WO2014208381A1 - 肝細胞増殖剤 - Google Patents
肝細胞増殖剤 Download PDFInfo
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- WO2014208381A1 WO2014208381A1 PCT/JP2014/065879 JP2014065879W WO2014208381A1 WO 2014208381 A1 WO2014208381 A1 WO 2014208381A1 JP 2014065879 W JP2014065879 W JP 2014065879W WO 2014208381 A1 WO2014208381 A1 WO 2014208381A1
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- acid
- hepatocyte
- agent
- liver
- hepatocytes
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- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 1
- 206010053219 non-alcoholic steatohepatitis Diseases 0.000 description 1
- 229960002748 norepinephrine Drugs 0.000 description 1
- SFLSHLFXELFNJZ-UHFFFAOYSA-N norepinephrine Natural products NCC(O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-UHFFFAOYSA-N 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 210000003240 portal vein Anatomy 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- WGJJROVFWIXTPA-OALUTQOASA-N prostanoic acid Chemical class CCCCCCCC[C@H]1CCC[C@@H]1CCCCCCC(O)=O WGJJROVFWIXTPA-OALUTQOASA-N 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 229940043131 pyroglutamate Drugs 0.000 description 1
- 229940109850 royal jelly Drugs 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- BZVJOYBTLHNRDW-UHFFFAOYSA-N triphenylmethanamine Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(N)C1=CC=CC=C1 BZVJOYBTLHNRDW-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/067—Hepatocytes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
- C12N2500/33—Amino acids other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
Definitions
- the present invention relates to a hepatocyte proliferating agent.
- the liver is an organ that plays an important role in metabolism, excretion, detoxification, maintenance of body fluid homeostasis, and the like. Since the liver is an organ having a high regenerative ability, it is known that its function and form can be recovered in a short period of time even if a part of the liver is excised.
- Recombinant human hessocicin stimulates propagation of hepatocytes in vivo and improve surviving influtant hep.
- EGF epidermal growth factor
- TGF- ⁇ transforming growth factor
- HGF hepatocyte growth factor
- FGF fibroblast growth factor
- IGF- ⁇ insulin, interleukin-6 (IL-6) Tumor necrosis factor- ⁇ (TNF- ⁇ ), norepinephrine and the like are known to promote the proliferation of hepatocytes in cell culture in vitro.
- EGF epidermal growth factor
- TGF- ⁇ transforming growth factor
- HGF hepatocyte growth factor
- FGF fibroblast growth factor
- IL-6 interleukin-6
- TNF- ⁇ tumor necrosis factor- ⁇
- norepinephrine norepinephrine
- EGF and TGF- ⁇ show proliferative activity against fibroblasts in addition to epithelial cells, and hepatocyte growth factor promotes proliferation of vascular endothelial cells and fibroblasts in addition to epithelial cells It has been known.
- Cancer cells express more receptors for EGF than normal cells. Therefore, when these growth factors described above are allowed to act on a cell population containing cancer cells and normal cells, the proliferation of not only normal cells but also cancer cells is enhanced.
- Patent Document 1 a glycoprotein consisting of royal jelly and having a molecular weight of 57 kilodaltons is known as a factor that promotes hepatocyte DNA synthesis, ie, promotes hepatocyte proliferation.
- a hepatocyte growth factor inducer containing a prostanoic acid derivative as an active ingredient (Patent Document 2) and a hepatocyte growth factor production inducer containing a processed product of Cordyceps sinensis (Patent Document 3) have been reported.
- the above-mentioned hepatocyte growth factor inducer or hepatocyte growth factor production inducer is not an agent that directly affects the proliferation of hepatocytes, but induces the production and secretion of hepatocyte growth factor and indirectly proliferates hepatocytes. It is a drug that promotes
- HPS Human hepasocin
- the present invention has been made in view of the above circumstances, and an object thereof is to provide a hepatocyte proliferating agent that can selectively proliferate normal hepatocytes without proliferating cancer cells.
- the inventors have conducted extensive research and found that a mixture containing pyroglutamic acid, aspartic acid and glutamic acid promotes the proliferation of hepatocytes, and completed the present invention. .
- the above mixture has no risk of growing cancer cells. And the said mixture shows the proliferation activity with respect to a hepatocyte equivalent to or more than the factor which grows the conventional hepatocyte.
- the above mixture makes it possible to proliferate hepatocytes in vitro, and is a very effective means in the fields of basic research, drug discovery research, bioreactors, liver disease treatment and regenerative medicine.
- the present invention provides the following [1] to [8].
- [1] A hepatocyte proliferating agent containing pyroglutamic acid, aspartic acid and glutamic acid as active ingredients.
- the hepatocyte proliferating agent according to [1] or [2] which is a liquid and has a pyroglutamic acid concentration of 0.5 to 20 mM.
- [4] The liquid preparation according to any one of [1] to [3], wherein the concentration of aspartic acid is 0.7 to 2.8 mM, and the concentration of glutamic acid is 0.1 to 1.6 mM.
- Hepatocyte growth agent [5] A therapeutic agent for liver disease comprising the hepatocyte proliferating agent according to any one of [1] to [4]. [6] The therapeutic agent according to [5], wherein the liver disease is liver cancer. [7] A liver regenerating agent comprising the hepatocyte proliferating agent according to any one of [1] to [4]. [8] A liver function recovery agent comprising the hepatocyte proliferating agent according to any one of [1] to [4].
- the present invention provides the following [9] to [20].
- [9] A method for growing normal hepatocytes, comprising a step of administering an effective amount of pyroglutamic acid, aspartic acid and glutamic acid to a subject.
- a method for regenerating a liver comprising a step of administering an effective amount of pyroglutamic acid, aspartic acid and glutamic acid to a subject.
- a method for restoring liver function comprising a step of administering an effective amount of pyroglutamic acid, aspartic acid and glutamic acid to a subject.
- a method for treating or preventing a liver disease comprising a step of administering an effective amount of pyroglutamic acid, aspartic acid and glutamic acid to a subject.
- Pyroglutamic acid, aspartic acid and glutamic acid for proliferating normal hepatocytes.
- Pyroglutamic acid, aspartic acid and glutamic acid for regenerating the liver.
- Pyroglutamic acid, aspartic acid and glutamic acid for treating or preventing liver disease.
- hepatocyte proliferating agent that can selectively proliferate normal hepatocytes without proliferating cancer cells.
- the hepatocyte proliferating agent according to the present embodiment contains pyroglutamic acid, aspartic acid and glutamic acid as active ingredients.
- a hepatocyte proliferating agent based on a combination of these amino acids is referred to as “PARI-S001”.
- the hepatocyte proliferating agent means an agent that promotes the proliferation of normal hepatocytes in both in vivo and in vitro environments.
- pyroglutamic acid aspartic acid and glutamic acid, free amino acids and / or salts thereof can be used, respectively.
- Pyroglutamic acid, aspartic acid and glutamic acid may be synthesized from known compounds by known methods, respectively, or may be obtained as commercial products.
- the salt of pyroglutamic acid, aspartic acid or glutamic acid is not particularly limited as long as it is a pharmacologically or physiologically acceptable salt.
- salts with inorganic bases eg, ammonium salts; salts with metals such as alkali metals (sodium, potassium, etc.), alkaline earth metals (calcium, magnesium, etc.), aluminum
- salts with organic bases for example, salts with organic amines such as methylamine, tritylamine, diethylamine, triethanolamine, morpholine, piperazine, pyrrolidine, tripyridine and picoline.
- Hydrated forms are also included in the above-mentioned free amino acids and / or salts thereof.
- the free amino acid and / or salt thereof may be either D-form or L-form.
- the above-mentioned free amino acids and / or salts thereof may be used alone or in any combination of two or more.
- pyroglutamic acid, aspartic acid and glutamic acid can be suitably used as L-form free amino acids and / or salts thereof, respectively, from the viewpoint of having hepatocyte proliferation activity.
- the concentration of pyroglutamic acid as the main active ingredient is not particularly limited, and aspartic acid and glutamic acid types and concentrations used in combination, as a hepatocyte proliferating agent It is appropriately set according to the use, formulation form, method of use and the like.
- the concentration of pyroglutamic acid in the solution is preferably 0.5 to 20 mM, more preferably 2 to 20 mM, and more preferably 10 to 20 mM, from the viewpoint of effectively promoting the proliferation of hepatocytes. More preferably, it is still more preferably 12 to 20 mM, and particularly preferably 14 to 20 mM.
- the viewpoint of effectively promoting the proliferation of hepatocytes while maintaining the shape (shape) of the hepatocytes well it is preferably 0.5 to 16 mM, more preferably 12 to 16 mM, Even more preferred is 14-16 mM.
- the concentration of aspartic acid is not particularly limited, and the types and concentrations of pyroglutamic acid and glutamic acid used together, use as a hepatocyte proliferating agent, formulation form, use It is set appropriately according to the method and the like.
- the concentration of aspartic acid in the solution is preferably 0.7 to 2.8 mM, more preferably 0.7 to 1.4 mM, from the viewpoint of effectively promoting hepatocyte proliferation.
- the concentration of glutamic acid is not particularly limited. It is set appropriately according to the method and the like.
- the concentration of glutamic acid in the solution is preferably 0.1 to 1.6 mM, more preferably 0.1 to 0.8 mM, from the viewpoint of effectively promoting the proliferation of hepatocytes. Even more preferred is ⁇ 0.8 mM.
- the molar ratio of pyroglutamic acid, aspartic acid and glutamic acid contained in the hepatocyte proliferating agent according to the present embodiment is not particularly limited, and the types of pyroglutamic acid, aspartic acid and glutamic acid used, uses of hepatocyte proliferating agent, and preparations It is appropriately set according to the form, usage method, and the like.
- the hepatocyte proliferating agent according to the present embodiment may contain only pyroglutamic acid, aspartic acid and glutamic acid, which are active ingredients, or may contain these active ingredients and other ingredients.
- examples of other components include stabilizers, preservatives, additives (for example, sodium bisulfite) and the like.
- the hepatocyte proliferating agent according to this embodiment can selectively proliferate normal hepatocytes, it can be used as a hepatic disease therapeutic agent, liver regenerating agent, and liver function restoring agent.
- liver diseases include liver cancer, acute hepatitis, chronic hepatitis, steatohepatitis, cirrhosis, fatty liver, alcoholic liver injury, nonalcoholic steatohepatitis, liver fibrosis and the like.
- the hepatocyte proliferating agent according to the present embodiment does not proliferate cancer cells, it is preferably used as a therapeutic agent for liver cancer.
- the therapeutic agent for liver disease according to this embodiment can be safely administered parenterally to subjects such as humans and animals.
- parenteral administration include intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, transdermal administration, pulmonary administration, nasal administration, and transmucosal administration.
- Examples of the dosage form of the therapeutic agent for liver disease according to this embodiment include injections (subcutaneous injections, intradermal injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.), and external preparations ( Transdermal preparations, ointments, etc.), liquids for external use (injections, poultices, coating agents, etc.), sustained-release preparations (eg sustained-release microcapsules), and the like.
- the liver disease therapeutic agent according to the present embodiment is encapsulated in a hydrogel or microcapsule of a bioabsorbable polymer such as collagen, gelatin, polylactic acid, and polyglycolic acid, and this is subcutaneously, in an organ, muscle, Alternatively, it can be used by injection or implantation in a local area such as the abdominal cavity.
- a bioabsorbable polymer such as collagen, gelatin, polylactic acid, and polyglycolic acid
- the hepatocyte proliferating agent may include pyroglutamic acid and may include aspartic acid or glutamic acid.
- L-pyroglutamic acid Nacalai Tesque
- L-aspartic acid Nacalai Tesque
- L-glutamic acid Nacalai Tesque
- Rapamycin Sigma
- PD98059 Sigma
- SB203580 Sigma
- LY294002 Sigma
- PARI-S001 refers to a mixture of pyroglutamic acid (10 to 20 mM), aspartic acid (1.4 mM), and glutamic acid (0.4 mM).
- ⁇ Cell> Isolation of normal rat hepatocytes (In situ collagenase perfusion method) SD rats (6-7 weeks old, male) (purchased from Kudo Co., Ltd.) were anesthetized by intraperitoneally administering 25% urethane (w / v) at a dose of 1.25 g / kg body weight. Thereafter, the anesthetized SD rat was laparotomized and perfused from the hepatic portal vein with buffer A (37 ° C.) for 7 minutes, and then buffer B was further perfused for 7 minutes.
- buffer A is a buffer obtained by adding ethylene glycol tetraacetic acid (EGTA) to 1M Hepes so that the final concentration is 0.05M.
- EGTA ethylene glycol tetraacetic acid
- Buffer B is a buffer obtained by adding a collagenase (Sigma) solution to buffer A so that the final concentration is 0.5 g / L. After the perfusion was completed, the liver of the SD rat was removed, and the liver cells were loosened with a scalpel in a dish placed on ice to make a single cell. Thereafter, ice-cooled DMEM medium (Dulbecco's Modified Eagle Medium) was added to the obtained single cells, pipetted, and filtered through gauze and a 100 ⁇ m-diameter mesh to obtain a cell suspension.
- DMEM medium Dulbecco's Modified Eagle Medium
- the filtered cell suspension was transferred to a 50 ml Falcon tube, and the supernatant was removed three times by washing with ice-cooled DMEM medium and centrifugation (50 ⁇ g for 1 minute). Thereafter, the cell suspension was centrifuged at 50 ⁇ g for 1 minute, the supernatant was removed, and ice-cooled DMEM medium was added again for pipetting.
- the cells thus obtained were used in the following experiments as rat normal hepatocytes (rat normal hepatocytes).
- HepG2 Independent Administrative Institution JCRB Cell Bank; hereinafter abbreviated as “JCRB”
- HLE HLE
- Huh-7 Huh-7 (JCRB) was cultured in RPMI medium containing 10% serum.
- ⁇ Normal cell proliferation test Rat normal hepatocytes (1.0 ⁇ 10 4 cells / well) were seeded in a 96-well plate, cultured for 3 hours, and then replaced with a medium supplemented with each PARI-S001. Thereafter, cell proliferation tests were performed on the cells cultured for 22 hours using the cell proliferation activity ELISA BrdU color development kit (Roche).
- the SRB assay is used for measuring cell density based on measuring the protein content of the cells.
- the specific procedure was as follows. Cells were seeded in 96-well plates and each PARI-S001 was added. 24 hours, 48 hours or 72 hours after the addition of PARI-S001, a 50% trichloroacetic acid (hereinafter sometimes referred to as “TCA”) solution was added at 25 ⁇ L / well, and the mixture was stored in a refrigerator at 4 ° C. Incubated for 1 hour. After 1 hour, the solution was removed and the TCA was removed by washing the well 4 times with 300 ⁇ L / well MilliQ water. Thereafter, the 96-well plate was inverted and tapped to remove excess water.
- TCA 50% trichloroacetic acid
- the 96-well plate was air-dried by leaving it at room temperature for 1 hour or more. 50 ⁇ L / well of sulforhodamine B solution was added to the dry well and allowed to stand at room temperature. After 20-30 minutes, the sulforhodamine B solution was removed and the wells were washed 4 times with 300 ⁇ L / well of 1% acetic acid solution. The plate was turned upside down and tapped to remove excess water. Further, the 96-well plate was air-dried by leaving it at room temperature for 1 hour or more. After the plate was air-dried, a lysis solution (10 mM Tris Base solution (pH 7.4)) was added at 100 ⁇ L / well. Thereafter, the 96-well plate was agitated with a shaker for 2 minutes to lyse the cells, and the absorbance at 565 nm was measured with a multiwell spectrophotometer ( ⁇ Quant).
- a lysis solution (10 mM Tris Base solution (pH 7.4)
- Intracellular signal transduction pathway analysis was performed to elucidate which intracellular signal transduction pathway PARI-S001 promotes cell proliferation activity.
- Cell proliferation activity was measured using PARI-S001 and various kinase inhibitors.
- the same method as in the above ⁇ Normal cell proliferation test> was used.
- FIG. 1 shows the results of the activity of DNA synthesis in normal rat primary hepatocytes (normal rat primary hepatocytes) when PARI-S001 in which the concentration of pyroglutamic acid was varied between 10 mM and 20 mM was added. It can be evaluated that the higher the activity of DNA synthesis (DNA synthesis activity), the higher the proliferation rate of normal rat primary hepatocytes.
- control As a control (hereinafter referred to as “control”), the DNA synthesis activity of normal rat primary hepatocytes to which nothing was added was used.
- the vertical axis in FIG. 1 is the relative value (percentage) of DNA synthesis activity relative to the control.
- the DNA synthesis activity of normal rat primary hepatocytes increased depending on the concentration of pyroglutamic acid contained in PARI-S001.
- concentration of pyroglutamate was 10 mM to 14 mM
- the morphology of normal rat primary hepatocytes was similar to that of control cells.
- FIG. 2 shows the results of the protein content in the hepatoma cell lines (HepG2, HLE and Huh-7) when PARI-S001 having a pyroglutamic acid concentration changed between 10 mM and 20 mM was added. It can be evaluated that the higher the protein content (protein amount), the higher the proliferation rate of the liver cancer cell line. On the other hand, if the amount of protein is small, it can be evaluated that the growth of the liver cancer cell line is suppressed. The amount of protein was quantified by the SRB assay described above. The control was the protein content of liver cancer cells to which nothing was added.
- SRB assay was performed 24 hours, 48 hours or 72 hours after the addition of each PARI-S001 to the cells.
- shaft of FIG. 2 was made into the relative value (percentage) of the quantity of the protein on the basis of the control
- the protein content in various hepatoma cell lines decreased compared to the control 48 hours after adding PARI-S001 to the cells. From this, it was found that depending on the concentration of pyroglutamic acid contained in PARI-S001, the proliferation of the hepatoma cell line was further suppressed with the passage of time in the presence of PARI-S001. From these results, it was found that a hepatocyte proliferating agent containing pyroglutamic acid, aspartic acid and glutamic acid as active ingredients is also useful as a therapeutic agent for liver diseases such as liver cancer.
- DMSO dimethyl sulfoxide
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Abstract
Description
[1]ピログルタミン酸、アスパラギン酸及びグルタミン酸を有効成分として含有する肝細胞増殖剤。
[2]各有効成分のモル比率が、ピログルタミン酸:アスパラギン酸:グルタミン酸=5~200:7~28:1~16である、[1]に記載の肝細胞増殖剤。
[3]液剤であって、ピログルタミン酸の濃度が0.5~20mMである、[1]又は[2]に記載の肝細胞増殖剤。
[4]液剤であって、アスパラギン酸の濃度が0.7~2.8mMであり、グルタミン酸の濃度が0.1~1.6mMである、[1]~[3]のいずれかに記載の肝細胞増殖剤。
[5][1]~[4]のいずれかに記載の肝細胞増殖剤を含む、肝疾患用治療剤。
[6]肝疾患が肝癌である、[5]に記載の治療剤。
[7][1]~[4]のいずれかに記載の肝細胞増殖剤を含む、肝臓再生剤。
[8][1]~[4]のいずれかに記載の肝細胞増殖剤を含む、肝機能回復剤。
[9]有効量のピログルタミン酸、アスパラギン酸及びグルタミン酸を対象に投与する工程を含む、正常な肝細胞を増殖させる方法。
[10]有効量のピログルタミン酸、アスパラギン酸及びグルタミン酸を対象に投与する工程を含む、肝臓を再生させる方法。
[11]有効量のピログルタミン酸、アスパラギン酸及びグルタミン酸を対象に投与する工程を含む、肝機能を回復させる方法。
[12]有効量のピログルタミン酸、アスパラギン酸及びグルタミン酸を対象に投与する工程を含む、肝疾患を治療又は予防する方法。
[13]正常な肝細胞を増殖させるための、ピログルタミン酸、アスパラギン酸及びグルタミン酸。
[14]肝臓を再生させるための、ピログルタミン酸、アスパラギン酸及びグルタミン酸。
[15]肝機能を回復させるための、ピログルタミン酸、アスパラギン酸及びグルタミン酸。
[16]肝疾患を治療又は予防するための、ピログルタミン酸、アスパラギン酸及びグルタミン酸。
[17]肝細胞増殖剤の製造における、ピログルタミン酸、アスパラギン酸及びグルタミン酸の使用。
[18]肝臓再生剤の製造における、ピログルタミン酸、アスパラギン酸及びグルタミン酸の使用。
[19]肝機能回復剤の製造における、ピログルタミン酸、アスパラギン酸及びグルタミン酸の使用。
[20]肝疾患治療剤の製造における、ピログルタミン酸、アスパラギン酸及びグルタミン酸の使用。
上述の遊離型のアミノ酸及び/又はその塩には、水和物の形態も含まれる。遊離型のアミノ酸及び/又はその塩は、D体、L体のいずれであってもよい。
液剤におけるピログルタミン酸の濃度としては、肝細胞の増殖を有効に促進するという観点から、0.5~20mMであることが好ましく、2~20mMであることがより好ましく、10~20mMであることが更に好ましく、12~20mMであることが更により好ましく、14~20mMであることが特により好ましい。さらに、肝細胞の形態(形状)を良好に維持しつつ、肝細胞の増殖を有効に促進するという観点から、0.5~16mMであることが好ましく、12~16mMであることがより好ましく、14~16mMであることが更により好ましい。
液剤におけるアスパラギン酸の濃度は、肝細胞の増殖を有効に促進するという観点から、0.7~2.8mMであることが好ましく、0.7~1.4mMであることがより好ましい。
液剤におけるグルタミン酸の濃度は、肝細胞の増殖を有効に促進するという観点から、0.1~1.6mMであることが好ましく、0.1~0.8mMであることがより好ましく、0.4~0.8mMであることが更により好ましい。
本実施形態に係る肝疾患治療剤の剤形としては、例えば、注射剤(皮下注射剤、皮内注射剤、静脈内注射剤、筋肉内注射剤、及び腹腔内注射剤等)、外用剤(経皮製剤、及び軟膏剤等)、外用液剤(注入剤、湿布剤、及び塗布剤等)、及び徐放性製剤(徐放性マイクロカプセル等)等が挙げられる。
さらに、本実施形態に係る肝疾患治療剤をコラーゲン、ゼラチン、ポリ乳酸、及びポリグリコール酸等の生体吸収性高分子のハイドロゲル又はマイクロカプセル中に封入し、これを皮下、臓器内、筋肉、又は腹腔等の局部に注射又は埋め込む等して用いることも可能である。
<試薬>
有効成分としてのアミノ酸は、L-ピログルタミン酸(ナカライテスク)、L-アスパラギン酸(ナカライテスク)、L-グルタミン酸(ナカライテスク)を用いた(以下、L体の標記は省略する。)。キナーゼインヒビターは、Rapamycin(Sigma)、PD98059(Sigma)、SB203580(Sigma)、LY294002(Sigma)を用いた。
以下ではPARI-S001とは、ピログルタミン酸(10~20mM)、アスパラギン酸(1.4mM)、グルタミン酸(0.4mM)の混合物をいう。
ラット正常肝細胞の分離(In Situコラゲナーゼ灌流法)
SDラット(6~7週齢、オス)(九動株式会社から購入)に、25%ウレタン(w/v)を1.25g/kg体重の用量で腹腔内投与することによって麻酔を施した。その後、麻酔されているSDラットを開腹し、肝門脈からバッファーA(37℃)で7分間灌流した後、更にバッファーBを7分間灌流した。ここで、バッファーAは、1M Hepesに、最終濃度が0.05Mになるようにエチレングリコール四酢酸(EGTA)を添加したバッファーである。バッファーBは、バッファーAにコラゲナーゼ(シグマ社)溶液を最終濃度が0.5g/Lになるように添加したバッファーである。灌流が完了した後、SDラットの肝臓を摘出し、氷上に置いたディッシュ内でメスを用いて肝臓の細胞をほぐして単一細胞とした。その後、得られた単一細胞に氷冷したDMEM培地(Dulbecco’s Modified Eagle Medium)を加えピペッティングし、ガーゼ及び100μm径のメッシュで濾過し、細胞懸濁液を得た。濾過した細胞懸濁液を50mlファルコンチューブに移し、氷冷したDMEM培地による洗浄及び遠心分離(50×gで1分間)による上清の除去を3回行った。その後、細胞懸濁液を50×gで1分間遠心分離し、上清を除去し、再び氷冷したDMEM培地を加えてピペッティングを行った。このようにして得られた細胞を、ラットの正常肝細胞(ラット正常肝細胞)として以下の実験に用いた。
分離した細胞懸濁液の上清を除去し、氷冷したWilliams’E培地(5%ウシ胎児血清(FBS)、10nM インスリン、10nM デキサメタゾンを含有)を加えることで細胞懸濁液を10mlに希釈した。得られた細胞懸濁液の一部を用いて、トリパンブルー染色を行い、生細胞の数を測定した。その後、1.0×104細胞/wellとなるように、ラット正常細胞を96穴コラーゲンコートプレートに播種し、37℃で培養した。
肝癌細胞は、以下の細胞株を使用した。HepG2(独立行政法人 医薬基盤研究所 JCRB細胞バンク;以下「JCRB」と略記する。)、HLE(JCRB)を10%血清含有DMEM培地で培養した。Huh―7(JCRB)を10%血清含有RPMI培地で培養した。
ラット正常肝細胞(1.0×104細胞/well)を96穴プレートに播種してから3時間培養した後、各PARI-S001を添加した培地に交換した。その後、22時間培養した細胞について、細胞増殖活性ELISA BrdU発色キット(Roche)を用いて、細胞増殖試験を行った。
HepG2(2.5×103細胞/well)、HLE(2.5×103細胞/well)、Huh―7(1.0×103細胞/well)を96穴プレートに播種してから24時間培養した後、各PARI-S001を添加した培地に交換した。その後、24時間、48時間又は72時間培養した細胞について、後述するSRBアッセイ法によって、各wellのタンパク質の量を定量した。その後、定量したタンパク質の量に基づいて細胞増殖活性を評価した。
SRBアッセイ法は、細胞のタンパク質含量の測定に基づいて、細胞密度の測定のために用いられる。具体的な手順は以下の通りに行った。
細胞を96wellプレートに播種し、各PARI-S001を添加した。PARI-S001の添加から24時間後、48時間後又は72時間後に、50% トリクロロ酢酸(以下「TCA」という場合がある。)溶液を25μL/wellで添加して、4℃の冷蔵庫にて、1時間インキュベートした。1時間後、溶液を除去し、300μL/wellのMilliQ水でwellを4回洗浄することによって、TCAを除いた。その後、96wellプレートを逆さにし、軽く叩いて余分な水を除去した。さらに、1時間以上室温に放置することによって、96wellプレートを風乾した。50μL/wellのスルホローダミンB溶液を乾いたウェルに加え、室温に静置した。20~30分後、スルホローダミンB溶液を除去し、300μL/wellの1%酢酸溶液でウェルを4回洗浄した。プレートを逆さにし、軽く叩いて余分な水を除去した。さらに、1時間以上室温に放置することによって、96wellプレートを風乾させた。プレートを風乾させた後、溶解液(10mM Tris Base solution(pH7.4))を100μL/wellで加えた。その後、96wellプレートをシェイカーで2分間攪拌して細胞を溶解し、マルチウェル分光光度計(μQuant)で565nmの吸光度を測定した。
細胞内シグナル伝達経路解析は、PARI-S001がどの細胞内シグナル伝達経路によって細胞増殖活性を促進するのかを解明するために行われた。PARI-S001と各種キナーゼインヒビターを用いて、細胞増殖活性を測定した。測定方法は、上述の<正常細胞増殖試験>と同様の方法を用いた。
<PARI-S001(ピログルタミン酸濃度10~20mM)におけるDNA合成活性>
ピログルタミン酸の濃度を10mM~20mMの間で変化させたPARI-S001を加えたときの正常ラットの初代肝細胞(正常ラット初代肝細胞)におけるDNA合成の活性の結果を図1に示す。DNA合成の活性(DNA合成活性)が高いほど、正常ラット初代肝細胞の増殖率が高いと評価できる。コントロール(以下、「対照」という。)は、何も添加していない正常ラット初代肝細胞のDNA合成活性を用いた。図1の縦軸は、対照を基準としたDNA合成活性の相対値(パーセント)とした。
ピログルタミン酸の濃度を10mM~20mMの間で変化させたPARI-S001を加えたときの肝癌細胞株(HepG2、HLE及びHuh―7)におけるタンパク質の含有量の結果を図2に示す。タンパク質の含有量(タンパク質の量)が多いほど、肝癌細胞株の増殖率が高いと評価できる。一方、タンパク質の量が少なければ、肝癌細胞株の増殖が抑制されていると評価できる。タンパク質の量は、上述したSRBアッセイ法によって定量した。対照は、何も添加していない肝癌細胞のタンパク質の含有量とした。各PARI-S001を細胞に添加してから24時間、48時間又は72時間経過した後に、SRBアッセイ法を行った。図2の縦軸は、対照を基準としたタンパク質の量の相対値(パーセント)とした。
ラット正常肝細胞を播種してから3時間培養した後、PARI-S001(ピログルタミン酸濃度10mM)及び、同時に各キナーゼインヒビターを添加した培地に交換した。Rapamycin(mTORのインヒビター)の濃度は、最終濃度が1.6ng/mL、3.1ng/mL、6.3ng/mL又は12.5ng/mLになるように調製した。PD98059(MEK1及びMEK2のインヒビター)、SB203580(MAPKのp38のインヒビター)及びLY294002(PI3Kのインヒビター)の濃度は、最終濃度が6.3μM、12.5μM、25μM又は50μMとなるように調製した。各キナーゼインヒビターの溶解を促進させるために、終濃度が0.5%となるようにジメチルスルホキシド(以下「DMSO」と略記する。)を培地に添加した。同様に対照にも終濃度が0.5%となるようにDMSOを培地に添加した。培地を交換してから更に22時間培養した後、細胞増殖活性ELISA BrdU発色キット(Roche)を用いて、DNA合成活性を評価した。
Claims (8)
- ピログルタミン酸、アスパラギン酸及びグルタミン酸を有効成分として含有する肝細胞増殖剤。
- 各有効成分のモル比率が、ピログルタミン酸:アスパラギン酸:グルタミン酸=5~200:7~28:1~16である、請求項1に記載の肝細胞増殖剤。
- 液剤であって、前記ピログルタミン酸の濃度が0.5~20mMである、請求項1又は2に記載の肝細胞増殖剤。
- 液剤であって、前記アスパラギン酸の濃度が0.7~2.8mMであり、前記グルタミン酸の濃度が0.1~1.6mMである、請求項1~3のいずれか一項に記載の肝細胞増殖剤。
- 請求項1~4のいずれか一項に記載の肝細胞増殖剤を含む、肝疾患用治療剤。
- 前記肝疾患が肝癌である、請求項5に記載の治療剤。
- 請求項1~4のいずれか一項に記載の肝細胞増殖剤を含む、肝臓再生剤。
- 請求項1~4のいずれか一項に記載の肝細胞増殖剤を含む、肝機能回復剤。
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JP2002000263A (ja) * | 2000-06-26 | 2002-01-08 | National Institute Of Advanced Industrial & Technology | 肝細胞の増殖方法 |
JP2002520013A (ja) * | 1998-07-10 | 2002-07-09 | エンセル,インコーポレイテッド | 細胞の長期増殖のための培地と基質 |
WO2012030217A2 (en) * | 2010-08-31 | 2012-03-08 | Friesland Brands B.V. | Culture medium for eukaryotic cells |
WO2014077289A1 (ja) * | 2012-11-15 | 2014-05-22 | 株式会社日本生物製剤 | 抗癌剤 |
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JP2003113095A (ja) | 2001-10-04 | 2003-04-18 | Pola Chem Ind Inc | Dna合成促進剤 |
JP2008201749A (ja) | 2007-02-21 | 2008-09-04 | Okayama Univ | 肝細胞増殖因子産生誘導剤及びその医薬品組成物 |
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JP2002520013A (ja) * | 1998-07-10 | 2002-07-09 | エンセル,インコーポレイテッド | 細胞の長期増殖のための培地と基質 |
JP2002000263A (ja) * | 2000-06-26 | 2002-01-08 | National Institute Of Advanced Industrial & Technology | 肝細胞の増殖方法 |
WO2012030217A2 (en) * | 2010-08-31 | 2012-03-08 | Friesland Brands B.V. | Culture medium for eukaryotic cells |
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