JP2003274963A - Gene recombinant cell strain and liver function supporting equipment using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a cell strain which is transformed with a drug metabolism enzyme gene and an ammonia metabolism enzyme gene such as glutamine synthase gene and has drug metabolism ability and a drug metabolism assay system which evaluates the function of the cell strain and uses the cell strain and to provide liver function supporting equipment such as a hybrid type artificial liver, etc., selectively removing toxic substances. <P>SOLUTION: The cell strain is transformed with the drug metabolism enzyme gene such as P450 3A4, etc., and the ammonia metabolism enzyme gene such as the glutamine synthase gene, etc., especially a human liver-derived cell strain such as HepG2 cell strain. The liver function supporting equipment such as the hybrid type artificial liver, etc., is obtained by using the cell strain. The drug metabolism assay system is obtained by using the cell strain. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell line transformed with a drug-metabolizing enzyme gene and an ammonia-metabolizing enzyme gene, and a liver function assisting device using the cell line.

[0002]

BACKGROUND OF THE INVENTION In Japan, there are many patients with acute liver failure including viral hepatitis. Acute liver failure is caused by rapid hepatocellular necrosis or hepatic dysfunction, resulting in hepatic encephalopathy.
If you have jaundice, ascites, bleeding tendency, renal failure, etc. For these, hemodiafiltration (continuous hemofiltration dialysis continuous a
rteriovenous hemofiltration (CHDF) and plasma exchange (p
Adjunctive liver function therapy combined with lasma exchange (PE) is currently being used clinically. This is a method in which a substance to be detoxified / metabolized by the liver is filtered from blood or is removed together with plasma to supplement the deficiency with human normal plasma. However, since it cannot compensate for all of various liver functions, it has not been able to achieve sufficient therapeutic results 1), 2), 3). Liver transplantation is an established treatment for acute liver failure, but lack of donors is a very serious problem4). Emergency response until donor liver is transplanted
As ((bridge use), the development of a liver function assisting device that can assist the liver function of a living body suffering from liver failure is an important issue.
Hepatocytes also have a very strong regeneration capacity5). From this point of view, it is highly important to assist the liver function by using the liver function assisting device during the period until the liver that has fallen into acute liver failure regenerates and recovers its function 6).

Under such a background, a hybrid artificial liver (bioartificial liver) is used as one of the liver function assisting devices.
Is mentioned. A hybrid artificial liver is a combination of a bioreactor (physical material) and animal cells (biological material) 7). In order to enhance the function as an artificial liver, various measures such as the structure of the bioreactor, the method of filling animal cells, the method of circulating blood have been made 8).
~14) .

[0004]

The animal cells used for the hybrid artificial liver are roughly classified into two types.
One is to use isolated hepatocytes derived from xenogeneic animals such as pigs. Although these have a high liver function, it takes time and labor to procure cells, and the liver function cannot be maintained for a long time. Moreover, since it is a heterogeneous animal, there is a risk of immune rejection and unknown virus infection. What is being used in confrontation with these is human-derived cell lines such as HepG2. Although it has low liver function, it has characteristics that it can be maintained for a long period of time and that cell procurement is easy. Therefore, we considered to add some of the liver functions to this human-derived cell line.

To date, the present inventors have found that a human liver-derived cell line
We have succeeded in imparting to HepG2 cells the ability to remove ammonia, which is one of the toxic substances15). Therefore, it was desired to construct a bioartificial liver as a selective removal system for toxic substances by imparting to human liver-derived cell line HepG2 cells a drug-metabolizing ability which is considered to be the next most important function of the bioartificial liver. Therefore, H having the ability to metabolize drugs
Our aim is to construct the epG2 cell line and evaluate its function. In addition to the purpose of treating liver failure, these animal cells capable of metabolizing drugs can be used as a model for studying drug metabolism in human liver (a model for drug toxicity test or identification of metabolic pathway).

[0006]

[Means for Solving the Problems] By the way, drug metabolism reactions are roughly classified into first phase reactions and second phase reactions 16). In the first phase reaction, polar groups such as a hydroxyl group, a carboxyl group, and an amino group are generated by oxidation, reduction, hydrolysis, or the like,
Refers to the reaction introduced. Although these functional groups are relatively small polar groups compared to the second phase reaction, their polarization generally causes the drug to lose its affinity for the site of action. As a result, the pharmacological action or physiological action is reduced and the form is easily excreted. The second phase reaction is a reaction in which a larger substituent is introduced than in the first phase reaction. It is a conjugation reaction in which glucuronic acid, sulfuric acid, some amino acids, glutathione and the like are introduced. Many compounds already have a functional group such as a hydroxyl group, a carboxyl group, or an amino group in advance, but a compound that undergoes the second phase reaction after these functional groups are generated or introduced by the first phase reaction Many. The substituent in the conjugation reaction has a higher polarity than the functional group introduced in the first phase reaction, and thus is more susceptible to excretion and loses its pharmacological action or physiological action.

Approximately 80% of drug metabolism in humans is mediated by cytochrome P450 present in liver microsomes. This P
450 is responsible for the first phase reaction and is composed of various subfamilies. In patients with fulminant hepatitis, it is said that the metabolic ability of the first-phase reaction is particularly reduced, and it is especially clinically important among them, and the adult liver has about 30
It is said that P450 3A4 occupies a high expression level and metabolizes many kinds of drugs 17), 18). Therefore, the present inventor first focused on this P450 3A4 activity in evaluating the drug metabolizing ability in animal cells. Also P450 3A4
Attempts were made to express P450 3A4 in animal cells by constructing the expression vector of and introducing it into animal cells. Then, the function of the obtained cell line was evaluated, and the possibility of clinical application as a cell used for a bioartificial liver was examined to complete the present invention.

That is, the present invention relates to the following aspects. 1. A cell line transformed with a drug-metabolizing enzyme gene and an ammonia-metabolizing enzyme gene. 2. The cell line according to 1 above, wherein the drug-metabolizing enzyme gene is P450. 3. The cell line according to 2 above, wherein the drug-metabolizing enzyme is P450 3A4. 4. 4. The cell line according to any one of 1 to 3 above, wherein the ammonia metabolizing enzyme gene is a glutamine synthetase gene. 5. The cell line according to any one of 1 to 4 above, wherein the cell is derived from a mammal. 6. 6. The cell line according to 5 above, wherein the cell is derived from human liver. 7. 7. The cell line according to 6 above, wherein the cells are derived from human hepatocytes. 8. The cell line according to 7 above, wherein the cell is HepG2. 9. 9. The cell line according to any one of 1 to 8 above, wherein the cell is a recombinant ammonia metabolism human hepatocyte cell line. 10. 9. The cell line according to any one of 1 to 8 above, wherein the drug metabolizing enzyme gene and the ammonia metabolizing enzyme gene are introduced into a common expression vector. 11. The cell line according to 10 above, wherein the expression vector is a plasmid. 12. The expression vector is a mammalian cell expression vector having two or more independent multiple cloning sites,
The cell line according to the above 10 or 11, which is a plasmid. 13. The cell line according to the above 12, which is the expression vector pBudCE4. 14. More than about 200 pmol / min P450 per mg protein
The cell line according to any one of 1 to 13 above, which exhibits activity. 15. More than about 490 pmol / min P450 per mg protein
15. The cell line as described in 14 above, which exhibits activity. 16. The cell line according to the above 14 or 15, wherein the P450 activity per mg of protein is maintained at about 450 pmol / min for 80 days. 17. A liver function assisting device, comprising the cell line according to any one of 1 to 16 above. 18. The cell line according to any one of 1 to 17 above, which is further transformed with another drug-metabolizing enzyme gene. 19. A liver function assisting device, which uses the cell line according to any one of 1 to 17 above. 20. Further, a liver function assisting device comprising the cell line according to the above 19, which uses a cell line of a different type. 21. 21. The hepatic function assisting device according to 20 above, wherein the different type of cell line is derived from human liver non-parenchymal cells. 22. 20 or 2, which is a hybrid artificial liver
1. The liver function assisting device according to 1. 23. 23. The liver function assisting device according to any one of 20 to 22 above, which comprises a circulating culture device. 24. A drug metabolism assay system using the cell line according to any one of 1 to 17 above. 25. a) The cell line according to any one of 1 to 17 above is cultured, b) the substance to be measured is added to the medium and further cultured for a predetermined period, c) the supernatant is collected, and d) in the collected supernatant. The concentration of each of the target substance to be measured and the metabolized target substance to be measured is determined and the concentration ratio between the two is determined, and e) the target substance to be measured and the test substance at an appropriate concentration are added to the medium and further cultured for a predetermined period, A drug metabolism assay system, which comprises repeating the above steps c) and d) and g) measuring the effect of the test substance on the metabolism of the substance to be measured from the change in the concentration ratio. 26. a) culturing the cell line according to any one of 1 to 17 above, b) adding nifedipine to the medium and further culturing for a predetermined period of time, c) collecting the supernatant, and d) nifedipine in the collected supernatant. And the concentration of oxidized nifedipine are determined and the concentration ratio between the two is determined.e) Nifedipine and the test substance at an appropriate concentration are added to the medium and the culture is continued for a predetermined period of time.f) Steps c) and d) above are repeated. , A) A drug metabolism assay system, which comprises measuring the effect of a test substance on the production of oxidized nifedipine from changes in the concentration ratio. 27. a) Culturing the cell line according to any one of 1 to 17 above, b) adding midazolam to the medium and further culturing for a predetermined period of time, c) collecting the supernatant, and d) midazolam in the collected supernatant. And the concentration of oxidized midazolam are determined and the concentration ratio between the two is determined.e) Midazolam and the test substance at an appropriate concentration are added to the medium and the culture is continued for a predetermined period of time.f) Steps c) and d) above are repeated. , B) A drug metabolism assay system, which comprises measuring the effect of a test substance on the production of oxidized midazolam from changes in the concentration ratio.

As the drug-metabolizing enzyme, any one known to those skilled in the art, for example, various enzymes belonging to cytochrome P450, specifically P450 3A4 (CYP3A4),
CYP2C, CYP1A2, CYP2E1, CYP2D
6 and CYP2A6 etc. can be used alone or in combination of two or more. Of these, P450 3A4 accounts for about 30% expression in the adult liver and can metabolize about half of the clinically used drugs. This P450
P450 2D6 and P450 2 which metabolize many drugs next to 3A4
It is also considered important to express C. Especially P4
50 2D6 is also attracting attention from the viewpoint of genetic polymorphism. Patients without this P450 2D6 activity (PM)
Is as low as 0.8% in Japanese, but 5 to 6 in white.
It is said that not only the idea of developing a bioartificial liver for treatment of patients with acute liver failure but also PM
It is also important from the perspective of developing a bioartificial liver for the treatment of Examples of ammonia metabolizing enzyme genes include:
For example, a glutamine synthetase gene derived from CHO cells can be mentioned. Any of these genes can be easily obtained or prepared by those skilled in the art from a commercially available or a cDNA library owned by a public institution, a vector or the like by a cloning means such as PCR.
The nucleotide sequences of these genes are disclosed in various documents. The cell line to be transformed is preferably derived from a mammal, for example, a cell line derived from human liver is more preferable, and an example of such a cell line is a HepG2 cell line. Such cells can be obtained from various public institutions (cell banks). Furthermore, for the purpose of enhancing drug metabolism activity, the cell line of the present invention obtained by transformation can be cloned,
The cell line thus obtained by cloning is also within the scope of the present invention. The drug-metabolizing enzyme gene and the ammonia-metabolizing enzyme gene may be carried in separate expression vectors and transformed separately, but it is efficient and convenient to carry them in a common expression vector. Although any vector known to those skilled in the art can be used as such an expression vector, a mammalian cell expression vector having two or more independent multiple cloning sites, for example, the expression vector pBudCE4 is preferable. In addition, each operation such as introduction of each gene into an expression vector and transformation of a cell line with the expression vector can be easily performed by any method and means known in the art. The cell line of the present invention may be further transformed with another type of drug-metabolizing enzyme gene. Examples of such a drug-metabolizing enzyme include a drug-metabolizing enzyme that is responsible for the second-phase reaction as well as the first-phase reaction by P450. For example, glucuronic acid conjugation is performed using UDP-GT (UDP-glucuronyltransferas
It is considered that it is effective to further introduce this UDP-GT expression vector because it is carried out according to e). The liver function assisting device of the present invention may have any configuration known to those skilled in the art, but a hybrid artificial liver type including a circulating culture device is preferable. In such a device, the cell line of the present invention is used as the biological material, but other types of cells can be further used as the biological material. The drug metabolism assay system of the present invention is characterized by using the above-mentioned cell line, and a metabolic reaction (for example, oxidation reaction, hydroxylation, etc.) of a measurement target substance (standard substance) such as nifedipine and midazolam by the cell line. reaction)
It can be carried out by measuring the effect of the test substance on.

Hereinafter, the present invention will be described in detail with reference to Examples, but the technical scope of the present invention is not limited to these.

[0011]

Example 1 Experimental Materials and Methods Unless otherwise specified, Wako Pure Chemical Industries or Nacalai Tesque special grade reagents were used. 1. Animal cell culture method 1. 1. Host animal cell HepG2 (RIKEN Cell Bank RCB0459) derived human hepatocellular carcinoma growth morphology epithelial-like

[0012] 1. 2． Animal cell culture medium Medium is 0.22 μM membrane filter (Falcon; 71
It was used after filter sterilization using 05).・ RDF (Gln +) RDF (Gln +) Medium composition RDF (HO) powder (NIPPON PHARMACEUTICAL) 8.44 g Glucose 2.58 g NaHCO 3 2.0 g Glutamine 0.333 g Penicillin G 58.8 mg Streptomycin 120 mg Milli-Q water 1 L Note 1) Use At the time of fetal bovine serum
(FBS); Gibco) was added in an amount corresponding to 10% (vol.%) Of the medium volume. Note 2) As needed, Zeocin (Invitrogen; R250-01)
Was added.・ RDF (Gln−) RDF (Gln−) Medium composition RDF (HO) powder (NIPPON PHARMACEUTICAL) 8.44 g Glucose 2.58 g NaHCO 3 2.0 g Glutamic acid 0.336 g NH 4 Cl 0.122 g Penicillin G 58.8 mg Streptomycin 120 mg Milli-Q water 1 L Note 1) The composition of glutamic acid and NH 4 Cl was determined so that the molar number was the same as that of glutamine in RDF (Gln +) medium. Note 2) RDF (HO) medium is a custom-made product of Nippon Pharmaceutical Co., Ltd.
Glucose and glutamine are removed from RDF medium. Note 3) Prior to use, FBS dialyzed in advance was added in an amount corresponding to 10% (vol.%) Of the medium volume. * 4) When MSX is required in the medium ((used for obtaining high MSX resistant strains), MSX (Sigma; M-5
379) was added. Note 5) If necessary, geneticin ((G418) (Sigma
G5013) and Zeocin were added.・ Serum culture medium Serum culture medium composition NaHCO 3 0.2 g Penicillin G (1700 units / mg) 5.88 mg Streptomycin 12 mg FBS 100 mL Dialysis serum Dialysis cellulosic tube (Sanko Junyaku ;; Size 27/32) 2
After soaking in% (w / v) NaHCO 3 and 1 mM EDTA (pH 8.0) for 10 minutes and autoclaving, cool to 4 ° C, add serum to it, and use 10 times the volume of dialysate for 30 minutes. → Dialysis was performed for 1 hour → 2 hours → 3 hours → 4 hours → overnight. Serum dialysate NaCl 8.0 g KCl 0.2 g Na 2 HPO 4 ・ 12H 2 O 2.9 g KH 2 PO 4 0.2 g Kanamycin 32 mg Streptomycin 120 mg Distilled water 1 L

1. 1. 3． Basic Items and Procedures for Animal Cell Culture 1. 1. 3． 1. Cell culture vessels As cell culture vessels, three types of T-flasks ((Sumitomo Bakelite; MS-20050 (bottom area 25 cm 2, volume 50 mL), MS-
21250 (75 cm2, 250 mL), MS-20800 (225 cm2, 800 m
L) (hereinafter abbreviated as small T-flask, medium T-flask, and large T-flask)) or a 100 mm dish (Corning; 25020). Now, for the small T-flask and 100 mm dish,
Usually, 10 mL of medium, 30 mL of medium T-flask, and 90 mL of medium T-flask were added. Small T-flasks were used for subculture of ordinary cell lines. A 100 mm dish was used in the culture experiment.

1. 3． 2． As a general rule, passaging the cells was done every other day (confirming that the cells were confluent). 1) Remove the old medium by pipetting and remove the following volume of 0.
A 25% trypsin solution was added. Small T-flask ... 5 mL, medium T-flask ... 15 mL, large T-flask ... 30 mL 2) Incubated at 37 ° C for about 10 minutes. 3) When the cells became detached and rounded, an equivalent amount of fresh medium was added and the mixture was pipetted well (the reaction to detach the cells is stopped at this point because trypsin inhibitor is normally contained in serum). 4) The cell suspension was transferred to a centrifuge tube, centrifuged at 80 × g. For 10 minutes, and the supernatant was removed by suction with a pipette. 5) Add fresh medium into the centrifuge tube to suspend the cells, and
-The flask was inoculated with the appropriate amount.

0.25% trypsin solution NaCl 8 g KCl 0.2 g Na 2 PO 4 .12H 2 O 2.9 g KH 2 PO 4 0.2 g TRYPSIN (Difco 1: 250) 2.5 g Milli-Q water 1 L

[0016] 1. 3． 3． Cryopreservation of cells For cryopreservation of cells, 10% dimethyl sulfone was added to the medium.
Suspend cells in a solution containing oxide (DMSO), dispense 1 mL each into a serum tube, place in BICELL (Nihon Freezer), freeze at -80 ° C overnight, and then store in liquid nitrogen. did. Thaw the cells in warm water at 37 ° C to dissolve the medium, then transfer the cell suspension to a centrifuge tube containing 10 mL of medium,
After centrifugation for 10 minutes at 80 × g., The supernatant was pipetted off and the cells were inoculated into a new T-flask.

1. 3． Four . Cell concentration measurement method (measurement by dye exclusion method) 19) The concentration of live cells and total cells was measured by the dye exclusion method using trypan blue. This is a trypan blue solution
0.2% (w / v) and 4.25% (w / v) NaCl aqueous solution were mixed at a ratio of 4: 1, and an equal volume of cell suspension was mixed with this solution.
Place one drop ((about 15 μL) on the hemocytometer ((ERMA 4296),
This is a method of immediately measuring the viable cell concentration and total cell concentration by microscopy. On the hemocytometer, the cells on the right and bottom sides were omitted at the time of measurement. Here, the depth of the hemocytometer was corrected according to the test value attached to the hemocytometer, the volume on the hemocytometer was calculated, and the cell concentration was calculated 20).

2. Drug metabolism activity using HPLC (P450 3A
4 Activity) Measuring system 2. 1. Method for measuring concentration of substrates and metabolites using HPLC 2
1) ~ 24) The drug metabolism activity (P450 3A4 activity) is a metabolite that uses the hormone testosterone (Daiichi Pure Chemicals; UC-339, molecular weight; 288.4), which is specifically metabolized only by P450 3A4, as a substrate. 6 β-hydroxytestosterone (Daiichi Pure Chemicals
;; UC-282, molecular weight; 304.4) was evaluated by HPLC. The internal standard method was used as the quantification method when performing HPLC. Here, androste is used as the internal standard substance.
nedione (4-Androstene-3,17-dione) (Daiichi Pure Chemicals
;; UC-300) was used. First, as standard concentration,
Metabolites and internal standard substances were prepared to 100 μM each, and the concentrations of substrates and metabolites were calculated based on the peak areas of the HPLC chart.

HPLC condition HPLC device ... Shimadzu Liquid Chromatograph System;
LC10AD; Column used ... C18 column Inertsil ODS-3V (4.
6 × 150 mm, 5 μm, GL Sciences Inc.); buffer used… solution A) methanol: distilled water = 45: 55 B
Liquid) Methanol: distilled water = 90:10. In the preparation of Buffer, methanol for HPLC (Nacalai Tesque) and distilled water were mixed at respective ratios, and then degassing was performed appropriately. Flow rate: 1.0 ml / min; Column temperature: Room temperature; Measurement wavelength: 254 nm; HPLC gradient condition for P450 3A4 activity

[0020]

[Table 1]

2. 2． Quantification of total cellular protein P450 3A4 activity is generally pmol 6 β-hydroxytestost
It is expressed in the unit of erone formed / min / mg protein. When the drug metabolism activity is determined using the cell culture supernatant, the total cell protein amount (mg protein) must be measured. Here, the total cell protein amount is calculated using the BCA Protein Assay Reagen.
t Kit (Pierce; 23225) was used for measurement.
The quantification method followed the attached manual. The procedure for adjusting the cell lysate (crude enzyme solution) in the measurement of total cell protein amount is shown below. The procedure is shown below. 1) Use a known amount of cells (medium for 100 mm dish culture)
5 ml was used as a standard), 500 μl of this was transferred to a centrifuge tube, and centrifuged at 1000 xg for 10 minutes at 4 ° C. 2) Remove the supernatant and remove 100 mM potassium phosphate buffer (p
H7.4) and the mixture was centrifuged at 1000 xg for 10 minutes at 4 ° C. 3) After ultrasonic treatment for 5 seconds using an ultrasonic cell destruction device (Kaiyo Denki T-A-4200), ice cooling was performed for 1 minute, and this was repeated 4 times. 4) After centrifugation at 14000 xg for 10 minutes at 4 ° C, the supernatant was used to quantify the total cell protein amount.

2. 3． Pretreatment of cell culture supernatant (sample) 25) When measuring substrate and metabolite concentrations in cell culture supernatant using HPLC, the sample pretreatment column Sep-Pak P
Samples were purified using lus C 18 (Waters). The procedure is shown below. The flow rate when applying to the column was set to 2 to 4 ml / min. 1) 2 ml of 100% methanol was applied to the column twice. 2) 2 ml of distilled water was applied to the column twice. 3) 2 ml of the sample was applied. 4) The column was washed by applying 2 ml of distilled water to the column. 5) Apply 2 ml of 100% methanol to elute the target substance.

3. P4 in animal cells
50 3A4 Activity measurement method 21), 23) 1) Using 10 mL of medium RDF (Gln + or Gln −),
1 × 10 7 cells were seeded in a 0 mm dish. Culture is 5
It was performed for 16 hours under% CO 2 and 37 ° C. 2) Replace the medium (10 mL of medium), and use testos as the substrate.
Terone (dissolved in 100% methanol to 100 mM) was added to a concentration of 100 μM. 3) After culturing at 37 ° C in 5% CO 2 for 2 hours, 2 mL of this culture supernatant was used to measure the activity. 4) After completion of the culture, the cells were peeled off by trypsin treatment and the total cell protein amount was measured.

[0024]

[Embodiment 2] 4. When applying cultured animal cells containing rifampicin, a human liver drug-metabolizing enzyme inducer for improving endogenous P450 3A4 activity, to the wild strain He
pG2 cells and genetically modified ammonia-metabolizing HepG2 cells (GS-HepG2) constructed by the present inventor.
Cells, endogenous P450 3A4 in MSX300 μM resistant strain)
Tried to improve the activity of. So far, in HepG2 cells, dexamethasone, phenobarbital, 3-methylcholanth
It has been reported that when drugs such as rene, prednisolone, carbamazepine and rifampicin are added to the medium, the drug-metabolizing ability is increased by drug induction26), 27), 28). Therefore, among these drug inducers, we aimed to improve the endogenous P4503A4 activity by adding rifampicin, which is said to be the most effective inducer of CYP3A. 1) RDF medium (containing 10% FBS) was added to 100 mm dish so that the final volume was 10 mL. 2) Wild strain HepG2 and genetically modified GS-HepG2 cells (HNAA-300A cell line) were seeded at 1 × 10 ₇ cells. 3) Incubated at 37 ° C, 5% CO ₂ for 12 hours. 4) Medium exchange. 10 mL of RDF medium containing 10% FBS or without serum was added. 5) The cells were cultured at 37 ° C and 5% CO ₂ for 24 hours. 6) Medium exchange. rifam dissolved in dimethylsulfoxide (DMSO)
Add picin to serum-free RDF medium to a final concentration of 10,50,100,200,300,500 μM. As a control, DMSO alone (0.1%) was added to serum-free RDF medium. 7) After culturing at 37 ° C in 5% CO ₂ for 24 hours, the medium was replaced and rifampicin at each concentration was added. 8) 7) was carried out for 6 days (medium was changed daily for 6 days), and then 200 μM testosterone was added to the medium, and P450 3
A4 activity was measured.

FIG. 1 shows the results obtained by the above-mentioned culture experiment in which the human liver drug-metabolizing enzyme inducer rifampicin was added to the wild strain HepG2 cells. This shows the P450 3A4 activity of wild-type HepG2 cells with and without FBS in the medium and at various rifampicin concentrations. The P450 3A4 activity of wild-type HepG2 cells showed a value of 0.6 pmol / min / mg-protein without drug induction.
The highest P450 3A4 activity was obtained by inducing rifampicin at 100 µM in the medium containing FBS, and the activity value was 2.3 pmol / min / mg-protein. By comparing the case of containing FBS in the medium and the case of not containing FBS, it was found that endogenous P450 3A4 was easily induced in the culture containing FBS. FBS (serum) contains various important factors such as growth factors.
Therefore, in FBS-free culture, important factors for survival have been depleted, and it was thought that this may be due to the slow response of cells to changes in the external environment.

Furthermore, genetically modified ammonia metabolism HepG2
FIG. 2 shows the results obtained by a culture experiment in which human liver drug-metabolizing enzyme inducer rifampicin was added to cells (GS-HepG2 cells, MSX300 μM resistant strain). This includes HNAA with and without dialyzed serum and at each rifampicin concentration.
P450 3A4 activity of −300A cell line. As can be seen from this figure, the HNAA-300A cell line had almost the same activity as the wild-type HepG2 cells in the state where drug induction was not applied. However, it was confirmed that drug induction by the addition of rifampicin was less likely to induce P450 3A4 than wild-type HepG2 cells. The HNAA-300A cell line is the wild type HepG2
After introducing pBK-CMV-GS vector into cells, MSX and
It was selected using G418. Considering that it can survive in such a harsh environment, HNAA
Since the −300A cell line is less sensitive to the outside world, P45 by rifampicin was compared to wild-type HepG2 cells.
We suspected that induction of 0 3A4 would be difficult.

[0027]

Third Embodiment 5. Construction of P450 3A4 expression vector 5． 1. P450 3A4 expression vector (pBudCE-GS-CYP3A4)
Outline of construction 5． 1. 1. Plasmid used pBudCE4 (Invitrogen; V532-
20) 29) was used. This is a multicloning site (MCS
) And owns humans upstream of each MCS.
cytomegalovirus (CMV) immediate-early promoter and human elongation 1 α-subsuit (EF-1 α) promote
has r. Zeocin can be selected as a marker gene in E. coli and animal cells. The outline of the construction of the P450 3A4 expression vector is shown in Table 2 below.

[0028]

[Table 2]

[0029] 5. 1. 2． Preparation of used plasmid Five . 1. 2． 1. Used strain E. coli TOP10 (invitrogen; C615−00)

5 1. 2． 2． Medium (LB-Zeocin) 1 g of Bacto-tryptone (Difco) in 100 mL of deionized water,
After dissolving 0.5 g of Bacto yeast extract (Difco) and 0.5 g of NaCl, it was adjusted to pH 7.0 and autoclaved. For plates, 2 g of Agar was added. After autoclaving, when the temperature reaches 55 ° C, Zeocin is added at 50 μg / m.
Added to be L.

5 1. 2． 3． Transformation of E. coli by the calcium chloride method and preparation of competent cells 1) E. coli to be transformed in 5 mL of LB medium overnight at 37 ° C.
The cells were shaken and cultured. 2) Inoculate 2 mL of the preculture solution into 40 mL of LB medium and incubate at 37 ° C.
Incubated for 2 hours. 3) The cells were allowed to stand for 10 minutes or longer on ice and then centrifuged at 4 ° C, 6000 xg for 5 minutes to collect the cells. 4) Suspend the precipitate in 20 mL of ice-cold 50 mM CaCl 2 and
It was left at ℃ for 20 minutes. 5) After collecting the cells by centrifugation, the cells were suspended in 4 mL of 50 mM CaCl 2 and dispensed in 200 μL aliquots.・ Appropriate amount (about 0.01 μg) of DNA solution was added to 200 μL of competent cells and left for 1 hour on ice. 42
After heat shock for 90 seconds at ℃, quench with ice water,
0.8 mL of LB medium was added, and the mixture was cultured at 37 ° C for 1 hour with shaking. Of this culture solution, 100 μL was spread on LB-Zeocin medium and cultured overnight at 37 ° C.

5. 1. 2． Four . Plasmid from E. coli
Preparation of DNA (alkaline extraction method) 5 mL of LB-Ampicilin liquid medium was inoculated with the Escherichia coli transformant and shake-cultured at 37 ° C for 16 hours. Culture medium 1.5
About 1 mL was collected and centrifuged at 12000 xg for 2 minutes to collect the cells. The precipitate was vortexed and then solutied.
on I 100 μL completely suspended, solution II 200 μL
Was added, gently mixed by inversion 3 to 4 times, and left for exactly 5 minutes on ice. 150 μL of pre-cooled solution III
Then, the mixture was vigorously mixed and left in ice for 5 minutes. 12000
After centrifugation at × g for 5 minutes, the supernatant was transferred to a new sample tube. This supernatant was subjected to phenol-chloroform extraction, ethanol precipitation, and then TE 50 μL containing 20 μg / mL Dnase free RNase.
Dissolved in. solution I: 50 mM Glucose, 25 mM Tris-Cl (pH
8.0), 10 mM EDTA solution II: 0.2 N NaOH, 1 %% SDS (prepared as needed) solution III: Acetic acid aqueous solution containing 5 M potassium acetate

5. 2． Gene-related basic operations 5． 2． 1. Agarose gel electrophoresis 0.8% agarose gel using 1/2 × TAE buffer (TaKaRa
I went to Agarose LO3). A small electrophoresis tank (Advance Co. Ltd .; Mupid2) was used as the electrophoresis tank. Samples were usually prepared by adding 1/10 volume of x10 electrophoresis stain solution to DNA solution. The electrophoresis was performed at a constant voltage of 100 V for about 40 minutes. After migration,
Gel is treated with ethidium bromide (EtBr) in water (0.5 μg / ml
) For 10 minutes and then transilluminator (Ultra
The DNA band was observed with Violet C62). The photographs were taken with a Polaroid® camera using a UV filter and a red filter.

5. 2． 2． To the solution containing ethanol-precipitated DNA, add 1/10 volume of 3 M sodium acetate solution (pH 5.2), and add 2.5 volume of 100 volume.
% Ethanol was added and mixed, and the mixture was left at room temperature for 20 minutes.
This was centrifuged at 18,000 xg for 10 minutes, and the supernatant was removed. Add an appropriate amount of 70% ethanol to this precipitate, and add 18000 xg.
After centrifuging for 10 minutes, remove the supernatant, dry the precipitate under reduced pressure, and wash with an appropriate amount of sterile water or sterile TE buffer (10 mM T
It was dissolved in ris-Cl (pH 7.5), 1 mM EDTA).

5 2． 3． The phenol-chloroform extracted DNA solution was saturated with TE buffer, and an equal volume of a phenol-chloroform-isoamyl alcohol (25: 24: 1) mixed solution was added and mixed well. This was centrifuged at 18000 xg for 10 minutes, and the upper layer was transferred to a new sample tube.

5 3． P450 3A4 gene by PCR and
Preparation of GS gene ・ Equipment used GeneAmpR PCR System 2400 (PERKIN ELMER) ・ template P450 3A4 gene: pLNCX-CYP3A4 (Els MD
(donated by Dr. e Grone), PBK- for the GS gene
CMV-GS was used. The primers used are shown in Table 3 below.

[0037]

[Table 3]

In designing the primers, the Kozak sequence (A / G NN ATG G) was referred to so that translation in animal cells could be initiated more correctly. The composition and conditions of each PCR reaction are shown in Table 4 below.

[0039]

[Table 4]

5 Four . pBudCE4 vector (invitrogen)
And P450 3A4 gene and GS gene which are inserts
ligation 5． 1. 2． Four . PBudCE4 vector prepared in Section 5 and 5.
3． It was decided to ligate using the P450 3A4 gene and the GS gene prepared in. This process was outsourced to Takara Shuzo Co., Ltd. Genetic Analysis Center. As a result,
Obtained P450 3A4 expression vector (pBudCE-GS-CYP3A4)
Are shown in Table 5 below. This vector contains pLNCX-CYP3A4 at two multicloning sites of pBudCE4 vector.
CYP3A4 (gene of P450 3A4) and pBK-CMV-GS-derived GS gene were inserted.

[0041]

[Table 5]

Furthermore, 5. 3． P450 3A4 gene and GS gene were amplified by PCR according to
Electrophoresis was performed using a% agarose gel. The result is shown in FIG. Lane is P450 3A4 gene (about 1.5 kbp),
Lane is GS gene (about 1.1 kbp), lane is λ / Hind
III marker. From this, the target gene P4
Acquisition of 50 3A4 gene and GS gene was confirmed. P450 3
P450 3A4 gene inserted in A4 expression vector (CYP 3
The sequence results of A4) are shown in Table 6 below. When compared with the nucleotide sequence of CYP3A4 registered in the database33), the 6th nucleotide after the start codon (ATG) was A (adenine) instead of C (cytosine). However, when translated into a protein, leucine was the constituent amino acid in both (CTC and CTA), so P450 3A4 inserted into the vector
Is considered to have the original activity.

[0043]

[Table 6]

Further, G inserted into the P450 3A4 expression vector
The results of S gene sequencing are shown in Table 7 below. GS
For genes, the GS of pBK-CMV-GS used as the template
It was exactly the same as the gene sequence 30). pBK-CMV-GS
-Transfected CHO cells (CN9-500-4 cell line) and HepG2
Since GS activity has been confirmed in cells (HNAA-300A cell line), it is considered that the GS inserted into this vector also has the function of the original protein.

[0045]

[Table 7]

[0046]

Fourth Embodiment 6. Introduction of P450 3A4 expression vector into animal cells The P450 3A4 expression vector was introduced into wild-type HepG2 cells (ATCC No.
HB-8065) and a recombinant ammonia metabolizing HepG2 cell (HNAA-300A strain) constructed by the present inventor were tried. When introducing a P450 3A4 expression vector into animal cells, this vector must be prepared in high purity. So Wizard PureFection Plasmid
A DNA Purification System (A2160; a method of removing high-purity plasmid DNA by adsorbing DNA on a magnet after removing endotoxin etc.) was used. The method was in accordance with the attached manual (method is omitted). Transfection using the high-purity P450 3A4 expression vector obtained above
I went. The transfection method is the liposome method (TaKaRa,
Trans IT Polyamine Transfection Reagents) was used. 1) 4 × 10 ⁵ cells were seeded on a 35 mm dish and cultured at 37 ° C. overnight. As a medium, in HepG2 ・ CHO-K1, FBS-containing RDF (Gln +) medium was used, and HNAA-300A ・ CN9-500 was used.
For -4, RDF (Gln −) medium containing dialyzed serum was used. 2) The next day, add 100 μl of serum-free medium to TransIT Transfectio.
20 μl of n Reagent was added and mixed well with a vortex. 3) Incubated at room temperature for 10 min. 4) Add 3 μg of vector DNA to this and gently pipetti.
ng 5) Incubated at room temperature for 10 min. 6) What was prepared in 5) was added to the cell culture medium prepared in 1). The dish was gently shaken to mix. 7) Incubated for 72 hours at 37 ° C. 8) The product prepared in 7) was trypsinized and peeled off, and the whole amount was seeded in a small-T flask (bottom area 25 cm 2). As a medium, RDF (Gln + and Gln −) medium containing no Zeocin was used for passage. 9) After becoming confluent in a small-T flask,
Subcultured in selective medium containing Zeocin. Zeo in selective medium
The cin concentration was 200 μg / ml for HepG2 and HNAA-300A.

The He expressing P4503A4 thus obtained was expressed.
Table 8 shows the results of measuring the P450 3A4 activity of pG2 cells (CYP3A4-GS-HepG2). In the table, the cells are referred to as "P4
50 3A4-HepG2 ”(hereinafter the same in each table of the present specification).

[0048]

[Table 8]

As is clear from Table 8, P450 3A4 introduced H
The activity level in epG2 cells is about 800 times higher than that of the wild type, 4
It was 90 pmol / min / mg-protein. Compared with the activity in P450 3A4-expressing CHO cells (P450 3A4-CHO cell line), the activity in P450 3A4-HepG2 cell line was about 20 times higher. P450 3A4 introduced CHO
P450 3A in P450 3A4-transfected HepG2 cells compared to cells
4 The activity was more than 20 times higher than that of CHO cells, which is an environment that supports the reaction mechanism of P450 in HepG2 cells (for example, in the oxidation / reduction reaction by P450, P
It is thought that this is because the electron supply to P450 from 450 reductase is actively carried out) and that it remains in the cell.

P450 of primary hepatocytes cultured here for 24 hours
The 3A4 activity value is reported to be 252.8 pmol / min / mg-protein 35). In the case of using rat primary hepatocytes, the P450 3A4 activity after culturing for 4 hours was 407 pmol / min / mg-pr.
otein, activity after culturing for 24 hours is 158 pmol / min / mg-prot
36). From this, P450 3A4 introduced Hep
It can be said that the G2 cell line had an activity equal to or higher than that of the primary hepatocytes. However, the P450 3A4 activity of human hepatocytes in vivo varies from 1000 to 1,000, although there are individual differences.
It is said to be about 1500 pmol / min / mg-protein37),
Furthermore, for clinical application to humans, P450 3A4-HepG2 cells
We wanted to further enhance P450 3A4 activity.

[0051]

[Fifth Embodiment] 7. Gene amplification method using the GS gene system in P450 3A4 expressing animal cells In order to increase the P450 3A4 activity in P450 3A4 expressing animal cells, gene amplification in the GS gene system was performed. 1) In a small-T flask (bottom area 25 cm 2), use Hep
For G2 cells, approximately 5 × 10 ⁵ cells were seeded in a selective medium containing 200 μg / ml Zeocin and each concentration of MSX, and the medium was exchanged until confluent. 2) When confluent, the cells are detached and MSX (Met
hionine Sulfoximine) approx.
The process of seeding 5 × 10 ⁵ cells was repeated to attempt gene amplification in a heterogeneous state.

As a result, resistant strains of each MSX concentration were obtained in P450 3A4-transfected HepG2 cells. Next, P450 3A4 activity was measured for each resistant strain (3). The result is shown in FIG. Since MSX may have affected P450 3A4 activity, MSX was not added to the medium in the passage before the activity measurement. In addition, MSX was not added to the medium when measuring the activity. We aimed to improve P450 3A4 activity using the GS gene amplification system by adding MSX, but all resistant strains showed almost the same activity value. Based on this, P45 using gene amplification in the GS gene was used.
It can be said that the 0 3A4 activity could not be increased.
Here, we considered as follows why each cell line acquired MSX resistance, but P450 3A4 activity did not increase. 1) P450 3A4 activity did not increase because gene amplification occurred not in the GS gene introduced into the P450 3A4 expression vector but in the endogenous GS gene (in this case, P450 3A4 activity was unchanged and GS activity was Will rise). 2) Gene amplification occurred in the GS gene introduced into the P450 3A4 expression vector, but the P450 3A4 gene was not amplified for some reason (in this case, P450 3A4 activity was unchanged and GS activity was increased). 3) Gene amplification itself in the GS gene system was not successful (in this case, neither P450 3A4 activity nor GS activity was changed). That is, it is considered that MSX resistance was acquired by a mechanism other than gene amplification (eg, membrane mutation).

[0053]

[Sixth Embodiment] 8. GS in P450 3A4-transfected HepG2 cells
Activity measurement method 32) To verify these things, each MSX concentration-resistant P450 3
We decided to evaluate the GS activity of A4-transfected HepG2 cells. 8． 1. GS activity measurement principle GS catalyzes the γ-glutamyl transfer reaction. That is,
γ-glutamylhydroxyamate is produced from hydroxylamine, and this γ-glutamylhydroxyamate is ferric ch
Addition of loride gives a characteristic brown color. This is hydroxy
This is the principle of measuring the activity of glutamine synthetase using lamine. Using this reaction, GS activity was measured.

8 2． GS activity measurement method 1) After washing the cell suspension with TBS once, 500 μL of Imid
Suspended in azole buffer. 2) Add 5 μL of 10% β-mercaptoethanol ((antioxidant). 3) Ultrasonic microbial cell disruption device (T-A-4200; Marine Electric Co., Ltd.) for 1 second after ultrasonic treatment for 10 seconds. The cells were disrupted by repeating ice-cooling 3 times. 4) 5 μL of 10% β-mercaptoethanol was added again. 5) 5 μL of 100 mM phenylmethylsulfonylfluoride (PMSF; ethanol solution) was added. 6) 5 μL of 200 mM Pepstatin A (ethanol solution) was added. (PMSF and Pepstatin A are protease inhibitors) 7) Centrifuged at 18,000 xg. For 5 minutes at 4 ° C. 8) The supernatant was transferred to a new eppen and the volume was measured. 9) After adding the reaction substrate solution to the crude enzyme solution and vortexing,
The reaction was carried out at 37 ° C for exactly 15 minutes. 10) Add 0.75 mL of FeCl ₃ solution, centrifuge at 18,000 × g. For 5 minutes, transfer the supernatant to a cuvette, and put it in A 535.
The absorbance was measured with. 11) The activity was determined by subtracting the blank value from the measured value. Under this measurement condition, when A 535 = 0.340, 1 unit was defined 32). Table 9 shows γ-glutamyl transfer
The composition of the activity measuring reagent is shown in Table 10, and the composition of the FeCl ₃ solution is shown in Table 10.

[0055]

[Table 9]

[0056]

[Table 10]

Since the purpose is to measure GS activity, the absorbance values other than the GS enzyme reaction should be excluded. Therefore, the blanks shown in Table 11 were taken.

[0058]

[Table 11]

Note) b 1, b 2 and b 3 indicate the type of blank. Further, in the above table, ◯ indicates that the addition was made, and x indicates that it was not added. b 1 is without substrate, b 2 is without enzyme, b 3 is without substrate and enzyme. The liquid volume is
Ultrapure water was added to make 500 μL. Finally, the blank during enzyme activity measurement was calculated as follows. blank = (b 1) ＋ (b 2) − (b 3)

FIG. 4 shows P450 in culture using a synthetic medium.
The results of GS activity (in synthetic medium) of 3A4-transfected HepG2 cells (each MSX concentration resistant strain) are shown. Each MSX concentration tolerance P450 3A4
No difference was found in GS activity in transfected HepG2 cells. This indicates that P450 3A in each MSX concentration resistant strain was
4 There was no difference in activity, probably because the gene amplification in the GS gene system did not occur successfully.
It is considered that each of these MSX concentration resistant strains acquired resistance by a mechanism other than gene amplification (for example, membrane mutation). In the future, if we consider increasing P450 3A4 activity using a gene amplification system, we must use a gene amplification system other than the GS gene (CAD gene system, etc.).

[0061]

Seventh Embodiment 9. Method for measuring P450 3A4 activity in HepG2 cells introduced with P450 3A4 in serum 30) By the way, in clinical application, porcine or human plasma comes into contact with transgenic animal cells in Cygnus (cell culture device). Therefore, as a form closer to that of application, the drug metabolizing capacity (P450 3A4 activity) of P450 3A4-introduced HepG2 cells is evaluated by the following method in culture using serum, which is a component closer to plasma, rather than synthetic medium. It was to be. The activity of each MSX concentration resistant strain was also measured in the same manner. 1) Seed 1 × 10 ⁷ cells in a 100 mm dish. Medium is RD
F (Gln +) was used. The medium volume is 10 mL. 5% CO 2, 37
It was incubated at ℃ for 16 hours. 2) The medium was removed, 10 mL of serum (FBS) was added, and the substrate, testosterone, was added so that the concentration was 100 μM. 3) Incubate for 2 hours in 5% CO 2 and 37 ℃. This culture supernatant
The activity was measured using 2 mL. 4) After completion of the culture, the cells were peeled off by trypsin treatment and the total cell protein amount was measured.

The obtained results are shown in FIG. P450 3A4 activity was 200 pmol / min / mg-prote in all cell lines.
The values before and after in are shown. This is P45 measured in synthetic medium.
The value was about 2/5 of 0 3A4 activity. However, there was no effect on the P450 3A4 activity of GS gene amplification by the addition of MSX.

[0063]

[Embodiment 8] 10. Method for measuring lidocaine metabolism in P450 3A4-transfected HepG2 cells 31) As a substrate for measuring P450 3A4 activity,
Hormonal substance testosterone (hydroxylation at 6 β-position)
Was used. When it is assumed that P450 3A4-transfected HepG2 cells are used as a bioartificial liver, it is necessary to evaluate (confirm) the metabolism of drugs other than testosterone. Therefore, we decided to measure the metabolic ability of lidocaine, which is widely used when evaluating the metabolic ability of bioartificial liver drugs.
Lidocaine is used as a model substrate for the first phase reaction and as a clinical index in liver failure.・ HPLC condition HPLC device… Shimadzu LC10AD liquid chromatograph system column… C18 column Inertsil ODS-3V (4.
6 × 150 mm, 5 μm, GL Sciences Inc.) Buffer: 20 mM NaClO 4 ((pH 2.5), 15% acetoni
trile (Deaerated) Flow rate… 2.0 ml / min Column temperature… Room temperature Measurement wavelength… 205 nm

The results are shown in Table 12. As can be seen from this table, the lidocaine metabolizing capacity in P450 3A4-transfected HepG2 cells was 5.1 nmol / min / mg-protein. still,
The value is the average value of two measurements, and each measured value is 4.7 and 5.5.

[0065]

[Table 12]

[0066]

Example 9 11. Maintenance of drug metabolic activity for a long period of time Further, it was examined whether or not the cell line of the present invention could maintain the activity for a long period of time. Therefore, HepG2 incorporating the cell line of the present invention, CYP3A4 and GS, was cultured for 80 days or more (about 20 generations or more), and its drug metabolism activity was measured. The results obtained are shown in Table 13 below. As a result, it was revealed that the high activity of about 426 pmol / min per mg of protein was maintained.

[0067]

[Table 13]

[0068]

[Embodiment 10] 12. Evaluation of drug metabolism as bioartificial liver based on clearance 12. 1 Theory of clearance calculation Here, based on the lidocaine metabolizing capacity of P450 3A4-transfected HepG2 cells, it was decided to evaluate the drug metabolizing capacity as a bioartificial liver when the cells were applied to Cygnus (cell culture device). In this evaluation, clearance was used as an index 38), 39), 40). Clearance generally indicates how much an organ has the ability to process a drug. The treatment here is a concept that includes not only metabolism but also all such as disappearance of a drug and movement through a biological membrane. Clearance (CL) (ml
/ min) is the speed of the drug at the concentration in blood (C) (mg / ml) Vel
When processed in (mg / min), it is defined by the following formula. Vel = CL ・ C Here, the clearance based on the blood (plasma) drug concentration C in (mg / ml) flowing into the bioartificial liver is defined as the bioartificial liver clearance (CL _{artificial liver} ) (ml / min). If defined, it is expressed by the following formula. Vel = CL _{artificial liver} , C in

12. 2． Calculation of clearance Here, plasma with a drug concentration C in (mg / ml) flows into the bioartificial liver at a flow rate Q (ml / min), and the drug concentration is C out (mg / ml) due to metabolism by P450 3A4-introduced HepG2 cells. Considering a model that becomes smaller and flows out, the inflow rate of the drug V in
(Mg / min) and outflow rate V out (mg / min) and bio artificial liver internal processing rate Vel (mg / min) are respectively drug inflow rate: V in = Q ・ C in Drug outflow rate: V out = Q ・ C out Bioartificial liver processing speed: Vel = CL _{artificial liver}・ C in From this, the rate of drug change in the bioartificial liver
(V) (mg / min) is expressed as V = Vin-Vout-Vel = Q (Cin-Cout) -CL artificial liver-Cin .... Here, in the steady state, since there is no change in the drug concentration in the bioartificial liver, V = 0 and CL _{artificial liver} = Q · (C in − C out) / C in. If (C in − C out) / C in = Er ……, then CL _{artificial liver} = Q · Er ……. Er is called the extraction efficiency, which represents the rate at which the drug is processed during the passage of plasma through the bioartificial liver.

12. 3． Relationship between bioartificial liver clearance and specific clearance When obtaining the bioartificial liver clearance (CL artificial liver), it is necessary to consider the specific clearance (CL int). First, the intrinsic clearance is based on the effective drug concentration (Ce) at the minute site where the drug in the bioartificial liver disappears. The processing capacity originally possessed by the bioartificial liver, so to speak, the treatment in P450 3A4-introduced HepG2 cells. Think of it as an ability. Here, as a hypothesis, the concentration of unbound drug (f
・ C) has a concentration equilibrium relationship with the concentration of the non-binding drug at the treatment site in the bioartificial liver, and only the non-binding drug is treated. Here, f represents the binding rate of the drug to plasma protein. Considering that the effective drug concentration (Ce) in the bioartificial liver is uniform and equal to the unbound concentration in plasma (fCout), Vel = dn / dt = CL _artificial rather than fCout = Ce _Liver・ C in = CL int ・ Ce = CL int ・ f ・ C out.

12. Four . In the steady state of bioartificial liver clearance, since V = 0 in the equation,
From the formula, Q (C in −C out) = CL int · f · C out. From this formula and the formula, the extraction efficiency Er is Er = f · CL int / (Q + f · CL int), and from the formula CL _{artificial liver} = Q · f · CL int / (Q + f · CL int) …… Becomes

12.5. Assuming that the drug metabolism by the intrinsic clearance P450 3A4-transfected HepG2 cells follows the Michaelis-Menten equation, the substrate concentration at the metabolic site, that is, the effective drug concentration in the bioartificial liver is Ce, Ce
Let V max be the maximum metabolic rate of the drug at infinity and K m be the Michaelis constant, Vel = V max · Ce / (K m + Ce). In addition, the intrinsic clearance is expressed as CL int = V max / (K m + Ce).

12. 6． Trial Calculation of Bioartificial Liver Clearance For example, as disclosed in Japanese Patent Laid-Open No. 06-113818, Cygnus (cell culture device: cell number of about 4 × 10 ⁹ cells)
, Volume 1 L) 41), 42) were filled with P450 3A4-transfected HepG2 cells, and bioartificial liver clearance was calculated. Lidocaine metabolizing capacity in previously measured P450 3A4-transfected HepG2 cells 5.1 nmol / min / mg-protein, K m = 65 μM in lidocaine metabolism (reference value) 43) and effective lidocaine concentration in bioartificial liver, that is, blood in steady state Lidocaine average concentration of Ce = 25 μM 43)
The value of CL int was calculated from the formula. As a result, CL int = 57m
It was calculated as l / min. Based on this value and the blood flow velocity Q = 15 ml / min 41) in Cygnus (cell culture device) and the binding rate of lidocaine to plasma proteins (reference value) f = 0.3 43),
Calculating bioartificial liver clearance, CL organ =
It was calculated as 8.0 ml / min. This value was almost the same as that of the bioartificial liver using rat primary hepatocytes (CL = 7.0 ml / min) 44). Therefore, it was found that the bioartificial liver using P450 3A4-transfected HepG2 cells has the same order of function in drug metabolism as the bioartificial liver using primary hepatocytes. Considering the fact that primary hepatocytes need to be procured from the living body, it is difficult to maintain liver function for a long period of time, and there is a risk of viral infection, etc., P450 3A4-transfected HepG2 cells with infinite proliferation ability are , It may be useful as a cell for bioartificial liver. However, in calculating the bioartificial liver clearance, it must be noted that it depends not only on the function of the cells themselves filled in the cell culture device but also on the blood flow velocity flowing into the bioartificial liver. For example, liver clearance in human liver is about 70
0 ml / min 45). This is about 100 times the value of the bioartificial liver calculated previously, but the blood flow rate in human liver is about 1600 ml / min 40), and the bioartificial liver (Kygnus:
It is about 100 times the blood flow velocity of 15 ml / min).

13. Artificial Liver Assistance Experiment An artificial liver assistance experiment was actually performed using the cell line of the present invention, CYP3A4-GS-HepG2, under the above-mentioned apparatus and culture conditions. FIG. 6 shows an actual liver failure model, and FIG.
Shows the definition of effective survival time used for the evaluation of artificial liver assist system. FIG. 8 shows the results of effective survival time in each experimental group. Here, "no treatment" is a group that does not unravel anything, "acellular human liver" is one in which an artificial liver system without cells is connected and circulated, "plasma exchange + CHDF (continuous hemofiltration)" Is a method generally used for the treatment of liver failure, “GS-HepG2” is a group using the cell line of the present invention, and “Wt-HepG2” is a wild type (cell line before gene transfer). Is a group using. From the results shown in FIG. 8, it can be seen that the liver function assisting device of the present invention provided an excellent life-prolonging effect. Furthermore, FIG. 9 and FIG.
As shown in 0, the occurrence frequency of cerebral hypertension, APTT active partial thromboslastin activity in blood coagulation system, ACT active coagulation time, HPT hepaplastin time,
And various improving parameters such as FVII factor 7 were observed.

[0075]

Example 11: Cell line of the present invention (CYP3A4-GS-H
Drug metabolism assay system using epG2) Nifedipine was added to cells, and the concentrations of nifedipine and hydroxide in the extracellular fluid were measured over time. At 100 nM, 5 minutes after the addition, 5% of the added amount of the hydroxide could be detected, and linearly increased to 50% at 30 minutes.
Further, it reached 75% at 60 minutes, and the parent drug was below the detection sensitivity at this point. On the other hand, in the cells of interest, the average was 7% even at 60 minutes. (FIG. 11) When 100 μM ketoconazole, cimetidine, and erythromycin, which are 3A4 inhibitors, were pretreated in this system for 10 minutes and then nifedipine was added, the amount of hydroxide detected after 30 minutes was 25-35% of that in the untreated case. became. (FIG. 12) On the other hand, even when hydroxide measurement was performed using midazolam instead of nifedipine, the amount of detected hydroxide was 35-45% of that in the case of no addition due to pretreatment with ketoconazole, cimetidine and erythromycin.
Became. (FIG. 13). Therefore, by adding an unknown drug to these cells and measuring the hydroxylation of nifedipine and midazolam, it is possible to easily evaluate whether or not the drug has a 3A4 inhibitory effect. In particular, this cell is characterized in that it has a higher metabolic ability so that the amount of the hydroxylated substance flowing into the culture medium can be measured.

Reference 1) Watanabe Meiji. 1994. Clinical liver failure. Nagai Shoten. Pp163-s.
167. 2) Kazuo Inoi. 1999. Liver function assisting device. Tokyo Women's Medical University Medical Engineering Research Facility. Advanced medical science that opens up the 21st century. Newton Press. P146 3) Enozawa Shin. 1999. Advanced Medical Research Project. Interviews. National Children's Hospital. 4) Naofumi Ito. 1994. Application form for new medical technology development research project. 5) Toshiji Oda. 1990. Biology of liver. The University of Tokyo Press. P38-
85. 6) Nagaki Masahito. 1994. Review Trends in research on hybrid artificial liver. Japanese clinical 52: 247-252. 7) Yoshida Fumitake. 1993. Chemical engineering and artificial organs. Kyoritsu Shuppan.
−274.8) Rozza J, Demetriou AA. 1995.Artificial liver evolu
tion and future perspectives.ASAIO Journal 41: 831
−839.7 9) Chen SC, Hewitt WR, Watanabe FD, Eguchi S, Kahaku E,
Middleton Y, Ronza J, Dometriou AA.1996.Clinical exp
erience with a porcine hepatocyte−based liver sup
port system.International Journal of Artificial Or
gans 19: 664−669. 10) Nyberg SL, Shatford RA, Peshwa MV, White JG, Cerra
FB, Hu WS.1993.Evaluation of ahepatocyte−entrapmen
t hollow fiber bioreacter: A potential bioartifici
al liver.Biotechnology and Bioengineering 41: 194-
203. 11) Nyberg SL, Misra SP. 1998. Hepatocyte liver-assis.
t systems? a clinicalupdate.Mayo Clinic Proceeding
73: 765− 771. 12) Friedman AL. 1998. Why bioartificial liver suppor
t remains the Holy Grail.ASAIO Journal 44: 241-24
3.13) Sussman NL, Killy JH.1993.Improved liver functio
n following treatmentwith an extracorporeal liver
assistant device.Artif.Organs 17: 27-30. 14) Enosawa S, Suzuki S, Kkefuda T, Amemiya H. 1996.Exa
mination of 7−ethoxycoumarin deethalation and amm
onia removal activities in 31 hepatocyte cell line
s.Cell-Transplant 5: 39-40 15) Yamanaka M. 1999. Master thesis of Osaka University. Development of liver-derived ammonia metabolizing cells for treatment of fulminant hepatitis. 16) Ryuichi Kato, Tetsuya Kamataki. 2000. Drug metabolism. G. Tokyo Kagaku Doujin. P41-43. 17) Yoshiro Imai, Tetsuya Kamataki. 1998. P450-Various functions and applications-Protein nucleic acid enzyme 43: 203-215. 18) Keizo Nakasa, Koichi Kitada. 2000. Cytochrome P450. Monthly Yakuji 42: 746-753. 19) Omasa, Takeshi. 1992. Culture engineering research on monoclonal antibody production using hybridoma. Osaka University doctoral thesis. 20) Kobayashi, S. 1996. Cell number counting method. Cell Culture Technology (3rd Edition, Chapter 1). Asakura Shoten. Pp.26-28. 21) Blake J, Pritchard M, Ding S, Smith G, Burchell B, W
olf C, Friedberg.T.1996.Coexpression of human P450 (C
YP3A4) and P450 reductase generates a highly functio
nal monooxygenase system in Escherichia coli .FEBS
Letters 397: 210−214. 22) Rauschenbach R, Gieschen H, Husemann M, Salomon B,
Hildebrand M. 1995.Stable expression of human cytoc
hrome P450 3A4 in V79 cells and its application fo
r metabolic profiling of ergot derivatives.Euro.J.
Pharm.293: 183−190.23) Ding S, Yao D, Burchell B, Wolf C, Friedberg. T. 199
7.High levels of recombinant CYP expression in chi
nese hamster ovary cells are modulated by coexpres
sed human P450 reductase and hemin 34.supplementat
ion.Arch.Biochem.Biophys 348: 403-410. 24) Gil L, Orellana M, Mancilla J, Garcia J, Vasquez H.
1986. Liver microsomalepoxide hydrase. Anal.Lette 1
9: 2261−275.25) Lionel G, Miriam O, Jerson M, Juan G, Hernan V. 198
6.Use of a new HPLC method in rat liver microsomal
testosterone monooxygenation and its application
to study the sex dependent expression of several h
ydroxylases.Analytical letters 19: 2261−2275. 26) Williams J, Chenery R, Hawksworth G. 1994. Inductio
n of CYP3A enzymes inhuman and rat hepatocyte cult
ures.Biochem.Society.Trans 22: 131.27) Dawson J, Adams D, Wolf C. 1985.Induction of drug
metabolizing enzymes in human liver cell line HepG
2.FEBS.Lette 183: 219-222. 28) Sumida A, Yamamoto I, Zhou Q, Morisaki T, Azuma J.1
999.Evaluation of induction of CYP3A mRNA using th
e HepG2 cell line and reverse transcription-PCR.B
iol.Pharm.Bull 22: 61-65. 29) pBudCE4 Catalog no.V532-20. invitrogen. 30) Naoko Tanimura .1998. Construction of cells with ammonia metabolism ability suitable for hybrid artificial liver and its functional evaluation. 31) Mario JB, Aoyama T, Frank JG, Urs AM.1989. Lidocain
e metabolism in humanliver microsomes by cytochrom
e P450 3A4.Clin.Pharmacol.Ther 46: 521−527. 32) Tabor H, Tabor CW.1970.Glutamine Synthetase.Meth
ods in Enzymolozy 129: 900−903. 33) Bork WR, Muto T, Beaune HP, Sribastava KP, Lloyd S
R, Guengerich PF.1989.Characterization of mRNA spec
ies related to human liver cytochrome P−450nifedi
pine oxidase and regulation of catalytic activity.
J. Biol. Chem 264: 910-919.34) Cokett MI, Bebbington CR, Yarranton GT.1990.High
level expression of tissue inhibitor of metallopro
teinases in CHO cells using GS gene amplification.
BIO / TECHNOLOGY 8: 662-667. 35) Donato MT, Castell JV, Gomez-Lechon MJ. 1999.Char
acterization of drug metabolizing activities in pi
g hepatocytes for use in bioartificial liver device
s: comparison with other hepatic cellular models.
J. Hepatol 31: 542−549. 36) Wortelboer H, de Kruif C, J. van Iersel A, Falke.H,
Noordhoek J, Blaauboer.B.1990.The isoenzyme pattern
of cytochrome P450 in rat hepatocytes in primary
culture, comparing different enzyme activities in m
icrosomal incubations and in intact monolayers.Bio
Chem.Pharmacol 40: 2525 ~ 2534. 37) Yamazaki H, Shimada T. 1997.Progesterone and test
osterone hydroxylation by cytochrome P450 2C19,2C9,
and 3A4 in human liver microsomes.Arch.Biochem.Bio
phys 346: 161-169. 38) Chikako Tanaka, Ryuichi Kato. 1993. Pharmacology (revised second edition). Nankodo.p14-23. 39) Kato Ryuichi. 1999. Clinical pharmacokinetics (revised second edition). Nankodo.p91-106. 40) Ryuichiro Nishigaki, Toshiharu Horie, Tomoo Ito. 1998. Pharmacokinetics. Maruzen. P86-99. 41) Miyashita T, Enosawa S, Suzuki S, Tamura A, Amemiya.
H, Matsumura T, Omasa T, Suga K, Aoki T, Koyanagi Y.2
000.Development of a bioartificial liver with glut
amine synthetase-transduced recombinant human hep
atoblastoma cellline, HepG2.Transplantation Proceed
ings 32: 2355−2358. 42) Enosawa S, Miyashita T, Suzuki S, Li XK, Tsunoda M,
Amemiya H, Yamanaka M, Hiramatsu S, Tanimura N, Omasa
T, Suga K, Matsumura T. 2000.Long−Term culture of g
lutamine synthetase−transfected HepG2 cells in ci
rculatory flow bioreactor for development of a bio
artificial 35.36 liver.Cell Transplantation 9: 711
−715.43) Hoener BA.1994.Predicting the hepatic clearance
of xenobiotics in humans from in vitro data.Biop
harmaceutics drug disposition 15: 295−304. 44) Nyberg SL, Mann HJ, Remmel RP, Hu WS, Cerra FB.199
3.Pharmacokinetic analysis verifies P450 function
during In Vitro and In Vivo application of abioart
ificial liver.ASAIO Journal 39: M252−256. 45) Iwata H, Sajiki T, Maeda H, Park YG, Zhu B, Satoh S,
Uesugi T, Ikai I, Yamaoka Y, Ikada Y. 1999.In Vitro ev
aluation of metabolic functions of a bioartificial
liver.ASAIO Journal 45: 299−306. 46) Shatford RA, Nyberg SL, Meier SJ, White JG, Payne W.
D, Hu WS, Cerra FB.1992.Hepatocytes function in a ho
llow fiber bioreactor: A potential bioartificial li
ver.Journal of Surgical Research 53: 549−557.

[Brief description of drawings]

FIG. 1 shows the results obtained by a culture experiment in which a human liver drug-metabolizing enzyme inducer rifampicin was added to wild-type HepG2 cells.

FIG. 2 0.8 of PCR products of P450 3A4 gene and GS gene
A photograph obtained by agarose gel electrophoresis is shown.

FIG. 3 shows P450 3A4 activity (in a synthetic medium) in P450 3A4-transfected HepG2 cells (resistant strains at each MSX concentration).

FIG. 4 shows the results of GS activity (in a synthetic medium) of P450 3A4-transfected HepG2 cells (each MSX concentration resistant strain).

FIG. 5 shows the results of measuring P450 3A4 activity in P450 3A4-transfected HepG2 cells with serum.

FIG. 6 shows an actual liver failure model.

FIG. 7 shows the definition of effective survival time used for evaluation of the artificial liver support system.

FIG. 8 shows the results of effective survival time in each experimental group.

FIG. 9 shows the results of the expression frequency of increased cerebral pressure.

FIG. 10 shows results regarding various parameters in the blood coagulation system.

FIG. 11 shows the metabolism of nifedipine by the cell line of the present invention.

FIG. 12 shows the effect of each inhibitor on nifedipine metabolism.

FIG. 13 shows the effect of each inhibitor on midazolam metabolism.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 1/16 A61P 43/00 101 7/08 105 43/00 101 C12N 15/00 ZNAA 105 5/00 B (72) Inventor Mitsumasa Kishimoto 1-24 Kakuda Kakudacho, Nishikyo-ku, Kyoto City, Kyoto Prefecture (72) Inventor Hiroo Katakura 4-15-16 Onohara Higashi, Minoh City, Osaka Prefecture (72) Inventor Shin Enozawa Kita-ku, Tokyo 4-26 Ukima 3-205 Ukima House (72) Inventor Shuichi Tsuruoka 1-9-4 Gion, Minamikawachi-cho, Tochigi Prefecture F-term (reference) 4B024 AA01 AA11 BA08 BA11 DA03 EA04 FA20 GA11 GA18 GA30 HA20 4B063 QA05 QA18 QA19 QQ08 QQ61 QQ91 QR02 QR20 QR77 QS24 QS39 QX01 4B065 AA93X AB01 AC20 BA02 BB40 CA27 CA28 CA44 CA46 4C077 AA07 BB10 EE01 PP29 4C087 AA02 BB52 BB64 BB65 DA04 DA08 NA05 NA10 ZA51 ZA75 ZC37

Claims (27)

[Claims] 1. A cell line transformed with a drug-metabolizing enzyme gene and an ammonia-metabolizing enzyme gene. 2. The drug-metabolizing enzyme gene is P450,
The cell line according to claim 1. 3. The cell line according to claim 2, wherein the drug-metabolizing enzyme is P450 3A4. 4. The cell line according to claim 1, wherein the ammonia metabolizing enzyme gene is a glutamine synthetase gene. 5. The cell according to claim 1, wherein the cell is derived from a mammal.
4. The cell line according to any one of 4 to 4. 6. The cell line according to claim 5, wherein the cell is derived from human liver. 7. The cell line according to claim 6, wherein the cells are derived from human hepatocytes. 8. The cell line according to claim 7, wherein the cell is HepG2. 9. The cell line according to claim 1, wherein the cell is a recombinant ammonia-metabolizing human hepatocyte cell line. 10. The cell line according to claim 1, wherein the drug metabolizing enzyme gene and the ammonia metabolizing enzyme gene are introduced into a common expression vector. 11. The cell line according to claim 10, wherein the expression vector is a plasmid. 12. The cell line according to claim 10, wherein the expression vector is a plasmid, which is a mammalian cell expression vector having two or more independent multiple cloning sites. 13. The cell line according to claim 12, which is the expression vector pBudCE4. 14. The cell line according to any one of claims 1 to 13, which exhibits a P450 activity of about 200 pmol / min or more per mg of protein. 15. The cell line according to claim 14, which exhibits a P450 activity of about 490 pmol / min or more per mg of protein. 16. P of about 420 pmol / min per mg of protein.
Cell line according to claim 14 or 15, characterized in that 450 activity is maintained for 80 days. 17. A liver function assisting device comprising the cell line according to any one of claims 1 to 16. 18. The cell line according to any one of claims 1 to 17, which is further transformed with another drug-metabolizing enzyme gene. 19. A liver function assisting device using the cell line according to any one of claims 1 to 17. 20. A liver function assisting device further comprising the cell line of claim 19, which uses a cell line of a different type. 21. The liver function assisting device according to claim 20, wherein the different type of cell line is derived from human liver non-parenchymal cells. 22. The liver function assisting device according to claim 20, which is a hybrid artificial liver. 23. The liver function assisting device according to claim 20, which comprises a circulating culture device. 24. A drug metabolism assay system using the cell line according to any one of claims 1 to 17. 25) A) culturing the cell line according to any one of claims 1 to 17, b) adding a substance to be measured to a medium and further culturing for a predetermined period, and c) collecting a supernatant, d) Determine the concentration ratio of the target substance and the metabolized target substance in the collected supernatant, and determine the concentration ratio between them. A drug metabolism assay system comprising culturing for a period of time, f) repeating steps c) and d) above, and g) measuring the effect of the test substance on the metabolism of the target substance from the change in the concentration ratio. 26. A) culturing the cell line according to any one of claims 1 to 17, b) adding nifedipine to the medium and further culturing for a predetermined period of time, c) collecting the supernatant, and d) The concentrations of nifedipine and oxidized nifedipine in the collected supernatant are measured to determine the concentration ratio of both, e) Nifedipine and the test substance at an appropriate concentration are added to the medium and the culture is continued for a predetermined period of time, and f) Step c above. ) And d) are repeated, and g) the effect on the production of oxidized nifedipine by the test substance is measured from the change in the concentration ratio, and a drug metabolism assay system. 27) A) culturing the cell line according to any one of claims 1 to 17, b) adding midazolam to the medium and further culturing for a predetermined period of time, c) collecting the supernatant, and d) The concentrations of midazolam and oxidized midazolam in the collected supernatant were measured to determine the concentration ratio of both, e) midazolam and the test substance at an appropriate concentration were added to the medium, and the culture was continued for a predetermined period of time, and f) step c above. ) And d) are repeated, and g) the effect on the production of oxidized midazolam by the test substance is measured from the change in the concentration ratio, and a drug metabolism assay system.