WO2021132586A1

WO2021132586A1 - Liver cell culture membrane, drug transport ability evaluation kit provided therewith, and drug transport ability evaluation method

Info

Publication number: WO2021132586A1
Application number: PCT/JP2020/048793
Authority: WO
Inventors: 琢男荻原; 健太溝井; 映子松本
Original assignee: 学校法人高崎健康福祉大学
Priority date: 2019-12-27
Filing date: 2020-12-25
Publication date: 2021-07-01
Also published as: JPWO2021132586A1; JP7136509B2

Abstract

Provided are a liver cell culture membrane and a drug transport ability evaluation kit that are to be used for in vitro, easily and directly evaluating drug transport ability, i.e., between the migration of an administered drug from the liver into blood vessels followed by whole body migration and the migration of the drug into bile in the bile duct followed by biliary excretion, which is more dominantly induced and to what extent, as one of important factors of distribution, metabolism and excretion. The liver cell culture membrane 14 comprises a membrane-type cell layer 12 which is formed by cells 12a, such as liver cancer cells, cultured in a membrane form on a porous plastic film 13, wherein the cells 12a have such an orientation property that the blood vessel side becomes the bottom side of the membrane-type cell layer 12 while the bile duct side becomes the top side of the membrane-type cell layer 12. The drug transport ability evaluation kit 1 is provided with: a liver cell culture membrane-holding insert container 10 containing the liver cell culture membrane 14, which is disposed on the bottom thereof, and housing a first test solution 15; and a well plate 20 comprising a well 21, into which the insert container is inserted, and housing a second test solution 25 in which the liver cell culture membrane 14 is to be immersed.

Description

Hepatocyte culture membrane, drug transport capacity evaluation kit equipped with it, and drug transport capacity evaluation method

The present invention is used to evaluate the drug transport capacity as to how predominantly either the systemic circulation in which the administered drug is transferred from the liver to the blood vessels or the bile excretion in the bile duct is caused. Hepatocyte culture membrane, drug transport capacity evaluation kit provided with it, drug transport capacity evaluation method, and orientation-enhanced hepatocyte medium that imparts vascular-side and bile duct-side orientation (polarity) to the hepatocyte culture membrane. It is about.

Drugs administered into the body, for example, drugs orally administered to patients, are absorbed from the gastrointestinal tract via gastrointestinal cells and reach the liver via the portal vein.

FIG. 3 schematically shows the state of absorption, distribution, metabolism, and excretion of a drug administered in vivo and absorbed in the gastrointestinal tract without being conjugated in the liver or after being conjugated in the enterohepatic circulation or systemic circulation. To explain with reference, the drug that reaches the liver is excreted unchanged from the liver together with bile, returns to the digestive tract via the bile duct (bile excretion), is absorbed again from the digestive tract, and partly from the liver. It enters the systemic circulation via blood vessels and reaches the target site (Fig. (A)). Alternatively, it is conjugated by the liver, excreted together with bile, returned to the digestive tract via the bile duct, then deconjugated and absorbed again via the gastrointestinal cells (enterohepatic circulation). In addition, a part of the liver enters the systemic circulation via blood vessels and reaches the target site (Fig. (B)). Alternatively, it is metabolized in the liver and excreted in urine and bile.

Drugs with a long half-life in the body, such as morphine, are greatly involved in enterohepatic circulation. Specifically, such drugs are absorbed from the gastrointestinal tract, such as the intestinal tract, and some of them enter the systemic circulation, but most of them undergo glucuronidation in the liver and are excreted in the intestinal tract together with bile before entering the intestine. Hydrolyzed by the bacteria, the glucuronidation is disengaged and absorbed again from the intestinal tract, part of which enters the systemic circulation. As a result of enterohepatic circulation by repeating this, the drug effect is exhibited for a long time. When used in combination with antibiotics, the action of intestinal bacteria is inhibited, making enterohepatic circulation difficult.

Among the drugs that cause enterohepatic circulation, organic anionic compounds such as azithromycin and atorvastatin, organic cationic compounds such as carvegylol and clomiphene, temocapril and moxifloxacin are drugs whose unchanged form is excreted in the bile. These include amphoteric compounds such as digitoxin and neutral compounds such as probucol, which are also greatly involved in enterohepatic circulation and take a long time to be excreted in manure.

On the other hand, it is known that many drugs having a molecular weight smaller than 300 to 500 have a low bile excretion rate, and the systemic circulation is more involved than the enterohepatic circulation. .. The larger the molecular weight is around 300 to 380 in rats, the larger the molecular weight is around 330 to 430 in guinea pigs, and the larger the molecular weight is around 410 to 500 in rabbits, the higher the bile excretion rate with respect to the dose. It is known to be expensive.

It has been found that bile excretion of these drugs or their metabolites plays an important role in the overall clearance of the drugs during the development of new drugs and the evaluation of existing drugs.

However, in the case of humans, the mechanisms of absorption, distribution, metabolism, and excretion are more complicated. Moreover, even with a low molecular weight, bile may be easily excreted. Furthermore, whether the administered drug is superior to bile excretion in the liver or systemic transfer to blood vessels depends on the physical properties of the drug, the species difference of the target animal, and the susceptibility to conjugation such as glucuronic acid conjugation. It differs greatly due to various factors such as the contribution of the transporter and its action, the susceptibility to the formation of micelles in bile, and the degree of transfer to bile.

In the past, in order to evaluate whether biliary excretion or systemic transfer in humans was superior, it was inferred from animal experiments in vivo, but in recent years, in vitro for easy evaluation. A method has been developed that can be inferred from the experiments in.

For example, Patent Document 1 provides an in-vitro method for characterizing the excretion of chemical entities in the gallbladder, a) a step of providing a cell culture containing hepatocytes forming at least one gallbladder canal, b) the above. Step of contacting the cell culture with the first chemical entity for a time sufficient to allow the hepatocytes of the culture to take up the chemical entity, c) at least one of the above without lysing the hepatocytes. The step of destroying one gallbladder and detecting the amount of the first chemical entity and / or its metabolite released by the at least one gallbladder, and d) lysing the hepatocytes and by the hepatocytes. A method comprising the step of detecting the amount of said first chemical entity and / or its metabolite released is disclosed.

Further, Patent Document 2 describes a method in which hepatocytes are cultured on an oxygen-permeable substrate to form a bile canaliculus, and the oxygen concentration in the medium is 4.0% or more and 12.5% or less. A method for assaying compound transport, in which hepatocytes are cultured to produce cultured hepatocytes in which hepatocytes are formed to form bile canaliculi, and the obtained cultured hepatocytes are used to evaluate the transport of compounds. , Is disclosed. In this method, after adding a compound and taking it into the bile canaliculus, the cultured hepatocytes are washed with a washing solution to remove the compound, and then subjected to a metabolic reaction in a reaction solution containing no compound. , The components in the reaction solution (cultured hepatocyte extracellular fluid) and / or the bile canaliculus fluid are analyzed to test the metabolic properties of the compound.

Both Patent Documents 1 and 2 form a bile canaliculus, and the amount of a drug whose transport capacity should be measured by destroying the bile duct and lysing hepatocytes is detected, or the amount of a drug taken into the bile canaliculus. Is to be detected. However, there has been a demand for a model that does not require destruction or lysis of cells or the like and can evaluate whether bile excretion or systemic transfer is superior by direct measurement of the concentration.

The present inventors have no orientation when culturing epithelial cells and parenchymal cells such as hepatocytes in a normal petri dish, but hepatocytes forming a membranous cell layer obtained by culturing hepatocytes on a porous plastic film. It was found that the culture membrane has an orientation in which the lower side in contact with the porous plastic film is on the blood side and the upper side not in contact with the porous plastic film is on the bile duct side, and further, drug transport having this hepatocyte culture membrane. The present invention has been completed by finding that the ability evaluation kit and the drug transport ability evaluation method using the kit provide an evaluation model as to which of bile excretion and systemic transfer is superior.

Special Table 2017-527283 Japanese Unexamined Patent Publication No. 2014-223061

The present invention has been made to solve the above-mentioned problems, and as one of the important factors for absorption, distribution, metabolism, and excretion of the target drug, the administered drug is transferred from the liver to the blood vessels in the whole body and the bile duct. Hepatocyte culture membrane, which is used to easily and directly evaluate the drug transport capacity of how predominantly any of the bile excretion transferred to bile is caused in vitro. It is an object of the present invention to provide a provided drug transport capacity evaluation kit, a drug transport capacity evaluation method, and an orientation-enhanced hepatocyte medium that imparts vascular-side and bile duct-side orientation to a hepatocyte culture membrane.

The hepatocyte culture membrane of the present invention made to achieve the above object is hepatocyte, bile duct cancer cell, free hepatocyte, animal-derived fresh humanized hepatocyte, and iPS cell induced by hepatocyte. And / or at least one of ES cells, organoids, and immortalized cells forms a membranous cell layer cultured in a membrane on a porous plastic film, and the cells form the membrane. It is characterized by having an orientation in which the lower side of the hepatocyte layer is on the blood vessel side and the upper side of the membranous cell layer is on the bile duct side.

The drug transport ability evaluation kit of the present invention made to achieve the above object includes a hepatocyte culture membrane-supporting insert container in which the hepatocyte culture membrane is provided at the bottom and stores the first test solution, and the hepatocyte culture membrane. It has a well into which a cell culture membrane-supporting insert container is inserted, and is provided with a well plate for accommodating a second test solution into which the hepatocyte culture membrane is immersed.

According to the drug transport ability evaluation kit, the porous plastic film is made of any plastic selected from, for example, fluororesin, polycarbonate, polyolefin, polyester, polyurethane, polyamide, polyimide, cellulose, and regenerated cellulose. It is a thing.

In this drug transport ability evaluation kit, the porous plastic film has a porous size smaller than the size of the hepatocytes, or the porous plastic film is sealed with an impermeable material and opened in a pattern. (Patterning).

In this drug transport ability evaluation kit, it is preferable that the porous plastic film is coated with collagen and / or fibronectin.

In this drug transport ability evaluation kit, it is preferable that at least one of the first test solution and the second test solution contains bile acid.

The drug transport capacity evaluation method of the present invention made to achieve the above object uses the drug transport capacity evaluation kit, the first test solution inside the hepatocyte culture membrane-supporting insert container, and the liver. The drug to be measured is added to any of the second test solution outside the cell culture membrane-supporting insert container and inside the well of the well plate to obtain the membranous cell layer and the porous plastic film. The drug transport ability is evaluated by measuring the transport amount of the drug to be measured over time and calculating the transport rate through the hepatocyte culture membrane.

Specifically, the drug transport ability evaluation method of the present invention made to achieve the above object is specifically described as follows.
Induced to hepatocytes, bile duct cancer cells, free hepatocytes, animal-derived humanized fresh hepatocytes, and hepatocytes on a porous plastic film provided at the bottom of the insert container tube containing the first test solution. By seeding and culturing at least one of iPS cells and / or ES cells, organoids, and immortalized cells to form a membranous cell layer, the membranous cell layer is formed. A hepatocyte culture membrane-bearing insert container is prepared by forming a hepatocyte culture membrane having an orientation that is on the blood vessel side below the membranous cell layer and on the bile duct side above the membranous cell layer. Process and
A step of preparing a well plate for containing a second test solution in which the hepatocyte culture membrane-supporting insert container is inserted and in which the hepatocyte culture membrane is immersed.
The first test solution containing the drug whose drug transport capacity is to be measured is contained in the hepatocyte culture membrane-supporting insert container, and the well of the well plate contains the second test solution which does not contain the drug to be measured. A blood-side transport amount measurement step of measuring the transport amount of the drug to be measured from the bile duct side to the blood side after containing the liquid and immersing the hepatocyte culture membrane in the second test solution.
The first test solution containing no drug to be measured is contained in the hepatocyte culture membrane-supporting insert container, and the second test solution containing the drug to be measured is contained in the well of the well plate. After immersing the hepatocyte culture membrane in the second test solution, the bile duct side transport amount measuring step for measuring the transport amount of the drug to be measured from the blood side to the bile duct side.
The process of calculating the transportation speed by measuring each transportation amount over time, and
A drug transport capacity calculation step for calculating the ratio of the blood transport rate and the bile duct transport rate to obtain the drug transport capacity of the drug to be measured.
It is to have.

This drug transport evaluation method is performed during the blood transport amount measurement step, and further by adding a transporter inhibitor to the first test solution and the second test solution, and during the bile duct side transport amount measurement step. Further, a transporter inhibitor was added to the first test solution and the second test solution, and the bile duct side transport amount measurement step was also performed for them, and the inhibitor was not added in the drug transport capacity calculation step. The ratio of the blood-side transport rate and the bile duct-side transport rate to the ratio of the blood-side transport rate and the bile duct-side transport rate with the addition of the inhibitor is calculated to determine the drug transport capacity of the drug to be measured. It may be something like that.

The orientation-enhanced hepatocyte medium of the present invention made to achieve the above object is induced into hepatocytes, bile duct cancer cells, free hepatocytes, animal-derived fresh humanized hepatocytes, and hepatocytes. At least one of iPS cells and / or ES cells, organoids, and immortalized cells is cultured on a porous plastic film to form a membranous cell layer, and below the membranous cell layer. It is a hepatocyte medium for expressing orientation on the blood vessel side and on the bile duct side on the upper side of the membranous cell layer, and is characterized by having a culture component and bile acid.

The hepatocyte culture membrane of the present invention forms a membranous cell layer in which cells derived from hepatocytes or induced by hepatocytes are cultured in a membranous manner on a porous plastic film, thereby forming a membranous cell layer below the membranous cell layer, that is,. The porous plastic film side has an orientation toward the blood vessel side, and the upper side of the membranous cell layer has the orientation toward the bile duct side, and is a monolayer film. Therefore, it does not form a three-dimensional mass or multiple layers of cells as in the case of culturing in a normal petri dish. Nor does it intentionally form bile ducts or bile canaliculi. Since this hepatocyte culture membrane has orientation, it is suitable for examining whether the drug to be measured is dominant in bile excretion or systemic transfer.

Therefore, if a hepatocyte culture membrane is used, for example, when developing a new drug or evaluating an existing drug, the drug to be measured, such as those drugs or their metabolites, is excreted in bile or is excreted in blood vessels (sinusoids). It is possible to construct a simple and highly reproducible evaluation system that can easily evaluate whether or not it is pushed back to the) side.

This hepatocyte culture membrane can express stable orientation at low cost by using easily available cells.

Since the drug transport ability evaluation kit of the present invention has an oriented hepatocyte culture membrane, the drug to be measured does not permeate into the bile canaliculus or bile canaliculus, but the membranous cell layer of the monolayer membrane. By measuring the degree of permeation of the drug, it is possible to evaluate whether the drug to be measured is superior in bile excretion or systemic transfer.

According to this drug transport ability evaluation kit and the drug transport ability evaluation method using the kit, the liver when the drug to be measured is administered to humans as one of the important factors of absorption, distribution, metabolism and excretion of the drug to be measured. Easily and directly evaluate in vitro the drug transport capacity of which circulation, the systemic circulation transferred from blood vessels to blood vessels or the enterohepatic circulation transferred to bile ducts, is caused predominantly. Can be used for. Furthermore, according to this drug transport capacity evaluation kit and drug transport capacity evaluation method, the function of the liver can be reflected because the hepatocyte culture membrane is used, so that it is not necessary to directly evaluate in vivo, but indirectly. The result in vivo can be estimated correctly.

In addition, this drug transport ability evaluation kit and drug transport ability evaluation method include the amount of the drug to be measured from the bile duct side to the blood side in the hepatocellular culture membrane and the amount of the drug to be measured from the blood side to the bile duct side. It is used to easily evaluate the drug transport capacity by comparing with.

In the orientation-enhanced hepatocyte medium of the present invention, the membranous cell layer in the hepatocyte culture membrane is oriented so that it is on the vascular side on the lower side, that is, on the porous plastic film side, and on the bile duct side on the upper side, at the cell culture stage. It can be enhanced. Therefore, if a hepatocyte culture membrane is prepared using an orientation-enhanced hepatocyte culture membrane, the physical properties of the drug to be measured can be determined by a drug transport capacity evaluation kit having the hepatocyte culture membrane and a drug transport capacity evaluation method using the kit. In particular, the pharmacokinetics in humans regarding whether bile excretion is likely to occur from the liver or systemic transfer is easily and accurately grasped from in vitro data that correctly reflects it.

It is a schematic cross-sectional view of the drug transport ability evaluation kit which has the hepatocyte culture membrane to which this invention is applied. It is a schematic cross-sectional view of the drug transport ability evaluation kit to which this invention is applied, and the graph which shows the measurement result by the drug transport ability evaluation method using it. It is a figure which schematically showed the state of absorption, distribution, metabolism, and excretion that a drug administered in a living body and absorbed in the gastrointestinal tract is not conjugated in the liver or is conjugated and then enterohepatic circulation or systemic circulation. ..

Hereinafter, embodiments for carrying out the invention will be described in detail, but the scope of the present invention is not limited to these embodiments.

The hepatocyte culture membrane of the present invention, the drug transport ability evaluation kit provided with the hepatocyte culture membrane, and the drug transport ability evaluation method will be described in detail with reference to FIG.

The hepatocyte culture membrane 14 has a porous plastic film 13 and a membranous cell layer 12 in which cells 12a are cultured in a membrane shape on the porous plastic film 13.

The drug transport capacity evaluation kit 1 is a hepatocyte culture membrane-supporting insert container in which the hepatocyte culture membrane 14 is provided at the bottom of a non-permeable plastic insert container cylinder 11 with the membranous cell layer 12 on the inner air side to close the container. A non-permeable plastic well plate 20 having a well 21 into which the hepatocyte culture membrane-supporting insert container 10 is inserted is provided. At the time of measurement by the drug transport ability evaluation method, the first test solution 15 is added to the hepatocyte culture membrane-supported insert container 10 and stored, and the second test solution 25 is added to and stored in the well 21 of the well plate 20. .. At that time, in the well 21, the hepatocyte culture membrane 14 of the hepatocyte culture membrane-supporting insert container 10 is immersed in the second test solution 25.

The cells 12a include, for example, hepatocyte cancer cells, bile duct cancer cells, free hepatocytes, animal-derived fresh humanized hepatocytes, hepatocyte-induced iPS cells and / or ES cells, organoids, and immortalized cells. At least one of the cells with. Precursor cells that become liver-like cells when differentiated are called liver organoids.

Examples of liver cancer cells include commercially available liver cancer cell lines, such as human liver cancer-derived cell line HepG2 (JCRB cell bank (National Research and Development Corporation Pharmaceutical Infrastructure, Health and Nutrition Research Institute), etc.), and well-differentiated human liver blastoma-derived cell line HuH. -7 (JCRB, etc.) is preferably used, but human liver cancer-derived cell line HepaRG (KAC Co., Ltd.), PLC-PRF-5 (Cosmo Bio Co., Ltd., etc.), human liver adenomas cell line SK-HEP -1 (Cosmo Bio Co., Ltd., etc.) may be used.
Examples of cholangiocarcinoma cells include commercially available liver cancer cell lines, for example, cholangiocarcinoma cell line TFK-1 (National University Corporation Tohoku University Institute of Aging Medicine Medical Cell Resource Center) and human immortalized cholangiocarcinoma cell line MMNK-1 (JCRB). ), Immortalized human hepatic endothelial cell line (JCRB), sinus endothelial cancer cells.
Examples of free hepatocytes include primary free hepatocytes (primary hepatocytes), for example, human hepatocyte strain Change-Live (Cosmo Bio Co., Ltd., etc.).
Examples of animal-derived fresh humanized hepatocytes include PXB-cell (PhoenixBio) and animal-derived hepatocytes (Public Interest Incorporated Foundation, Experimental Animal Central Research Institute) in which human cells are engrafted in immunodeficient animals.
In addition, in the case of precursor cultured cells induced by hepatocytes, examples of induced pluripotent stem cells: iPS cells include iCell hepatocytes (Fujifilm Co., Ltd.) and Cellartis (Takara Bio Inc.).

These hepatocytes (parenchymal cells) are seeded on the porous plastic film 13, preferably on the horizontal porous plastic film 13, and cultured in a medium, whereby the cells are cultured on the porous plastic film 13. It becomes a monolayer film of cultured cells 12a that have grown uniformly and without gaps on the surface, and covers the porous plastic film. Compared to endothelial cells, hepatocytes are less likely to form a membrane having orientation (polarity) like endothelial cells even when cultured on a porous plastic film, and the membranous cell layer 12 having excellent orientation is It was not known. However, this membranous cell layer 12 is a monolayer membranous one in which cells 12a proliferate in a monolayer without gaps, and each cell is a blood vessel in the membranous cell layer 12 on the porous plastic film side. By expressing the transporter on the side and the transporter on the bile duct side on the opposite side of the porous plastic film, a specific drug can be preferentially transferred to the blood vessel side or the bile duct side. It has become. Also, like the liver, it selectively transfers a specific drug or glucose to the blood vessel side, or exhales the specific drug without allowing it to permeate. As a result, it acts like the first phase (detoxification system), the second phase (conjugation system), and the third phase (excretory system) in the same manner as the liver. As a result, the blood vessel side is located below the membranous cell layer 12, and the bile duct side is located above the membranous cell layer 12, which is a substrate for the transporter MRP2, which is a protein exhaled by the liver. Drugs such as BSP (bromosulfophthaline: bile excretion marker), which enter the enterohepatic circulation at a rate of almost 100%, exhibit high orientation because they are transferred more to the bile duct side than to the vascular side. Is shown. When the cultured cells 12a are cultured on the porous plastic film 13, particularly when they are cultured in a bile acid-containing culture medium, the orientation becomes more remarkable. Due to such orientation by using the hepatocyte culture membrane 14, excretion in bile, hepatic metabolism, and hepatotoxicity can be investigated.

The hepatocyte culture membrane 14 composed of these membranous cell layers 12 and the porous plastic film 13 is a test target drug for which it should be examined whether bile excretion from the liver or systemic transfer is dominant. In the liquid, the transport rate of the membranous cell layer 12 from the blood side B to the bile duct side A (P _BtoA ) and the transport rate from the bile duct side A to the blood side B ( _PAtoB ). The ratio may vary depending on the drug to be measured, but is 1.3 or more, preferably 1.8 or more, more preferably 2.0 or more, and even more preferably 5.0 or more. This ratio is expressed by the following mathematical formula (1).

In addition to the above indicators, when determining the ratio of the transport rate when an inhibitor (inhibitor) such as a transporter inhibitor is added, the transport rate from the blood side B to the bile duct side A (P). and _BtoA), transport speed from the canalicular a the ratio between the transport rate of the orientation of the blood side B (P _AtoB), to further inhibit the orientation of the material from the blood side B upon addition of the canalicular a ( and P _{BtoA ^i),} the value obtained by dividing the ratio of the rate of transport of the canalicular a to the orientation of the blood side B (P _{AtoB ⁱ⁾} is, may vary by the measurement target drug, 1.3 or more, It may be preferably 1.8 or more, more preferably 2.0 or more, and even more preferably 5.0 or more. This ratio is expressed by the following formula.

When the value of the formula (1) is 1 or more and the orientation of both is recognized, when an inhibitor is added to obtain the transport rate ratio (P _BtoA ⁱ ) / ( _PAtoB ⁱ ), the value is It can be seen that the transporter is functioning in the direction of orientation of movement because it becomes smaller and the orientation is not recognized or decreased.

On the other hand, when it is better to add an inhibitor to obtain the transport rate ratio, when _{the value of (P BtoA} ) / ( _PAtoB ) in the formula (1) becomes less than 1, for example, the drug to be measured is blood. There are cases where it is easy to go in the direction. Specifically, when the transport rate is measured by adding an inhibitor, when the value of the formula (2) is large, it seems that the blood is heading toward the blood, but bile excretion is also functioning. Is shown. In this case, it is necessary to use an inhibitor that inhibits the movement of the drug to be measured. More specifically, bromosulfophthalene (BSP) migrates in HepG2 cells by a multidrug resistance-associated protein 2 (MRP2) excretory transporter expressed on the bile duct side, and thus inhibits MK-571. Is used as an inhibitor. As another example, in the case of P-glycoprotein (P-gp) instead of MRP2, rhodamine 123 (Rho123) is used instead of BSP, and verapamil is used as an inhibitor.

Even if these hepatocytes are cultured in a normal petri dish or a plastic well plate, they do not show orientation because they are randomly present, and they gradually form colonies or form a three-dimensional mass or multiple layers. It will form.

Therefore, in order to prepare this hepatocyte culture membrane 14, it is important to culture hepatocytes on the porous plastic film 13.

When preparing the hepatocyte culture membrane 14, hepatocytes are used in a normal liquid medium or a solid medium, preferably a commercially available or time-prepared liquid medium, for example, Dalbecco-modified Eagle's medium as a culture component, and FBS (bovine) as a conditioning component. A liquid medium containing fetal serum) is used. Examples of the liquid medium include commercially available DMEM (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.).

It is more preferable that such a liquid medium further contains bile acid as an additive because the orientation of the hepatocyte culture membrane 14 is further improved. Bile acids are steroids with a tetracyclic structure that are widely found in the bile of mammals and the like, and have a basic structure in which 1 to 3 hydroxy groups are bonded to the mother nucleus of colacic acid. Specifically, cholic acid and Primary bile acids such as kenodeoxycholic acid, secondary bile acids such as deoxycholic acid and lithocholic acid that are converted by intestinal bacteria, conjugated bile acids in which they are amide-bonded to glycine or taurine, taurocholic acid, taurokenodeoxycholic Examples include acids, glycodeoxycholic acids, tauroursodeoxycholic acids, and / or lithocholic acids. Bile acids may be single or plural.

The bile acid is contained in the liquid medium at a concentration of, for example, preferably 1 μM to 300 μM, more preferably 100 μM.

To improve the orientation of the hepatocyte culture membrane 14, other additives such as dimethyl sulfoxide (DMSO), active vitamin D, azacitidine, corhitin, riphanpicin in liquid medium, in place of bile acids or with bile acids. , And a differentiation promoter that expresses enzymes and transporters, such as dexamethasone, and additives that induce orientation, such as vitamin A and cobalt chloride, may be contained.

Examples of the material of the porous plastic film 13 in the hepatocellular culture membrane 14 include a fluorine-containing resin such as polytetrafluoroethylene (PTFE), polycarbonate, polyolefin such as polyethylene, polypropylene and polystyrene, and polyester such as polyethylene terephthalate. , Polyurethane, polyamide, polyimide, cellulose, and regenerated cellulose. The porous plastic film 13 may be a plastic film or a non-woven fabric.

The porosity of the porous plastic film 13 is smaller than the size of individual hepatocytes, which is about 10 μm, and hepatocytes cannot pass through, while the molecule of the drug to be measured or its hydrate in the test solution permeates. The pores are larger than the molecular diameter of the molecule or hydrate so that it can be formed. Specifically, the porous pore diameter is, for example, 0.1 to 10 μm, preferably 0.3 to 8 μm, and more preferably the average pore diameter of the porous plastic film 13 attached to a commercially available empty kit is 0.3 μm. It is 0.4 μm, 1.0 μm, 3.0 μm, 5 μm or 8.0 μm. The thickness of the porous plastic film 13 is, for example, 1 to 100 μm, preferably 10 to 30 μm.

The porous plastic film 13 may have been subjected to tissue culture treatment that optimizes cell adhesion and proliferation, and may be coated with collagen and / or fibronectin. Further, the porous plastic film may be patterned. In the collagen-coated porous plastic film, the adhesiveness of cells 12a is improved by collagen.

As the insert container cylinder 11 having the porous plastic film 13 attached to the bottom of the cylinder and the well plate 20 having the wells 21 used for producing the drug transport ability evaluation kit 1, commercially available ones are used. The porous plastic film 13 closes the bottom of the insert container cylinder 11 by heat fusion or adhesive adhesion. The upper end peripheral edge of the insert container cylinder 11 is a collar that is wider than the upper end edge of the well 21. As a result, these are suspended so that the insert container cylinder 11 comes to the center of the insert container cylinder 11 when it is inserted into the well 21. The insert container cylinder 11 has a cylindrical shape with the same diameter in the lower half and a tubular shape and / or rib in which the upper half slightly expands toward the upper side so as to prevent the capillary phenomenon of the liquid medium from the side wall surface of the well 21. It has a tubular shape. Examples of the insert container cylinder 11 and the well plate 20 to which the porous plastic film 13 is attached and the lid 30 for closing the well plate 20 from contamination with germs during culturing include transwells, snapwells, and falcon culture inserts (all of them). (Product name manufactured by Corning Co., Ltd.) and ad-MED Vitrigel (trade name manufactured by Kanto Chemical Co., Inc.) can be mentioned.

It is difficult to store and maintain the membranous cell layer 12 in the hepatocyte culture membrane 14 in the drug transport ability evaluation kit 1 for a long period of time. Therefore, it is preferable to prepare the hepatocyte culture membrane 14 at the time of use to prepare the drug transport capacity evaluation kit 1 immediately before the measurement of the drug transport capacity evaluation method.

The drug transport capacity evaluation method using this drug transport capacity evaluation kit 1 will be described as follows with reference to FIG. 2 (a).

In the drug transport ability evaluation kit 1, the first test solution 15 is poured into the inside of the hepatocyte culture membrane-supporting insert container 10. Further, the second test solution 25 is poured into the well 21 of the well plate 20 outside the hepatocyte culture membrane-supporting insert container 10.

The basic composition liquid of the first test liquid 15 and the second test liquid 25 is a buffer solution. The buffer solution is not particularly limited as long as the hepatocellular culture membrane is not damaged, but is a phosphate buffer solution having a pH of 7.4, a phosphate buffered saline solution having a pH of 7.4, and a physiological isotonic buffer salt solution (Hanks balanced salt solution:). WBSS-HEPES (pH 7.4)).

When performing the drug transport capacity evaluation method, the first test solution 15 should be examined as to whether the administered drug is likely to enter bile excretion on the bile duct side or systemic transfer on the vascular side. And the second test solution 25, and after a while, the amount of the drug to be measured transferred through the hepatocyte culture membrane 14 composed of the membranous cell layer 12 and the porous plastic film 13. Measure. The measurement may be quantified by measuring the concentration by an optical method, such as fluorescence intensity, or transmittance or absorbance at a specific measurement wavelength such as ultraviolet light, or high performance liquid chromatography (HPLC) or liquid. It may be measured by a chromatograph tandem mass spectrometer (LC-MS / MS). If necessary, it may be quantified using a calibration curve of different standard concentrations and their transmittances and absorbances in advance.

Regarding the drug transport capacity evaluation method using this drug transport capacity evaluation kit 1, an example of a series of steps of a hepatocyte culture membrane 14 preparation step, a drug transport capacity evaluation kit 1 production step, and a subsequent drug transport capacity evaluation step. More specifically, it is as follows.

The first step is the step of preparing the hepatocyte culture membrane-supported insert container. Specifically, first, a porous plastic film 13 provided at the bottom of an empty insert container cylinder 11 for accommodating the first test liquid 15 at the time of drug transport capacity evaluation, for example, Transwell (manufactured by Corning Co., Ltd.). Cell 12a, which is a hepatocyte such as hepatocyte cell, for example, HepG2 cell, is seeded on a collagen-coated polytetrafluoroethylene membrane having a pore size of 3.0 μm at the bottom of the insert container tube of (trade name). To do.

While covering with a lid 30 so as not to be contaminated with various germs, put a normal culture solution, for example, DMEM, into the inside of the insert container cylinder 11 on the porous plastic film 13 together with the seeded cells 12a, and culture the cells. One day later, the culture medium was replaced to include, for example, bile acids such as cholic acid, deoxycholic acid, taurocholic acid, kenodeoxycholic acid, taurokenodeoxycholic acid, glycodeoxycholic acid, tauroursodeoxycholic acid, or lithocholic acid at a concentration of 100 μM. Cells 12a were cultured in the same culture medium for 1 to 4 weeks, for example, 7th day, 14th day, or 21st day, except that they were contained, and appropriately, for example, 4th day, 7th day, 10th day 14 When the cells 12a are continuously cultured by exchanging with the same bile acid-containing culture medium at an appropriate time such as the day, the cells 12a proliferate and become a membranous cell layer 12 with the porous plastic film 13, and the hepatocellular culture is performed. The film 14 is formed.

The membranous cell layer 12 is a monolayer membrane in which cells 12a are grown in a monolayer without gaps. As a result, a hepatocyte culture membrane 14 composed of the membranous cell layer 12 and the porous plastic film 13 is formed. The membranous cell layer 12 is oriented so that the lower side of the membranous cell layer 12, that is, the side of the porous plastic film 13 is the blood vessel side, and the upper side of the membranous cell layer 12, that is, the side opposite to the porous plastic film 13 is the bile duct side. Has sex. Whether the porous plastic film 13 is superior to bile excretion from the liver or systemic transfer is to be examined. From the test solution in which the drug to be measured is dissolved, the drug is either front or back of the porous plastic film 13. However, due to the orientation of the membranous cell layer 12, the direction from the blood side B to the bile duct side A is easier to permeate than the direction from the bile duct side A to the blood side B. ing. As a result, the hepatocyte culture membrane-supporting insert container 10 on which the oriented hepatocyte culture membrane is formed is prepared.

Another step is the step of preparing and preparing the well plate 20. Specifically, it has a well 21 into which the hepatocyte culture membrane-supporting insert container 1 is inserted, and is empty for containing a second test solution in which the hepatocyte culture membrane 14 is immersed in the evaluation of drug transport capacity. Well plate 20 is prepared. Such a well plate 20 is a commercially available well plate having multiple wells such as 1 well, 6 wells, 12 wells or 24 wells of Transwell (trade name manufactured by Corning Inc.).

As a result, the drug transport ability evaluation kit 1 including the hepatocyte culture membrane-supported insert container 10 and the well plate 20 is produced.

Subsequently, in order to evaluate the drug transport capacity, the blood side transport volume measurement step and the bile duct side transport volume measurement step are performed, and then the drug transport capacity calculation step is performed. Specifically, on the 7th day of culture, which is the time to perform the drug transport ability evaluation method, the culture solution collected in the hollow of the insert container cylinder 11 of the hepatocyte culture membrane-supporting insert container 10 is taken out.

Next, the blood transport volume measurement step is performed as follows. The drug to be measured for drug transport ability is contained in a buffer solution to prepare the first test solution 15. The first test solution 15 containing the drug whose drug transport capacity is to be measured is slowly poured into the hepatocyte culture membrane-supporting insert container 10 so that the membrane cell layer 12 does not come off, and is contained therein. On the other hand, a second test solution 25 composed of only a buffer solution containing no drug to be measured is prepared. The second test solution 25 is poured into the well 21 of the well plate 20, and the hepatocyte culture membrane 14 is immersed in the second test solution 25. Then, the amount of the drug to be measured from the bile duct side A to the blood side B is measured. For the measurement, the concentration of the drug to be measured in the first test solution 15 and the concentration of the drug to be measured in the second test solution 25 are measured, and the amount transported to the blood side is quantified. For example, when the drug to be measured is Rho123, which is a standard substrate of transporter P-gp and is a fluorescent substance, the concentration is measured from the amount of fluorescence.

Next, the bile duct side transport amount measurement step is performed as follows. Using the same hepatocyte culture membrane-supporting insert container 10 and well plate 20, the hollow and well 21 of the insert container cylinder 11 are washed with a buffer solution, or a new hepatocyte culture membrane-supporting insert container 10 and well plate are used. Prepare 20. Another first test solution 15 consisting of only a buffer solution containing no drug to be measured is prepared, and slowly poured into the hepatocyte culture membrane-supporting insert container 10 so that the membranous cell layer 12 does not peel off, and the container is contained. On the other hand, another drug to be measured for drug transport capacity is contained in a buffer solution to prepare another second test solution 25. The second test solution 25 is poured into the well 21 of the well plate 20, and the hepatocyte culture membrane 14 is immersed in the second test solution 25. Then, the amount of the drug to be measured from the blood side B to the bile duct side A is measured in the same manner. The measurement of the transport amount may be a quantification by high performance liquid chromatography using a concentration-area calibration curve, a quantification using LC-MS / MS, or a specific color quantification, for example, a ratio by fluorescence absorption. It may be color quantitative. The measurement may be performed by a radioactive isotope. The measurement sensitivity depends on the measurement method.

Finally, the drug transport capacity calculation step is performed as follows. The ratio of the amount of the drug to be measured from the bile duct side A to the blood side B and the amount of the drug to be measured from the blood side B to the bile duct side A is calculated. Specifically, in a plurality of cases (for example, n = 3), the amount of the drug to be measured from the bile duct side A to the blood side B and the amount of the drug to be measured from the blood side B to the bile duct side A are determined. _{Measured every predetermined time, for example, every 15 minutes, the inclination AtoB} is obtained from a linear approximation formula with the horizontal axis as time and the vertical axis as the amount of transport from the bile duct side A to the blood side B, and the transport rate per unit area of the cell. _{(Cm / sec) is calculated, while in the same way, the slope BtoA} is calculated from a linear approximation formula with the horizontal axis as time and the vertical axis as the amount of transport from the blood side B to the bile duct side A, and per unit area of the cell. Calculate the transport speed (cm / sec). The ratio of these slopes, that is, (slope _BtoA ) / (slope _AtoB ) is calculated to determine the drug transport capacity of the drug to be measured.

Hereinafter, an example in which the hepatocyte culture membrane of the present invention and the drug transport ability evaluation kit provided with the hepatocyte culture membrane were prepared and the drug transport ability evaluation method was performed using the kit will be described in detail below.

(Example 1)
As shown in FIG. 2, specific examples of the hepatocyte culture membrane 14, the drug transport ability evaluation kit 1, and the drug transport ability evaluation method are shown below with respect to an example using HepG2 cells (JCRB).
[material]
DMEM (high glucose) (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.), FBS (manufactured by biosera), deoxycholic acid (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.), BSP (manufactured by MP Biochemicals) were used as reagents. ..
[Cell culture]
HepG2 was cultured in DMEM supplemented with 10% FBS. The 12-well inserts were ^{seeded at approximately 2 × 10 5} pieces / cm ² , and the medium was replaced with a medium supplemented with 100 μM bile acid only on the bile duct side on the 1st and 4th days after seeding, and the medium was replaced on the blood vessel side. It was used in the following experiments on the 7th day of culture.
[Transportation experiment]
HBSS-HEPES (pH 7.4) was used as the transport buffer.
The substrate drug was 24 μMBSP.
The membrane resistance value of the cultured plate was measured using Millicell ERM2 (manufactured by Merck Millipore) to confirm the formation of the cell layer on the membrane.
Well inserts were washed with 0.9% NaCl.
The substrate drug (BSP) of the transporter was used as the transport substance, and in the case of transport from the A side to the B side, the substrate drug was used as the first test solution and the transport buffer was used as the second test solution. In the case of transport from the B side to the A side, the transport buffer was used as the first test solution and the substrate drug was used as the second test solution. The work was carried out on a 37 ° C. water bath.
In the case of a 12-well insert, the amount of buffer was 0.5 mL for the first test solution and 1.5 mL for the second test solution.
After 15, 30, 45 and 60 minutes, 10% of the liquid volume was collected from the opposite side to which the transport drug was added. An equal amount of buffer collected each time was added.
The concentration of the collected sample was measured by HPLC (manufactured by Shimadzu Corporation, trade name: DIOD E ARRAY DETECTOR SPD-M20A as a detector set).
[Calculation method]
From the obtained concentration, the transport speed (P (cm / sec)) in each transport direction (A to B, B to A) is calculated, and the Efflux ratio (P of B to A / P of A to B) is calculated. Calculated. The calculation method is as follows.

(In formula (3), Q: amount of substance (mol), t: time (sec), A: hepatocyte culture membrane area (cm ² ), C ₀ : addition concentration (μM))

The results are shown in Table 1.

(Example 2)
The examples using HuH-7 cells (JCRB) and the examples performed according to Example 1 are shown in detail below.
[material]
As reagents, DMEM (low glucose) (manufactured by SIGMA), Rho123 (manufactured by SIGMA), verapamil (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.), and other materials were in accordance with Example 1.
[Cell culture]
HuH-7 was cultured in DMEM (low glucose) supplemented with 10% FBS.
The 24-well inserts were ^{sown at approximately 2 × 10 5} pcs / cm ² , and the medium was changed 1 day and 4 days after sowing. It was used in the following experiments on the 7th day of culture.
[Transportation experiment]
The substrate drug was 10 μM Rho123 and the inhibitor was 100 μM verapamil.
The membrane resistance value of the cultured plate was measured using Millicell ERM2 (manufactured by Merck Millipore) to confirm the formation of the cell layer on the membrane.
Well inserts were washed with 0.9% NaCl.
When no inhibitor was added, the transport buffer was used as the first test solution and the second test solution, and incubated at 37 ° C. for 10 minutes.
Then, the substrate drug (Rho123) of the transporter was used as a transport substance, and in the case of transport from the A side to the B side, the substrate drug was used as the first test solution and the transport buffer was used as the second test solution. In the case of transport from the B side to the A side, the transport buffer was used as the first test solution and the substrate drug was used as the second test solution.
When the inhibitor was added, the solution containing the inhibitor was used as the first test solution and the second test solution, and incubated at 37 ° C. for 10 minutes.
Then, in the case of transport from the A side to the B side, the solution containing the substrate drug and the inhibitor was used as the first test solution, and the transport buffer containing the inhibitor was used as the second test solution. In the case of transport from the B side to the A side, the transport buffer containing the inhibitor was used as the first test solution, and the solution containing the substrate drug and the inhibitor was used as the second test solution.
All work was done on a 37 ° C water bath.
The collected sample was measured with a fluorescence plate reader (manufactured by Perkin Elmer, product name: ARVO MX) at an excitation wavelength of 485 nm and a fluorescence wavelength of 535 nm.
Other conditions were in accordance with Example 1.
[Calculation method]
From the obtained concentration, the transport speed (P (cm / sec)) in each transport direction (A to B, B to A) is calculated, and the Efflux ratio (P of B to A / P of A to B) is calculated. Calculated. Further, the value obtained by dividing the Efflux ratio (ER _n ) when only the substrate drug was used by the Efflux ratio (ER _{i) in the presence of the inhibitor was calculated.} The calculation method is as follows.

(In formula (3), n: no inhibitor added, i: inhibitor added)
The results are shown in Table 1.

As is clear from Table 1, according to the hepatocyte culture membranes of Examples 1 and 2, the drug transport capacity evaluation kit provided with the hepatocyte culture membrane, and the drug transport capacity evaluation method using the hepatocyte culture membrane, bile excretion from the liver and systemic transfer were observed. It was confirmed in an in vitro experimental system that it can be investigated appropriately, easily, and quickly as a model corresponding to in vivo dynamics, especially in humans, to determine which of these is superior.

The hepatocyte culture membrane of the present invention, a drug transport ability evaluation kit provided with the hepatocyte culture membrane, and a drug transport ability evaluation method using the same are used for examining the pharmacokinetics of a drug.

1 is a drug transport capacity evaluation kit, 10 is a hepatocyte culture membrane-supporting insert container, 11 is an insert container tube, 12 is a membranous cell layer, 12a is a cell, 13 is a porous plastic film, 14 is a hepatocyte culture membrane, 15 Is the first test solution, 20 is the well plate, 21 is the well, 25 is the second test solution, 30 is the lid, A is the bile duct side, and B is the blood vessel side.

Claims

At least one of hepatocyte, bile duct cancer cell, free hepatocyte, animal-derived fresh humanized hepatocyte, iPS cell and / or ES cell induced by hepatocyte, organoid, and immortalized cell. The cells form a membranous cell layer cultured in a membranous manner on a porous plastic film, and the cells are on the vascular side below the membranous cell layer and on the upper side of the membranous cell layer. A hepatocyte culture membrane characterized by having orientation on the bile duct side.
The hepatocyte culture membrane according to claim 1 is provided at the bottom and has a hepatocyte culture membrane-supporting insert container for containing the first test solution and a well into which the hepatocyte culture membrane-supporting insert container is inserted. A drug transport ability evaluation kit comprising a well plate containing a second test solution in which the hepatocyte culture membrane is immersed.
The second aspect of claim 2, wherein the porous plastic film is made of any plastic selected from a fluororesin, polycarbonate, polyolefin, polyester, polyurethane, polyamide, polyimide, cellulose, and regenerated cellulose. Drug transport capacity evaluation kit.
The drug transport ability evaluation kit according to claim 2, wherein the porous plastic film has a porous size smaller than the size of the hepatocyte, or the openings of the porous structure are arranged in a pattern.
The drug transport ability evaluation kit according to claim 2, wherein the porous plastic film is coated with collagen and / or fibronectin.
The drug transport ability evaluation kit according to claim 2, wherein at least one of the first test solution and the second test solution contains bile acid.
Using the drug transport ability evaluation kit according to any one of claims 2 to 6, the first test solution inside the hepatocyte culture membrane-supporting insert container and the outside of the hepatocyte culture membrane-supporting insert container. The drug to be measured is added to any of the second test solution inside the well of the well plate, and the hepatocyte culture membrane of the membranous cell layer and the porous plastic film is passed through. A method for evaluating a drug transport ability, which comprises evaluating the drug transport capacity by measuring the transport amount of the drug to be measured over time and calculating the transport speed.
Induced to hepatocytes, bile duct cancer cells, free hepatocytes, animal-derived humanized fresh hepatocytes, and hepatocytes on a porous plastic film provided at the bottom of the insert container tube containing the first test solution. By seeding and culturing at least one of iPS cells and / or ES cells, organoids, and immortalized cells to form a membranous cell layer, the membranous cell layer is formed. A hepatocyte culture membrane-bearing insert container is prepared by forming a hepatocyte culture membrane having an orientation that is on the blood vessel side below the membranous cell layer and on the bile duct side above the membranous cell layer. Process and
A step of preparing a well plate for containing a second test solution in which the hepatocyte culture membrane-supporting insert container is inserted and in which the hepatocyte culture membrane is immersed.
The first test solution containing the drug whose drug transport capacity is to be measured is contained in the hepatocyte culture membrane-supporting insert container, and the well of the well plate contains the second test solution which does not contain the drug to be measured. A blood-side transport amount measurement step of measuring the transport amount of the drug to be measured from the bile duct side to the blood side after containing the liquid and immersing the hepatocyte culture membrane in the second test solution.
The first test solution containing no drug to be measured is contained in the hepatocyte culture membrane-supporting insert container, and the second test solution containing the drug to be measured is contained in the well of the well plate. After immersing the hepatocyte culture membrane in the second test solution, the bile duct side transport amount measuring step for measuring the transport amount of the drug to be measured from the blood side to the bile duct side.
The process of calculating the transportation speed by measuring each transportation amount over time, and
A drug transport capacity calculation step for calculating the ratio of the blood transport rate and the bile duct transport rate to obtain the drug transport capacity of the drug to be measured.
A drug transport ability evaluation method characterized by having.
During the blood-side transport amount measurement step, a transporter inhibitor is further added to the first test solution and the second test solution, and during the bile duct-side transport amount measurement step, the first test is further performed. A transporter inhibitor was added to the solution and the second test solution, and the bile duct side transport amount measurement step was also performed for them. In the drug transport capacity calculation step, the blood transport rate without the inhibitor added. The claim is characterized in that the ratio of the bile duct side transport rate to the ratio of the blood side transport rate and the bile duct side transport rate with the addition of the inhibitor is calculated to obtain the drug transport capacity of the drug to be measured. 10. The drug transport evaluation method according to 10.
At least one of hepatocyte, bile duct cancer cell, free hepatocyte, animal-derived fresh humanized hepatocyte, iPS cell and / or ES cell induced by hepatocyte, organoid, and immortalized cell. In order to culture cells on a porous plastic film to form a membranous cell layer, and to express an orientation in which the lower side of the membranous cell layer is the blood vessel side and the upper side of the membranous cell layer is the bile duct side. An orientation-enhanced hepatocyte medium, which comprises a culture component and bile acid.