line that has been confirmed to induce CYP3A4 among the 7 FLCs is more preferable. In addition, since culture in a serum-free medium is possible, it is possible to exclude the influence of serum proteins in the in vitro metabolic test described later.
Although human-derived hepatocytes are accompanied by ethical problems associated with collection and are difficult to obtain, human-derived hepatocytes subcultured according to the present invention maintain cytochrome P450 activity high, Useful.
[0012]
The method for producing hepatocytes according to the present invention includes three-dimensional high-density culture of hepatocytes, and further culturing the hepatocytes after subculture in a medium containing a cytochrome P450 inducer. For example, a P450 inducer may be added to the culture solution used for subculture, or the cells after subculture may be transferred to a medium containing the P450 inducer. As described above, the three-dimensional high-density culture preferably uses a holofiber bioreactor or a radial flow bioreactor. When these are used, after high-density culture, for example, 50 μM rifampicin as a P450 inducer is used as the culture solution. Add and reflux. If the cytochrome P450 inducer is not water-soluble, it is dissolved in an organic solvent such as DMSO and diluted so that the final concentration of DMSO is 0.25%. The culture temperature is 0 ° C. to 40 ° C., preferably about 37 ° C., and the pH is preferably about 6.5 to 7.5. The culture in the medium containing the cytochrome P450 inducer is preferably performed at least for a time when the effect of the inducer is sufficiently exhibited. For example, in the case of rifampicin, the effect is maximized 48 to 72 hours after the addition, so it is desirable to use it for in vitro tests or for the preparation of liver microsomes during this period. If the effect does not last once the inducer is added and the effect reaches a peak, the induction effect is maintained by repeated addition after an appropriate period of time. Further, when not used immediately after induction, it can be stored frozen when the effect reaches a peak.
[0013]
By contacting the test compound with hepatocytes or liver microsomes in which the expression of cytochrome P450 according to the present invention is induced, the sensitivity of the test compound to metabolism by cytochrome P450 can be evaluated. The test compound is not particularly limited, for example, peptides, proteins, non-peptidic natural products, synthetic compounds, fermentation products, etc. These compounds are active ingredients, solutions, other cell extracts, plant extracts, animal tissue extracts. It can be used in the present method as a composition such as liquid or plasma. The evaluation method can be performed in the same manner as or in conformity with the known method. For example, a sufficient time is allowed for the test compound to be metabolized by adding the test compound to the medium of hepatocytes in which the expression of P450 is induced. Can be carried out by detecting metabolites in the cells or the culture solution after elapse of time. When the test compound is not water-soluble, it can be dissolved in an organic solvent such as DMSO and added to the medium under the condition that it is diluted so that the final concentration of DMSO is sufficiently low. CYP3A4, which is particularly significantly induced in the present invention, is known to metabolize about 50% of commercially available pharmaceuticals. If hepatocytes or liver microsomes from which CYP3A4 has been induced are used, the sensitivity of the test compound to CYP3A4 can be reduced. Since it becomes possible to evaluate, it is very useful.
[0014]
Further, by contacting cells or liver microsomes in which the expression of cytochrome P450 according to the present invention is induced with at least two test compounds, the interaction between these test compounds can be evaluated. Specifically, for example, a plurality of test compounds can be simultaneously added to the medium of hepatocytes in which expression of P450 is induced, and metabolites by P450 of each test compound can be detected and quantified. .
Currently, multi-drug combinations are used in clinical practice, so it is necessary to evaluate the interaction of multiple drugs at the preclinical and clinical trial stages. For example, when a drug (A) metabolized by CYP3A4 and a CYP3A4 inhibitor (B) are used in combination, the metabolism of the drug (A) is inhibited and the blood concentration increases. When the drug (A) metabolized by CYP3A4 and the CYP3A4 inducer (C) are used in combination, the metabolism of the drug (A) is promoted and the blood concentration decreases. Further, when a plurality of drugs metabolized by CYP3A4 are used in combination, metabolism may be competitively inhibited and the blood concentration of any drug may increase. The above-mentioned interactions can have unexpected effects on the human body and are required to be fully evaluated by in vitro studies at an early stage of preclinical studies in order to conduct safe clinical studies. ing. The hepatocytes or hepatic microsomes according to the present invention, which can be subcultured, supplied stably, and maintain the ability of drug metabolism, are very useful for these evaluation tests.
[0015]
Examples of the present invention will be described in detail below, but the present invention is not limited thereto.
[Example 1] Production and maintenance of human liver cancer cell line Human liver cancer cell line FLC-5 derived from human hepatocellular carcinoma (HCC) cell line was suspended in ASF-104 serum-free medium (Ajinomoto Co., Inc.) The culture apparatus was maintained at 37 ° C. in very humid air containing 5% CO ₂ . During cell passage, cells were separated by adding 25 USP unit / ml trypsin (Difco co., Ltd.) to a 2% EDTA solution.
[0016]
[Example 2] Three-dimensional high-density culture radial flow bioreactor using a radial flow bioreactor (hereinafter sometimes abbreviated as "RFB") RA-15 (Able Co., Ltd.) and mass flow controller RAD925 (Able stock) 3) high-density culture was performed according to the instructions using hydroxyapatite beads (PENTAX) having a diameter of 1-2 mm and a pore size of 200 μm or less. Inject 5 × 10 ⁷ FLC-5 cells into RFB and add the medium first to ASP-104 serum-free medium, then 2% FBS and 3 g / l to help the cells adhere to the beads, and after attachment, ASF -Returned to 104. This enables high-density culture at 10 ⁸ cells / ml or higher, and observation with a scanning electron microscope confirms that the cultured FLC-5 cells are encapsulated on the surface of hydroxyapatite beads. It was. Moreover, it was observed by the transmission electron microscope that the cells were in close contact with each other, and that a capillary-like structure and an intercellular junction device were also developed. The cells were spherical or cubic, and a three-dimensional three-dimensional culture was realized.
[0017]
[Example 3] Analysis of mRNA expression by real-time PCR Total RNA was extracted from cells cultured by the above-described method using SV total RNA extraction kit (PROMEGA). Rifampicin (Sigma) was added to the FLC-5 cell culture solution at 50 μM 48 hours before analysis. Primers and TaqMan probes for YP3A4 were designed and prepared with Primer Express ™ software (Perkin-Elmer Applied Biosystems) using GenBank database according to the cDNA sequence registered by NCBI with the number of AF182273. Based on the genomic DNA sequence information registered under the AF209389 number, the probe was designed to be applied to the Exon / Intron junction to completely eliminate genomic DNA contamination. Primers and probes are shown below.
Forward Primer Sequence: 5 'GCAGGAGGAAATTGATGCAGTT
Reverse Primer Sequence: 5 'GTCAAGATACTCCATCTGTAGCACAGT
TaqMan Probe Sequence: 5 'TACCCAATAAGGCACCACCCACCTATGA
Using these primers and probe, reverse transcription reaction and signal detection were performed using ABI Prism 7700 Sequence Detector (Perkin-Elmer Applied Biosystems) according to the attached instructions. For the calibration curve, total RNA collected from FLC-5 cells cultured in monolayer was used, and the quantitative range from 1000 ng total RNA / 50 μl reaction system to 0.32 ng total RNA / 50 μl reaction system with a 5-fold common ratio was examined. . Specificity was GAPDH mRNA as an endogenous control. The results are shown in FIG. The expression level of CYP3A4 mRNA was increased 7.7 times in the three-dimensional high-density culture compared to the monolayer culture without rifampicin induction. In monolayer culture, the expression level increased only 2.4-fold with rifampicin induction compared to non-induced rifampicin, whereas it increased 33.8-fold with rifampicin induction compared with non-induced rifampicin in three-dimensional high-density culture. . Compared with the rifampicin induction group in monolayer culture, the expression level of CYP3A4 mRNA was increased 108.4 times in the rifampicin induction group in three-dimensional high-density culture.
[0018]
[Example 4] Preparation of microsomes and Western blotting
After FLC-5 cells were three-dimensionally cultured at high density with RFB, they were cultured under rifampicin induction and non-induction, and cryopreserved at −80 ° C. while attached to the beads. To 0.5-1.0 g of hydroxyapatite beads with FLC-5 cells attached, add twice the amount of homogenate buffer (10% sucrose, 0.01 MK ₂ HPO ₄ , 1 mM DTT, pH 7.30) and shake. Stir. The beads are allowed to settle spontaneously, and the supernatant containing the broken cell components is centrifuged at 3000 rpm for 5 minutes, and the supernatant is recovered and further centrifuged at 3500 g for 13 minutes. The supernatant obtained here is centrifuged at 24000 g for 30 minutes, and the precipitate is resuspended in a buffer (0.15 MK ₂ HPO ₄ , 1 mM DTT, pH 7.30) to obtain rifampicin-induced and non-induced microsomal fractions. After the protein quantification of the prepared microsomal suspension was performed by the Raleigh method, a 4-20% gradient gel was prepared, 10 μg of microsomal protein was arranged in each lane, and SDS polyacrylamide gel electrophoresis was performed. After the completion of electrophoresis, the protein was transferred from the gel to the PVDF membrane. The PVDF membrane was shaken in a reaction solution (0.025M Tris, 0.15M NaCl, pH 7.4) containing 3% BSA for 30 minutes for blocking. Anti-human CYP3A4 antibody (Gentest Corp.) was reacted at room temperature for 1 hour, and anti-rabbit IgG alkaline phosphatase conjugate (Gentest Corp.) was further reacted at room temperature for 1 hour. After washing, the plate was immersed in an alkaline phosphatase buffer solution (0.1 M NaCl, 0.05 M MgCl ₂ , 0.1 M Tris, pH 9.5) and reacted with a substrate solution in which NBT (Sigma) and BCIP (Sigma) were mixed to cause color development.
The results are shown in FIG. In the rifampicin-induced cell microsome (right), CYP3A4 expression was clearly high even at the protein level.
[0019]
[Example 5] Metabolism experiment of testosterone using three-dimensional high density cultured FLC-5 cells
Testosterone, a representative substrate of 50 μM CYP3A4, was added to 15 ml of a culture solution of FLC-5 cells cultured three-dimensionally at high density with RFB and refluxed. After 6 hours, the culture medium and the beads with the cells attached thereto were collected and stored frozen at −40 ° C. In the rifampicin induction group, 50 μM rifampicin (Sigma) was added to the culture 48 hours before adding testosterone. Testosterone (Wako) was dissolved in DMSO so that the final concentration of DMSO in the culture was 0.25% or less. Testosterone concentration was measured by HPLC from the cryopreserved sample. Nitrazepam (Wako) as an internal standard for HPLC is appropriately added to the culture medium, and ethyl acetate is further added and shaken for 10 minutes. After centrifuging at 3200 rpm for 5 minutes, 4.5 ml of the upper organic layer was transferred to a glass tube and dried by an evaporator, and then dissolved by adding 250 μl of HPLC moving bed solvent. The moving bed was DW: methanol (40%: 60%), adjusted to pH 3.4 with H ₃ PO ₄ and degassed. The column was a reverse-phase CAPCELLPAK C18 UG120 (Shiseido), and the detector was an SPD-10AVP UV absorption detector (Shimadzu Corporation). 70 μl of the sample was injected, the moving bed solvent was passed at a flow rate of 0.7 ml / mn, and the absorbance at 244 nm was measured.
The result of the metabolic experiment using the rifampicin induction group is shown in FIG. 3A, and the result of the rifampicin non-induction group is shown in FIG. In the rifampicin induction group, the peak of 6β-hydroxytestosterone, which is a metabolite of testosterone by CYP3A4, could be detected in the cells and in the culture solution. On the other hand, in the rifampicin non-induction group, intracellular 6β-hydroxytestosterone was detected but very little, and the culture broth was below the detection sensitivity.
[0020]
【The invention's effect】
According to the present invention, it was possible to provide subcultured hepatocytes containing high concentrations of cytochrome P450 and liver microsomes derived from the hepatocytes. The hepatocytes and liver microsomes according to the present invention maintain the ability of drug metabolism by cytochrome P450 in vitro and are useful for drug metabolism tests.
[Brief description of the drawings]
FIG. 1 shows the results of measuring the expression of CYP3A4 mRNA using rifampicin treatment of FLC-5 cells cultured in a monolayer and FLC-5 cells cultured in three dimensions at high density using a real-time PCR method.
FIG. 2 shows the results obtained by measuring the amount of CYP3A4 by Western blotting with and without rifampicin treatment on FLC-5 cells cultured in three-dimensional high density.
FIG. 3 shows the results of a testosterone metabolism experiment in rifampicin-induced or non-induced FLC-5 cells cultured in three dimensions.

Claims (7)

A method for evaluating a drug interaction between two or more test compounds,
(A) FLC cells or HepG2 cells are three-dimensionally cultured at a high density, and further cultured after culturing in a medium containing an inducer that induces expression of CYP3A4, prepared from FLC cells, HepG2 cells, or these cells Contacting the incubated liver microsomes with the two or more test compounds, and incubating;
(B) detecting or quantifying a reaction product of the test compound . The method according to claim 1, wherein the density of the FLC cells or HepG2 cells cultured in three-dimensional high density is 10 ⁶ cells / ml or more. The method according to claim 2, wherein the density of FLC cells or HepG2 cells cultured in three-dimensional high density is 10 ⁸ cells / ml or more.   The inducing agent that induces the expression of CYP3A4 is at least one of phenytoin, carbamazepine, rifampicin, dexamethasone, sulfinpyrazone, or a barbituric acid drug and a salt thereof, according to any one of claims 1 to 3. the method of.   5. The method of claim 4, wherein the inducer that induces expression of CYP3A4 is rifampicin.   6. The method according to any one of claims 1 to 5, wherein the FLC cells are FLC5 cells.   The method according to any one of claims 1 to 6, wherein in (a), three-dimensional high-density culture of cells is performed using a holofiber bioreactor or a radial flow bioreactor.

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