Cholesterol-free high-density lipoprotein particles

Abstract

To provide a particle that has high cholesterol uptake capacity and can be utilized as a cholesterol transporter, and a method for producing the particle.SOLUTION: The present invention provides a high-density lipoprotein particle that does not contain, at least, cholesterol, the particle being collected from a culture of hepatocytes; and a method for producing the particle, characterized by culturing hepatocytes and collecting the high-density lipoprotein particle that does not contain, at least, cholesterol, from the obtained culture.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to cholesterol-free high-density lipoprotein particles. The particles have cholesterol-removing ability and are used as cholesterol carriers.

Background Art

[0002] High-density lipoprotein (HDL) has been reported to be deeply related to the suppression of arteriosclerosis (Non-Patent Documents 1 to 3). For this reason, the development of therapeutic agents aimed at controlling the amount of HDL in the blood has been promoted, but cases where satisfactory drug effects have not been obtained in clinical trials have been reported (Non-Patent Documents 4 to 7). In recent years, it has become clear that there are differences in the ability to remove cholesterol from peripheral cells, so-called Cholesterol Uptake Capacity (CUC), depending on the type of HDL (Non-Patent Document 8). From this, the recognition that increasing HDL with high CUC has become an important point required for therapeutic agents has been increasing.

[0003] HDL is mainly newly produced in the liver and then secreted into the blood, and extracts and takes up free cholesterol from peripheral tissues by a transporter called ABCA1 (HDL-CUC). The free cholesterol in HDL is esterified by lecithin-cholesterol acyltransferase to become cholesterol ester. This cholesterol ester is exchanged with neutral fat contained in VLDL and LDL, and HDL matures. Mature HDL is taken up via the HDL receptor in the liver and then regenerated. Thus, paying attention to lipid metabolism such as CUC in HDL is an urgent matter for the development of the above-mentioned therapeutic agents, and further, it is expected to greatly contribute to understanding the whole picture of the lipid metabolism function of hepatocytes (especially human hepatocytes) responsible for the de novo synthesis, uptake, and regeneration of HDL. As cells having a lipid metabolism function equivalent to or similar to that of human hepatocytes, chimeric mice (PXB mice) in which 70% or more of the liver is replaced with human hepatocytes are known (Non-Patent Document 9). In PXB mice, lipid transport between HDL and low density lipoprotein (LDL) is carried out by Cholesterol Ester Transport protein secreted by human hepatocytes, and thus it has been confirmed that the lipoprotein profile in the blood shows a configuration similar to that of humans (Non-Patent Document 10). That is, it has been proven that hepatocytes (PXB-cells) isolated from PXB mice also have a lipid metabolism ability similar to that of fresh human hepatocytes (Non-Patent Document 11).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

[0005] Against this background, there has been a demand for the development of particles having a high cholesterol extraction ability and used as cholesterol carriers. As a result of intensive studies to solve the above problems, the present inventors have found that high-density lipoprotein particles contained in a culture of human fresh hepatocytes or cells having a similar lipid metabolism function (for example, hepatocytes derived from PXB mice) have cholesterol extraction ability, and have completed the present invention. [Means for Solving the Problems]

[0006] That is, the present invention is as follows. [1] At least cholesterol-free high-density lipoprotein particles collected from a culture of hepatocytes. [2] The particles according to [1], wherein the hepatocytes are human hepatocytes or non-human-derived hepatocytes having a lipid metabolism function equivalent to that of the human hepatocytes. [3] The particles according to [2], wherein the non-human-derived hepatocytes are hepatocytes derived from a non-human animal in which at least 70% of the liver has been replaced with human hepatocytes. [4] The particles according to [1], which have cholesterol extraction ability. [5] The particles according to [3], wherein the non-human animal is a mouse. [6] A cholesterol carrier comprising the particles according to any one of [1] to [5]. [7] The carrier according to [6], which extracts cholesterol in a tissue and transports it to the liver. [8] The carrier according to [7], wherein the tissue is a cardiovascular tissue. [9] The carrier according to [8], wherein the cholesterol in the cardiovascular tissue is cholesterol in atherosclerotic plaques.

[10] A method for producing the particles, comprising culturing hepatocytes and collecting at least cholesterol-free high-density lipoprotein particles from the resulting culture.

[11] The method according to

[10] , wherein the hepatocytes are human hepatocytes or non-human-derived hepatocytes having a lipid metabolism function equivalent to that of the human hepatocytes.

[12] The method according to

[10] , wherein the non-human-derived hepatocytes are hepatocytes derived from a non-human animal in which at least 70% of the liver has been replaced with human hepatocytes.

[13] The method according to

[10] , wherein the high-density lipoprotein particles have cholesterol extraction ability.

[14] The method according to

[12] , wherein the non-human animal is a mouse.

[15] A pharmaceutical composition for circulatory diseases, comprising the particles according to any one of [1] to [5].

[16] A pharmaceutical composition for circulatory diseases, comprising the carrier according to [6].

[17] The pharmaceutical composition according to

[15] , wherein the circulatory disease is at least one disease selected from the group consisting of hypercholesterolemia, familial hypercholesterolemia, atherosclerosis, peripheral vascular disease, dyslipidemia, angina pectoris, ischemia, stroke, myocardial infarction, hypertension, and vascular complications of diabetes.

[18] The pharmaceutical composition according to

[16] , wherein the circulatory disease is at least one disease selected from the group consisting of hypercholesterolemia, familial hypercholesterolemia, atherosclerosis, peripheral vascular disease, dyslipidemia, angina pectoris, ischemia, stroke, myocardial infarction, hypertension, and vascular complications of diabetes. [Advantages of the Invention]

[0007] According to the present invention, cholesterol-free HDL is provided. The HDL of the present invention is characterized by not containing cholesterol and has a high cholesterol recovery ability, so it is useful as a cholesterol carrier. [Brief Description of the Drawings]

[0008]

Figure 1

Figure 2

Figure 3

Figure 4

[0009] 1. Summary The present invention relates to at least cholesterol-free high-density lipoprotein particles. The particles of the present invention are collected from a hepatocyte culture and have the ability to extract cholesterol. High-density lipoprotein (HDL), which is a type of lipid particle, has the function of extracting (recovering) cholesterol contained in tissues and organs in the body. In recent years, restoring the cholesterol recovery function of HDL has been becoming a target for drug discovery. And HDL contained in the blood of animals including humans always contains cholesterol, although there are some differences.

[0010] However, in the present invention, a hepatocyte culture, particularly the culture supernatant, was fractionated into very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and HDL fractions, and as a result of confirming the amount of cholesterol contained in each fraction, almost no cholesterol was contained in the HDL fraction (Figure 4). In Figure 4, the upper part shows the cholesterol content and the lower part shows the triglyceride content. It was confirmed that there were almost no peaks for both cholesterol and triglyceride in the HDL fraction. It was unclear whether the absence of cholesterol in the HDL fraction in the above culture supernatant was due to the absence of HDL itself or the absence of the function of HDL to absorb cholesterol although HDL exists.

[0011] Therefore, in the present invention, the protein (APOA1) constituting HDL was semi-quantified and the cholesterol recovery function of HDL was measured. As a result, it was confirmed that HDL capable of extracting cholesterol exists in the above culture supernatant.

[0012] The present invention has been completed based on the above findings. Figure 1 is a schematic diagram of the high-density lipoprotein particles of the present invention (also referred to as "the particles of the present invention"). In Figure 1, Panel A is a schematic diagram of HDL that normally exists. HDL is a spherical particle as a whole, forming two regions: a surface layer 10 (outer shell) and an interior 20 (inner shell). The surface layer 10 of HDL is a membrane composed of apolipoprotein, phospholipid, and free cholesterol, and its interior 20a is mainly occupied by cholesterol, fat-soluble vitamins, and neutral fat. Panel B is a schematic diagram of the HDL particles obtained by the present invention.

[0013] In the particles of the present invention, the surface layer 10 is a membrane composed of apolipoprotein, phospholipid, and free cholesterol, and the interior 20b contains fat-soluble vitamins but does not contain at least cholesterol (cholesterol ester). In one aspect of the present invention, neither cholesterol nor neutral fat is contained. Here, the terms "not containing" or "free of" for neutral fat and cholesterol mean not containing at all (content rate is zero) or substantially not containing, meaning that the neutral fat in the particles is 5% or less, preferably 1% or less, and the cholesterol in the particles is 10% or less, preferably 7% or less.

[0014] 2. Cultured cells The cells used for culturing to obtain the particles of the present invention are human hepatocytes or cells having lipid metabolism functions similar to or analogous to the human hepatocytes. (1) Human hepatocytes Human hepatocytes (liver parenchymal cells) can be collected from liver tissues collected by biopsy or surgery. It is also possible to use established human hepatocyte cell lines. Liver tissues collected by biopsy or surgery are made into a cell dispersion by a conventional method such as collagenase perfusion method. The obtained dispersed cells are replaced into a culture medium such as DMEM and then filtered using gauze or mesh. Since the obtained cell suspension contains Kupffer cells, stellate cells, bile duct epithelial cells, sinusoidal endothelial cells, liver stem cells, etc. in addition to liver parenchymal cells, the target fresh hepatocytes can be obtained by centrifugation. For example, since the specific gravity of liver parenchymal cells is heavy, they can be recovered by centrifugation at 50×g for about 2 minutes. The recovered fresh human hepatocytes are used for the culture described below.

[0015] (2) Cells having a lipid metabolism function equivalent or similar to that of human hepatocytes As another aspect of the present invention, in addition to the above human fresh hepatocytes, cells having a lipid metabolism function the same as or similar to that of human hepatocytes (referred to herein as "equivalent hepatocytes") can be used. "Equivalent or similar" means a function that can produce the same value within at least 3-fold range, for example, within 3-fold range, within 2.5-fold range, within 2-fold range, or within 1.5-fold range, compared with the lipid metabolism function of human fresh hepatocytes. For example, consider a system for measuring lipoproteins as a lipid metabolism function derived from human hepatocytes. If the secretion amount of lipoproteins of human fresh hepatocytes is x mg, cells that secrete within 1 / 3 to 3 times of x mg can be said to be equivalent hepatocytes. Examples of such equivalent hepatocytes include cells derived from non-human animals in which at least 70% of the liver has been replaced with human hepatocytes (hereinafter referred to as "human hepatocyte chimeric animals"), or cells obtained by introducing a gene into somatic cells and differentiating them into hepatocytes.

[0016] (i) Cells derived from human hepatocyte chimeric animals The cells used for the culture to obtain the particles of the present invention are hepatocytes derived from non-human animals in which part or all of the hepatocytes in the liver have been replaced with human hepatocytes. In the present specification, the non-human animal is referred to as a "human hepatocyte chimeric non-human animal". The "non-human animal" is preferably a mammal, and more preferably a rodent. Examples of rodent animals include mice, rats, guinea pigs, squirrels, hamsters, etc., but mice or rats, which are widely used as experimental animals, are preferred. The human hepatocyte chimeric non-human animal can be obtained by transplanting human hepatocytes into a liver-disabled immunodeficient non-human animal according to a known method (for example, Japanese Patent Application Laid-Open No. 2002-45087).

[0017] Hepatic injury immunodeficient non-human animals can typically be produced by subjecting genetically immunodeficient non-human animals to a hepatic injury induction treatment or subjecting animals with genetic hepatic injury to an immunodeficiency induction treatment. Examples of genetically immunodeficient animals include SCID mice, nude mice, RAG2 knockout mice, NOD mice, NOG mice, etc. As animals with genetic hepatic injury, transgenic animals into which a hepatic injury-inducing protein gene (such as uPA gene, tPA gene, etc.) linked under the control of an enhancer and / or promoter of a protein specifically expressed in hepatocytes are introduced can be utilized.

[0018] In the present invention, the "human hepatocytes" transplanted into hepatic injury immunodeficient non-human animals may be human-derived hepatocytes. For example, human hepatocytes isolated from human liver tissue according to conventional methods such as the collagenase perfusion method can be used. The human liver tissue may be from a healthy human or from a patient suffering from a disease such as fatty liver or liver cancer, but preferably it is from a healthy human. The isolated human hepatocytes can be used as they are or can be further purified before use.

[0019] Transplantation of human hepatocytes into hepatic injury immunodeficient non-human animals is carried out by transplanting them into the liver via the spleen of the non-human animal or directly from the portal vein. The non-human animals obtained by the above method are animals in which at least 70% of the liver has been replaced by human hepatocytes. As mice in which at least 70% of the liver has been replaced by human hepatocytes, commercially available products ("PXB mice", Phoenix Bio Co., Ltd.) can also be used.

[0020] Recovery of human hepatocytes from chimeric non-human animals can be carried out according to conventional methods such as the collagenase perfusion method. The recovered human hepatocytes can be used as they are, but they may also be purified using monoclonal antibodies that specifically recognize human hepatocytes or hepatocytes of non-human animals. In this specification, the hepatocytes collected from the above-mentioned "PXB mice" are also referred to as "PXB-cells". In the present invention, hepatocytes collected from the above-described human hepatocyte chimeric animal are used for the following culture.

[0021] (ii) Cells differentiated into hepatocytes In another aspect of the present invention, genes can also be introduced into somatic cells to differentiate them into hepatocytes. Methods for differentiating somatic cells into hepatocytes by gene transfer into somatic cells are known, and methods using induced pluripotent stem cells (iPS cells) or direct reprogramming methods for directly reprogramming somatic cells into hepatocytes can be employed. For example, by artificially expressing four transcription factors, Oct4, Sox2, Klf4, and c-Myc, which are highly expressed in embryonic stem cells, in somatic cells, the somatic cells can be reprogrammed to obtain induced pluripotent stem cells (iPS cells). After obtaining iPS cells, they can be differentiated into hepatocytes by culturing them in a medium containing fibroblast growth factor (FGF), bone morphogenetic protein (BMP), hepatocyte growth factor (HGF), etc. alone or in an appropriate combination. The hepatocytes obtained as described above are used for the following culture. Also, as a direct reprogramming method, a method of introducing two transcription factors (Hnf4α and Foxa1, Hnf4α and Foxa2, or Hnf4α and Foxa3) into somatic cells is known.

[0022] 3. Cell culture The culture vessel used for cell culture is not particularly limited as long as it is suitable for adherent culture. Examples include dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, tubes, culture flasks, culture bags, etc. In the present invention, the culture of human hepatocytes or equivalent hepatocytes can be carried out using a medium generally used for the culture of animal cells. Examples of such media include Dulbecco's modified Eagle's medium (DMEM), Williams' medium E, RPMI-1640 medium, etc., and preferably DMEM. However, the medium is not limited to these.

[0023] To the medium, fetal bovine serum, buffer, antibiotic, pH adjuster, etc. can be appropriately added as needed. Hepatocytes are 4 cells / cm 2 ~2.1×10 6 cells / cm 2 , preferably 1.9×10 5 cells / cm 2 ~2.3×10 5 cells / cm 2 seeded on the culture vessel at a cell density of. The culture can be carried out at 30 - 40 °C, preferably about 37 °C, in an atmosphere with a CO2 concentration of 1 - 10%, preferably about 5% in a CO2 incubator.

[0024] The culture period of human hepatocytes or equivalent hepatocytes may be a period sufficient for the cells to secrete and / or accumulate the particles of the present invention, for example, 6 days to 3 weeks, preferably 2 weeks. The medium is partially or completely replaced with fresh medium every 1 - 4 days. After the completion of the culture, the particles of the present invention are collected from the culture. The culture means any of the culture supernatant, cultured cells, and disrupted products of the cultured cells. To collect the particles of the present invention, centrifugation method, precipitation method, direct method, gel filtration method, etc. can be employed.

[0025] 4. Cholesterol-free HDL particles (1) Structure of the HDL particles of the present invention As described above, the HDL particles of the present invention have a membrane in the surface layer portion composed of apoprotein, phospholipid, and free cholesterol, and the inside has a structure that contains fat-soluble vitamins but contains at least no cholesterol, or contains neither cholesterol nor neutral fat.

[0026] Examples of the apolipoprotein constituting the HDL particles of the present invention include apolipoprotein A1 (also referred to as "APOA1". Other apolipoproteins can be expressed in the same way.), apolipoprotein A-II, apolipoprotein A-VI, apolipoprotein A-V, apolipoprotein C-I, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein C-IV, apolipoprotein D, apolipoprotein E, apolipoprotein F, apolipoprotein H, apolipoprotein J, apolipoprotein L-I, and apolipoprotein M.

[0027] (2) Confirmation that the HDL particles of the present invention do not contain cholesterol Confirmation (test method) that the HDL particles of the present invention do not contain cholesterol can be performed by gel filtration HPLC (LipoSEARCH) or the like.

[0028] (3) Cholesterol uptake ability For the cholesterol uptake ability, after contacting labeled cholesterol with HDL, HDL is captured by magnetic beads to which an HDL capture antibody is immobilized. Next, a detection antibody that binds to the labeled cholesterol is reacted with the complex of the magnetic beads, the HDL capture antibody, and HDL, and the signal is detected. The larger the signal amount, the more it indicates that cholesterol has been taken up by the HDL particles of the present invention.

[0029] In addition, although the HDL particles of the present invention are contained in the cell culture solution, it is possible to evaluate the cholesterol uptake ability using the same procedure as HDL contained in animal serum. That is, after reacting the cell culture solution with polyethylene glycol 4000, APOB is removed by centrifugation, and cholesterol is taken up into HDL by incubating after mixing the remaining supernatant solution fraction containing HDL and labeled cholesterol. The method for capturing HDL and detecting labeled cholesterol is as described above.

[0030] 5. Cholesterol carrier The particles of the present invention can be used as carriers that extract cholesterol from tissues and transport it to the liver. As the particle size of the particles of the present invention, those having an average particle diameter of 7.5 to 20 nm, preferably about 7.6 to 15 nm can be used. Those having a particle size within this range serve as carriers suitable for movement within capillaries in organs and tissues while ensuring a sufficient cholesterol loading amount.

[0031] The lower limit value of the average particle diameter is 7.5 nm or more, and the upper limit value is 20 nm or less. Therefore, the range of the average particle diameter is, for example, 7.5 nm to 18 nm, preferably 7.6 nm to 15 nm.

[0032] Since the particles of the present invention are substances constituting HDL, they can be safely used as cholesterol delivery carriers. Therefore, in the medical field, cholesterol can be extracted from cardiovascular tissues, other organs and tissues in the body, accumulated in the particles, and transported to the liver. In particular, in cardiovascular tissues, it is excellent in the ability to extract cholesterol from atherosclerotic plaques.

[0033] 6. Pharmaceutical composition The particles of the present invention can further be used as a pharmaceutical composition for cardiovascular diseases. Examples of the target cardiovascular diseases include at least one disease selected from the group consisting of hypercholesterolemia, familial hypercholesterolemia, atherosclerosis, peripheral vascular diseases, dyslipidemia, angina pectoris, ischemia, stroke, myocardial infarction, hypertension, and vascular complications of diabetes.

[0034] As administration forms of the pharmaceutical composition of the particles of the present invention, there are parenteral administration and oral administration. Examples of parenteral administration routes include intravenous injection (IV) such as drip infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. Examples of oral administration include tablets, capsules, granules, powders, syrups, etc. These forms are manufactured by known methods and contain carriers, diluents, and excipients commonly used in the pharmaceutical field. For example, an injection is prepared by dissolving, suspending, or emulsifying the particles of the present invention in a sterile aqueous or oily liquid usually used for injections. As the aqueous liquid for injection, physiological saline, glucose, and other isotonic solutions containing auxiliary drugs are used, and appropriate solubilizing agents such as polyalcohols such as alcohol and propylene glycol, and nonionic surfactants can be used in combination.

[0035] The pharmaceutical composition containing the particles of the present invention is administered in a sufficient amount to cure or at least partially prevent the symptoms of the disease in patients with cardiovascular diseases. The administration method can be appropriately selected according to the age, symptoms, etc. of the patient. The effective dose of the pharmaceutical composition of the present invention is determined by reaching a total serum HDL particle number of 27.0 μmol / L or more, or a serum concentration of small HDL or very small HDL defined by a particle diameter of 7.6 nm to 9.8 nm reaching 23.0 μmol / L or more. However, the pharmaceutical composition of the present invention is not limited to these doses. The administration time may be after the disease occurs, or may be prophylactically administered for symptom relief at the time of onset when the onset of the disease is predicted.

Example

[0036] Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these examples.

[0037] 1. Culture of PXB-cells Human hepatocytes were isolated from PXB mice prepared by transplanting JFC (Bio reclamation IVT: Caucasian, 1Y, Male) into cDNA-uPA wild / + / SCID mice using the collagenase perfusion method. The cell viability was over 80%.

[0038] DMEM + 10% FBS was added to PXB-cells to prepare a cell suspension with a cell density of 8×10 5 cells / mL (2.1×10 5 cells / cm 2 ). The cell suspension was added to a 24-well plate (BioCoat TM Collagen I 24 well plate; Corning Japan) at 0.5 mL per well. The plate with the added cell suspension was left standing at room temperature for 15 - 30 minutes. After confirming that the cells had lightly adhered to the bottom of the well, they were cultured in an incubator (37°C, 5% CO2). The start day of the culture was designated as Day0.

[0039] <HDL-CUC measurement from Day6 to 8> The medium was changed to dHCGM at 0.5 mL per well on Day1 and Day2. Then, on Day6, it was changed to William’s E + CM-4000 at 0.5 mL per well, and the culture supernatant was collected on Day8, 2 days later. The collected supernatant samples were used for the analysis of the cholesterol uptake ability by HDL (HDL-CUC). (Corresponding to 8 days in Figure 3)

[0040] <HDL-CUC measurement from Day13 to 15> The medium was changed to dHCGM at 0.5 mL per well on Day1, Day2, Day6, and Day9. Then, on Day13, it was changed to William’s E + CM-4000 at 0.5 mL per well, and the culture supernatant was collected on Day15, 2 days later. The collected supernatant samples were used for the analysis of the cholesterol uptake ability by HDL (HDL-CUC). (Corresponding to 15 days in Figure 3)

[0041] <HDL-CUC measurement from Day 20 to 22> The medium was changed to dHCGM at 0.5 mL per well on Day 1, Day 2, Day 6, Day 9, Day 13, and Day 16. Then, on Day 20, it was changed to William’s E + CM-4000 at 0.5 mL per well, and the culture supernatant was collected on Day 22, two days later. The collected supernatant samples were used for the analysis of the cholesterol uptake ability by HDL (HDL-CUC). (Corresponds to 22 days in Figure 3) In addition, to obtain the background value, the cholesterol uptake ability by HDL (HDL-CUC) of non-cultured William’s E + CM-4000 was analyzed.

[0042] <Apolipoprotein measurement from Day 13 to 15> The medium was changed to dHCGM at 1.0 mL per well on Day 1. Then, on Day 2, Day 6, and Day 9, it was changed to dHCGM at 0.5 mL per well. Then, on Day 13, it was changed to William’s E + CM-4000 at 0.5 mL per well, and the culture supernatant was collected on Day 15, two days later. The collected supernatant samples were used for the measurement of apolipoprotein. (Corresponds to PXB in Figure 2)

[0043] 2. Culture of human fresh hepatocytes Human non-frozen hepatocytes (HEP220-MW24; BIOPREDIC International) seeded in a 24-well plate and culture medium (Incubation Medium 100 mL; HIL214-100M; BIOPREDIC International) were obtained. With the acquisition date as Day 0, they were cultured in the culture medium for one day. On Day 1, it was changed to William’s E + CM-4000 at 0.5 mL per well, and the culture supernatant was collected on Day 3, two days later. The collected supernatant samples were used for the measurement of apolipoprotein (corresponds to FHH in Figure 2) and the analysis of the cholesterol uptake ability by HDL (HDL-CUC). (Corresponds to human fresh hepatocytes in Figure 3)

[0044] 3. Measurement of Apolipoprotein Apolipoproteins (APOA1, APOA5, APOB100, APOC3) in the culture supernatant were quantified using ELISA (enzyme-linked immunosorbent assay).

[0045] The quantification results of apolipoproteins in the culture supernatant by ELISA are shown in Figure 2. Figure 2 shows the measurement results of apolipoproteins contained in the culture supernatants of PXB-cells (corresponding to PXB), human fresh hepatocytes (corresponding to FHH), HepG2 (a human hepatoma-derived cell line), and HuH-7 (a human hepatoma-derived cell line). APOA1, which constitutes HDL particles, is abundantly present in the culture supernatant of PXB-cells. This indicates the presence of HDL particles containing APOA1 as a constituent component in the culture supernatant.

[0046] Measurement of cholesterol uptake ability For the purpose of removing APOB protein contained in the culture supernatant, a 22% PEG4000 solution was added to the sample in an equal volume to the sample, and pelleted by centrifugation. Since HDL composed of APOAI remains in the supernatant, the HDL function (cholesterol uptake ability; HDL-CUC) was measured using this supernatant fraction. The process of PEG treatment is as follows.

[0047] 30 μL of the sample was dispensed into a 1.5 mL proteo save tube, and 30 μL of 22% PEG4000 was added. This was left standing at room temperature for 20 minutes and centrifuged at 3000 rpm and 25°C for 15 minutes. Then, after leaving the sample standing on ice for 10 minutes, it was centrifuged at 10000 rpm and 4°C for 3 minutes. The supernatant after centrifugation was collected and diluted to 200-fold (corresponding to a sample volume of 0.025 μL), 50-fold (corresponding to a sample volume of 0.1 μL), and 10-fold (corresponding to a sample volume of 0.5 μL) for the culture supernatant derived from PXB-cells, and diluted to 4-fold (corresponding to a dilution factor of 4) and 2-fold (corresponding to a dilution factor of 2) for the culture supernatant of human fresh hepatocytes for measurement.

[0048] The measurement was carried out using a research-use fully automated high-sensitivity immunoassay device HI-1000 (Sysmex Corporation). The reagent addition procedure and reaction time are as follows. · 90 μL of R1 reagent (biotin-labeled cholesterol) · 10 μL of sample · 30 μL of R2 reagent (Anti-human APOAI antibody-labeled magnetic beads) · React at 37 °C for 369 seconds · Washing (B / F separation) · 100 μL of R3 reagent (Streptavidin-ALP) · React at 37 °C for 568 seconds · Washing (B / F separation) · 50 μL of R4 reagent (luminescent substrate) · 100 μL of R5 reagent (buffer) · React at 42 °C for 305 seconds · Luminescence measurement

[0049] The results are shown in FIGS. 3 and 4. FIG. 3 is a diagram showing the results of testing HDL-CUC. FIG. 4 is a diagram showing the lipids contained in the lipoprotein fraction. The HDL-CUC test method specifically selects HDL particles incorporating biotin-labeled cholesterol with Anti-human APOAI antibody, and then quantifies the cholesterol in HDL by making the biotin-labeled cholesterol incorporated into the HDL particles emit light. Since an increase in luminescence amount dependent on the dilution ratio was confirmed in the culture supernatants used in the test, the presence of HDL particles having a cholesterol recovery function was shown in the culture supernatants of fresh human hepatocytes and PXB-cells.

Explanation of symbols

[0050] 10: Surface part 20: Inside

Claims

1. At least cholesterol-free high density lipoprotein particles obtained from cultures of hepatocytes.

2. The particle according to claim 1, wherein the hepatocytes are human hepatocytes or non-human hepatocytes having lipid metabolic function equivalent to that of the human hepatocytes.

3. The particle according to claim 2 , wherein the non-human derived hepatocytes are hepatocytes derived from a non-human animal in which at least 70% of the liver has been replaced with human hepatocytes.

4. The particle according to claim 1 , having cholesterol efflux capacity.

5. The particle according to claim 3 , wherein the non-human animal is a mouse.

6. A cholesterol carrier comprising the particle according to any one of claims 1 to 5.

7. The carrier according to claim 6, which extracts cholesterol from tissues and transports it to the liver.

8. The method of claim 7, wherein the tissue is a circulatory system tissue.

9. The carrier according to claim 8, wherein the cholesterol in the circulatory system tissue is cholesterol in atherosclerotic plaque.

10. A method for producing high density lipoprotein particles which comprises culturing hepatocytes and collecting at least cholesterol-free high density lipoprotein particles from the resulting culture.

11. The method according to claim 10, wherein the hepatocytes are human hepatocytes or non-human hepatocytes having lipid metabolic function equivalent to that of the human hepatocytes.

12. The method according to claim 10, wherein the non-human derived hepatocytes are hepatocytes derived from a non-human animal in which at least 70% of the liver has been replaced with human hepatocytes.

13. The method of claim 10, wherein the high density lipoprotein particles have cholesterol efflux capacity.

14. The method of claim 12, wherein the non-human animal is a mouse.

15. A pharmaceutical composition for cardiovascular disease, comprising the particles according to any one of claims 1 to 5.

16. A pharmaceutical composition for treating cardiovascular diseases, comprising the carrier according to claim 6.

17. The pharmaceutical composition according to claim 15, wherein the cardiovascular disease is at least one disease selected from the group consisting of hypercholesterolemia, familial hypercholesterolemia, atherosclerosis, peripheral vascular disease, dyslipidemia, angina pectoris, ischemia, stroke, myocardial infarction, hypertension, and vascular complications of diabetes.

18. The pharmaceutical composition according to claim 16, wherein the cardiovascular disease is at least one disease selected from the group consisting of hypercholesterolemia, familial hypercholesterolemia, atherosclerosis, peripheral vascular disease, dyslipidemia, angina pectoris, ischemia, stroke, myocardial infarction, hypertension, and vascular complications of diabetes.

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