JP2011126792A - Activated macrophage-specific cell death inducer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicament effective for intractable inflammatory disease such as septicemia, and to provide an application thereof. <P>SOLUTION: It is discovered that the dehydrocorydaline of one component of Corydalis turtschaninovii macrophage-specifically induces the cell death. As a result, the activated microphage-specific cell death inducer contains the dehydrocorydaline or a salt thereof as an active ingredient. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to drugs. Specifically, the present invention relates to a drug that specifically acts on activated macrophages and induces cell death and uses thereof.

  In sepsis and inflammatory bowel disease (ulcerative colitis, Crohn's disease), bacterial components, particularly macrophages abnormally activated by lipopolysaccharide (hereinafter LPS), are deeply involved in pathogenesis. Antibiotics and anti-inflammatory agents are used to treat these diseases, but sufficient effects have not been obtained in severe cases. Various antibiotics have been developed so far, but there are cases where sufficient effects cannot be exhibited due to the occurrence of resistant bacteria, etc., and side effects such as fungi substitution are problematic. Many drugs that control inflammation have been developed, but these are classified into steroidal drugs and non-steroidal drugs. Steroidal drugs have various pharmacological actions other than anti-inflammatory action, have low specificity, and have many side effects such as Cushing's syndrome due to long-term use. Nonsteroidal drugs exhibit anti-inflammatory effects mainly by inhibiting the action of COX, but the effects are not sufficient, and are not used for diseases with severe inflammation such as sepsis. Furthermore, it has side effects such as causing peptic ulcer.

  On the other hand, many traditional treatments use herbal medicines (herbs). The components of herbal medicine are diverse and their structures are also various (Non-patent Document 1). A crude drug with such characteristics can be said to be a treasure trove of drug candidates. In fact, several herbal ingredients have been identified and utilized as active pharmaceutical ingredients. However, in order to identify effective herbal components, it is necessary to construct a screening system suitable for evaluating the activity after identifying the activity that provides the desired therapeutic effect. ), It is still difficult to identify effective herbal ingredients for specific diseases and conditions.

Japanese Patent Application Laid-Open No. 7-10964 JP 2002-212074 A JP 2007-1112738 A

Bent S, Ko R (2004) .Commonly used herbal medicines in the United States: a review. Am J Med 116: 478-485. Kazuo Watanabe, Yoshiaki Goto, Manabu Murakami, "Applied Pharmacology", 1974, Vol. 8, No. 8, 1105-1113 Shoji Yukinobu, Kawashima Katsuyoshi, Shimizu Tosho, "Nippon Yakushi", 1974, Volume 70, 425-437 Kubo M, Matsuda H, Tokuoka K, Ma S, Shiomoto H. Anti-inflammatory activities of methanolic extract and alkaloidal components from Corydalis tuber. Biol Pharm Bull 17: 262-265.

  An object of the present invention is to provide a drug effective for intractable inflammatory diseases such as sepsis and its use.

In sepsis and inflammatory bowel disease (ulcerative colitis, Crohn's disease), macrophages abnormally activated by LPS are deeply involved in pathogenesis. The present inventors have established an unprecedented therapeutic strategy in which cell death is specifically induced for the activated macrophages and the pathological conditions are improved by eliminating the activated macrophages. In order to find the compounds necessary for the realization of this therapeutic strategy, screening was conducted focusing on the components of crude drugs. As a result, dehydrocorydaline (dehydrocorydaline, compound name 5,6-dihydro-2,3,9,10-tetramethoxy-13-methyl-dibenzo [a, g] quinolizinium, see Fig. 7), which is a component of engosaku, is LPS. Was found to induce significant cell death against activated macrophages. As a result of investigating the mechanism, activated macrophages showed an increase in mitochondrial membrane potential, and dehydrocorridin may induce a cell death by depleting intracellular ATP by inhibiting this increase in mitochondrial membrane potential. all right. On the other hand, the effect of dehydrocorridin on the mitochondrial membrane potential, intracellular ATP level and cell viability of non-activated macrophages was very mild. From the above results, it was found that inhibiting the increase in mitochondrial membrane potential of activated macrophages is an effective means for inducing cell death specifically for activated macrophages. Moreover, the knowledge that it was effective to use as an index whether or not to inhibit the increase in mitochondrial membrane potential was obtained in screening compounds effective for the above therapeutic strategies. The inventions listed below are mainly based on the above results. In addition, it has been reported that dehydrocorridin has an antiallergic action, gastrointestinal motility activation action, gastric juice secretion inhibitory action, antitumor action, and the like (see Patent Documents 1 to 3, Non-Patent Documents 2 and 3). It is also known to be effective against compound-induced inflammation (Non-patent Document 4). The new action of dehydrocorridin found by the present inventors is to “inhibit the increase in mitochondrial membrane potential in macrophages activated by LPS” and to deplete intracellular ATP (as a result, cell death Is different from the previously reported effects.
[1] An activated macrophage-specific cell death inducer containing dehydrocorridin or a salt thereof as an active ingredient.
[2] The activated macrophage-specific cell death inducer according to [1], wherein the activated macrophages are macrophages activated by LPS.
[3] The activated macrophage-specific cell death inducer according to [1] or [2], wherein the macrophage activated by LPS is a therapeutic agent for a disease in which pathogenesis is involved.
[4] The activated macrophage-specific cell death inducer according to [3], wherein the disease is sepsis, ulcerative colitis or Crohn's disease.
[5] Use of dehydrocorridin or a salt thereof for producing an activated macrophage-specific cell death inducer.
[6] A therapeutic method comprising a step of administering a therapeutically effective amount of dehydrocorridin or a salt thereof to a patient having a disease in which macrophages activated by LPS are involved in the pathogenesis.
[7] The treatment method according to [6], wherein the disease is sepsis, ulcerative colitis or Crohn's disease.
[8] A screening method for an activated macrophage-specific cell death inducer, comprising examining whether or not a test substance exhibits an activity of inhibiting an increase in mitochondrial membrane potential of macrophages by LPS.
[9] The screening method according to [8], comprising the following steps (1) to (3):
(1) culturing macrophages in the presence of LPS and in the presence of a test substance;
(2) a step of measuring the mitochondrial membrane potential of macrophages; and (3) a step of determining the effectiveness of the test substance based on the measurement results, wherein LPS is effective in inhibiting the increase in mitochondrial membrane potential ( Steps that serve as an index of activated macrophage-specific cell death induction).
[10] A macrophage (control group 1) cultured under the same conditions as in step (1) except that LPS is present and no test substance is present, and the mitochondrial membrane potential measurement result of the control group 1 The screening method according to [9], wherein efficacy is determined in step (3) using as a comparison target.
[11] Macrophages cultured under the same conditions as in step (1) except for the absence of LPS and the absence of a test substance (control group 2), and the absence of LPS and the presence of a test substance Except for the above, prepare macrophages (control group 3) cultured under the same conditions as in step (1), and compare the measurement results of mitochondrial membrane potential between control group 2 and control group 3 to test for non-activated macrophages The screening method according to [9] or [10], wherein the toxicity of the substance is also determined.
[12] When the mitochondrial membrane potential in the measurement result of the control group 3 is lower than the mitochondrial membrane potential in the measurement result of the control group 2, it is determined that the test substance has nonspecific cytotoxicity. [11] A screening method according to 1.

Effect of dehydrocorridin on cell viability of RAW264 cells (macrophage-derived cultured cell lines) activated with LPS. (A) 1μg / ml LPS and 24μM Albiflorin (Alb), Aucubin (Auc), Barbaloin (Bar), Benzoylmesaconine (Ben), Catalpol (Cat), Dehydrocorydaline (Deh), Epihesperidin (Epi), Geniopicroside (Gen) , RAW264 cells were cultured for 24 hours in the presence of 6-Gingerol (Gin), Liquidin (Liq), Paeoniflorin (Pae), or Swertiamarin (Swe), and the cell viability was evaluated. Data are shown as mean ± standard deviation. N = 3. (B) RAW264 cells were cultured for 24 hours in a medium containing 0-24 μM dehydrocorridin in the presence or absence of LPS, and cell viability was evaluated. (C) After culturing RAW264 cells for 20 hours in a medium containing 24 μM dehydrocorridin or 1 μg / ml LPS, or both, Annexin-V-FITC and 7-amino-actinomycin D (7-AAD) And evaluated by flow cytometry. Annexin-V + / 7-AAD + means induction of cell death. Effect of dehydrocorridin on apoptosis induction (A, B). RAW264 cells were cultured in a medium containing 24 μM dehydrocorridin and 1 μg / ml LPS. Ultraviolet (UV) light was irradiated for 30 seconds as an apoptosis positive group. The control group did not receive any treatment. Activation of caspase 9 (A) and caspase 3/7 (B) was evaluated after 4, 8 and 16 hours. N = 3. (C) RAW264 cells were irradiated with UV for 0 to 30 seconds, and cell viability was evaluated after 24 hours. (D) RAW264 cells were cultured in a medium containing 24 μM dehydrocorridin and 1 μg / ml LPS. UV light was irradiated for 30 seconds as an apoptosis positive group. The control group did not receive any treatment. After 16 hours, the release of cytochrome c from the mitochondria into the cytoplasm was examined. (E) RAW264 cells were cultured in a medium containing 24 μM dehydrocorridin and 1 μg / ml LPS. The apoptosis positive group was irradiated with UV light for 30 seconds. The control group did not receive any treatment. After 24 hours, genomic DNA fragmentation was assessed. Effect of dehydrocorridin on mitochondrial membrane potential and intracellular ATP content. (A) RAW264 cells were cultured in a medium containing 24 μM dehydrocorridin, 1 μg / ml LPS, or both. After 4, 8, and 16 hours, the amount of rhodamine 123 stored in the mitochondria was examined, and the mitochondrial membrane potential was evaluated. N = 3. (B) RAW264 cells were cultured in a 1 μg / ml LPS-containing medium in the presence and absence of 24 μM dehydrocorridin, and the amount of intracellular ATP was evaluated after 16 hours. Effect of dehydrocorridin on cytokine production. RAW264 cells were cultured in a medium containing 0-24 μM dehydrocorridin and 1 μg / ml LPS for 24 hours, and the concentrations of IL-1β and IL-6 in the culture were measured. N = 3. In vivo effect of dehydrocorridin. LPS (dose 10 mg / kg) and dehydrocorridin (dose 0 to 4.8 μmol / kg) were administered to mice by subcutaneous injection on day 0, and changes in body weight were monitored from day 1 to day 5. did. N = 6. *: P <0.05 (according to Student's t-test with respect to the control group [dosage of dehydrocorridin is 0 μmol / kg)). **: p <0.01. One control group died on the third day. (B) LPS (dose 10 mg / kg) and dehydrocorridin (dose 0 or 4.8 μmol / kg) were administered to mice by subcutaneous injection. After 24 hours, plasma IL-6 concentration was measured. N = 9. (C) Dehydrocorridin (9.6 μmol / kg) alone was administered to mice by subcutaneous injection. After 24 hours, plasma ALT concentration and BUN were measured. N = 5. Effect of dehydrocorridin on peritoneal macrophages. (A) Mouse peritoneal macrophages were cultured for 24 hours in a medium containing 0-24 μM dehydrocorridin in the presence or absence of 1 μg / ml LPS, and cell viability was evaluated. N = 3. (B, C) In the absence or presence of 24 μM dehydrocorridin, peritoneal macrophages were cultured in a medium containing 1 μg / ml LPS, and 16 hours later, the amount of rhodamine 123 in the mitochondria (B) and the amount of intracellular ATP ( C) was evaluated. (D) RAW264 cells were cultured in a medium containing 0-24 μM dehydrocorridin and 1 μg / ml LPS for 24 hours, and the IL-6 concentration in the culture was measured. Model of action mechanism of dehydrocorridin on LPS activated macrophages. LPS stimulation activates transcription factors such as NF-κB and induces the production of cytokines (IL-1β, IL-6, etc.). Intracellular ATP is consumed by this cytokine production, but the amount of ATP in the cell is maintained by increasing ATP production due to an increase in mitochondrial membrane potential (upper stage). Dehydrocorridin inhibits the increase in mitochondrial membrane potential, prevents its increase in ATP production and depletes the amount of intracellular ATP, resulting in decreased cell viability (bottom). Moreover, further overproduction of cytokines is suppressed by deficiency of intracellular ATP amount and decreased cell viability.

  The first aspect of the present invention provides an activated macrophage-specific cell death inducer (hereinafter abbreviated as “cell death inducer of the present invention”). In the present invention, “activated macrophage-specific” refers to a remarkable and excellent effect on activated macrophages compared to non-activated macrophages. Macrophage activation is preferably caused by stimulation with LPS. On the other hand, the “cell death inducer” refers to a drug or a drug that induces cell death. The cell death according to the present invention is caused by a deficiency in the amount of intracellular ATP by inhibiting an increase in mitochondrial membrane potential accompanying activation.

  The cell death inducer of the present invention contains dehydrocorridin or a salt thereof as an active ingredient. The kind of salt is not particularly limited. However, since dehydrocorridin is known to exist stably as a nitrate, it is preferably a nitrate.

  The purity of the active ingredient of the present invention is not particularly limited as long as it exhibits the effect essential to the present invention, that is, “inducing cell death specifically for activated macrophages”. Therefore, it is not limited to a high-purity substance that does not substantially contain impurities, and may exist, for example, as a component of a herbal extract. The active ingredient of the present invention can be prepared from Engosaku (Corydalis yanhusuo) by a conventional method (using extraction with a polar solvent such as methanol, ethanol, water, ether, chloroform, recrystallization, chromatography, etc.) (for example, patent) Reference 2 is helpful).

  The cell death inducer of the present invention can be used as a therapeutic agent for a disease in which macrophages activated by LPS are involved in the pathogenesis. Typical examples of “diseases in which macrophages activated by LPS are involved in pathogenesis” are sepsis, ulcerative colitis, and Crohn's disease. The cell inducer of the present invention is preferably applied to the treatment of these diseases. On the other hand, since activated macrophages are also involved in the pathogenesis of autoimmune diseases (such as rheumatoid arthritis and systemic lupus erythematosus) and systemic inflammatory response syndrome, they are specific to macrophages activated in these diseases. If cell death can be induced and eliminated, it is expected to be effective in improving the disease state. It should be noted that allergies and compound-induced inflammations reported in the past that dehydrocorridin is effective do not fall under “diseases in which macrophages activated by LPS are involved in the pathogenesis”.

  Formulation of the cell death inducer of the present invention can be performed according to a conventional method. In the case of formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

  The dosage form for formulation is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories.

  The cell death inducer of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (that is, a therapeutically effective amount). The amount of the active ingredient in the cell death inducer of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set within a range of, for example, about 0.1 wt% to about 95 wt% so that a desired dose can be achieved.

  The cell death inducer of the present invention is administered orally or parenterally (intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) according to the dosage form. Applied to the subject. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed). Local administration may be used instead of systemic administration. As local administration, direct injection or application to a target tissue can be exemplified.

  The dose of the cell death inducer of the present invention is set so as to obtain the expected therapeutic effect. In setting a therapeutically effective dose, the patient's symptoms, age, sex, weight, etc. are generally considered. A person skilled in the art can set an appropriate dose in consideration of these matters. For example, the dose can be set so that the amount of the active ingredient per day is about 1 mg to about 240 mg, preferably about 12 mg to about 120 mg, for an adult (body weight: about 60 kg). As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the patient's symptoms and the duration of effect of the active ingredient can be taken into consideration.

  As is apparent from the above description, the present application also provides a therapeutic method for a specific disease, characterized by administering a therapeutically effective amount of the cell death inducer of the present invention to a patient. The specific disease here is “a disease in which macrophages activated by LPS are involved in the pathogenesis”. As mentioned above, typical examples of the disease are sepsis, ulcerative colitis, and Crohn's disease.

(Screening method)
The second aspect of the present invention relates to a method for screening for a substance that induces cell death specifically for activated macrophages. The compound selected by the screening method of the present invention is promising as an activated macrophage-specific cell death inducer, and can be used for the treatment of sepsis, ulcerative colitis, Crohn's disease and the like. In the screening method of the present invention, based on the knowledge that “inhibiting the increase in mitochondrial membrane potential of activated macrophages is an effective means for realizing activated macrophage-specific cell death induction”, it is caused by LPS. The effectiveness of the test substance is judged using as an index whether or not it exhibits the activity of inhibiting the increase in mitochondrial membrane potential of macrophages. That is, the screening method of the present invention is characterized by examining whether or not a test substance exhibits an activity that inhibits an increase in mitochondrial membrane potential of macrophages by LPS. In one aspect of the invention, the following steps are performed.
(1) a step of culturing macrophages in the presence of LPS and a test substance (2) a step of measuring the mitochondrial membrane potential of macrophages (3) a step of determining the effectiveness of the test substance based on the measurement results, and using LPS Steps that indicate that inhibition of increased mitochondrial membrane potential is an indicator of effectiveness (induction of activated macrophage-specific cell death)

  In step (1), macrophages are prepared and cultured in the presence of LPS and the presence of a test substance. As the macrophages, RAW264 cells, peritoneal macrophages and the like can be used. RAW264 cells can be easily obtained from the RBC cell bank (RIKEN BioResource Center), American Type Culture Collection, and the like through a predetermined procedure. In addition, peritoneal macrophages can be prepared by conventional methods. For example, for the preparation of mouse peritoneal macrophages, macrophages can be obtained by administering a thioglyconate-containing solution into the abdominal cavity of the mouse, washing the abdominal cavity after 4 days, and collecting the washing solution. The species of macrophage is not particularly limited. Examples of biological species are mice, rats, and humans.

  LPS is a component constituting the outer cell wall membrane of Gram-negative bacteria, and has a structure in which a sugar chain is bound to lipid (lipid A). The origin (bacterial species) and serotype of LPS are not particularly limited, but Escherichia coli O111: B4 or the like may be used. Various LPSs are commercially available, and can be purchased from, for example, Sigma-Aldrich Japan Co., Ltd.

  As a test substance, organic compounds or inorganic compounds having various molecular sizes can be used. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, vitamins (B1 B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.) The test substance may be derived from a natural product or may be synthetic. In this case, an efficient screening system can be constructed using, for example, a combinatorial synthesis technique, and plant extracts, cell extracts, culture supernatants, etc. may be used as test substances. Existing drugs may be used as test substances.

  In order to culture macrophages in the presence of LPS and in the presence of a test substance, for example, macrophages are seeded on a culture dish and, after a predetermined time (for example, 24 hours) has elapsed, LPS and the test substance are added to the culture solution. What is necessary is just to replace | exchange for the culture solution which added LPS and the test substance. Immediately after sowing, addition of LPS and the test substance or replacement with a culture solution containing LPS and the test substance may be performed. In addition, a culture solution to which LPS and a test substance are added in advance may be used so that a “state where LPS and a test substance are present in the culture solution” is formed simultaneously with sowing.

  The culture time in the presence of LPS and in the presence of the test substance is preferably 8 to 24 hours, more preferably 14 to 18 hours, in order to sufficiently raise the mitochondrial membrane potential due to LPS. The optimum culture time can be determined by a preliminary experiment in which macrophages are cultured in the presence of LPS and the relationship between the culture time and the increase in mitochondrial membrane potential is examined (see Examples described later).

  Matters that are not mentioned in the present specification (medium, culture temperature, etc.) may be in accordance with general culture conditions for culturing macrophages. For example, if it is a medium, a medium suitable for culturing macrophages (for example, RPMI1640, Dulbecco's modified Eagle medium (D-MEM), D-MEM / F-12, etc.) as needed, serum, plasma, Serum albumin, antibiotics, L-glutamine, etc.) may be used. The culture temperature is usually 37 ° C.

In step (2), the mitochondrial membrane potential of the macrophages passed through step (1), that is, macrophages cultured in the presence of LPS and in the presence of a test substance for a predetermined time is measured. The method for measuring the mitochondrial membrane potential is not particularly limited. One preferred measurement method is a method of detecting fluorescence with a flow cytometer or a fluorescence microscope after staining with a fluorescent dye that recognizes mitochondrial membrane potential-dependent retention (accumulation). As fluorescent dyes that can be used in the method, rhodamine 123 (Rhodamine 123), dihydrorhodamine 123 (Dihydrorhodamine 123), JC-1 (5,5 ', 6,6'-tetrachloro-1,1', tetraethylbenzimidazolocarbo-cyanine iodide ), TMR (Tetramethyl rhodamine), MitoTracker ^(TM) Orange CMTMRos (Molecular Probes (registered trademark), Invitrogen), MitoTracker ^(TM) Orange CM-H2TMRos (Molecular Probes (registered trademark), Invitrogen), MitoTracker ^(TM) Red CM-H2XRos (Molecular Probes (registered trademark), Invitrogen) can be exemplified.

  In step (3), the effectiveness of the test substance is determined based on the measurement result in step (2). In the present invention, “inhibition of increase in mitochondrial membrane potential by LPS” is adopted as an indicator that the test substance is effective. That is, the test substance is determined to be effective when it is found that LPS inhibits the increase in mitochondrial membrane potential, and the test substance is effective when it is not found that LPS inhibits increase in mitochondrial membrane potential. It is determined that it is not. When a plurality of test substances are used, the effectiveness of each test substance can be compared and evaluated based on the degree of inhibition.

  Usually, as a comparison target, macrophages (hereinafter referred to as “control group 1”) cultured in the presence of LPS and in the absence of a test substance (other conditions are the same as in step (1)) are prepared. The mitochondrial membrane potential is also measured in parallel. Then, by comparing the mitochondrial membrane potential of the control group 1 and the mitochondrial membrane potential of the test group, it is determined whether the test substance has inhibited the increase in mitochondrial membrane potential by LPS. Thus, if the effectiveness of a test substance is determined by comparison with the control group 1, a more reliable determination result can be obtained.

  On the other hand, in order to confirm that an increase in mitochondrial membrane potential due to LPS occurs, macrophages cultured in the absence of LPS and in the absence of a test substance (other conditions are the same as in step (1)) (hereinafter, “ It is preferable to prepare a control group 2 ”) and measure the mitochondrial membrane potential in parallel.

  In addition, in order to be able to simultaneously determine nonspecific toxicity of the test substance, macrophages cultured in the absence of LPS and in the presence of the test substance (other conditions are the same as in step (1)) (hereinafter referred to as “ It is preferable to prepare a control group 3 ”) and measure its mitochondrial membrane potential. If the mitochondrial membrane potential in the control group 3 is low compared to macrophages cultured in the absence of LPS and in the absence of the test substance (that is, the control group 2), the test substance is an LPS-dependent mitochondrial membrane. It does not inhibit the increase in potential but acts independently of LPS to lower the mitochondrial membrane potential, and can be determined to have nonspecific cytotoxicity.

  When a substance selected by the screening method of the present invention has a sufficient drug effect, the substance can be used as it is as an active ingredient of a cell death inducer. On the other hand, when it does not have a sufficient medicinal effect, it can be used as an active ingredient of a cell death inducer after its medicinal effect is improved by modifying it, such as chemical modification. Of course, even if it has a sufficient medicinal effect, the same modification may be applied for the purpose of further increasing the medicinal effect.

  Focusing on macrophages that play an important role in both innate and acquired immunity, especially to realize an unprecedented therapeutic strategy that induces cell death specifically against activated macrophages and eliminates activated macrophages, Screening was performed by the following method.

1. Materials and Methods (1) Reagents All crude drug-derived components were purchased from Wako Pure Chemical Industries, Ltd. (purity is 98% or higher for all). LPS (Escherichia coli serotype O111: B4) was purchased from Sigma-Aldrich.

(2) Cells Mouse macrophage-derived RAW264 cells were provided by RIKEN RBC Cell Bank (RIKEN BioResource Center). Peritoneal macrophages were collected 4 days after injection from the abdominal cavity of ICR mice injected intraperitoneally with 2.5 ml of 3% thioglycolic acid. Two hours after cell seeding, the cells were washed with a phosphate buffer to remove non-adherent cells.

(3) Cell viability assay
RAW264 cells (2 × 10 ⁴ ) or peritoneal macrophages (8 × 10 ⁴ ) were seeded in 96-well plates. After 24 hours, 6-24 μM herbal medicine-derived components were added in the presence or absence of 1 μg / ml LPS. On the other hand, UV transilluminator Model TFX-20M (wavelength 290-330 nm, peak wavelength 312 nm) (Vilber Lourmat, France) was used for RAW264 cells in the apoptosis positive group, and ultraviolet rays (UV) were applied from the bottom side of the plate. Irradiated (10-30 seconds). After 24 hours, cell viability was assessed using a colorimetric CellTiter96 cell proliferation assay (Promega, USA).

(4) Annexin V-FITC / 7-amino-actinomycin D (7-AAD) assay.
RAW264 cells (1 × 10 ⁵ ) were seeded in 24-well plates. After 24 hours, the cells were cultured for 20 hours in the presence of dehydrocorridin (24 μM), in the presence of LPS (1 μg / ml), or in the presence of dehydrocorridin (24 μM) and LPS (1 μg / ml). Subsequently, staining was performed with Annexin V-FITC (BioVision, USA) and 7-AAD (BD Pharmingen, USA). A total of 1 × 10 ⁴ cells were analyzed with a flow cytometer (Beckman, USA).

(5) Caspase activity assay
RAW264 cells (2 × 10 ⁴ ) were seeded in 96 well plates. After 24 hours, dehydrocorridin (24 μM) and LPS (1 μg / ml) were added. On the other hand, the apoptosis positive group was irradiated with ultraviolet rays in the same manner as described above (30 seconds). After 4 to 16 hours, caspase 3/7 activity and caspase 9 activity were detected using an assay kit (Caspase-Glo 3/7 assay kit, Caspase-Glo 9 assay kit, Promega).

(6) Cytochrome c release assay
RAW264 cells (6 × 10 ⁵ ) were seeded in 6-well plates. After 24 hours, dehydrocorridin (24 μM) and LPS (1 μg / ml) were added. On the other hand, the apoptosis positive group was irradiated with ultraviolet rays in the same manner as described above (30 seconds). After 16 hours, the cytoplasmic fraction was extracted and analyzed by Western blotting using an anti-cytochrome c antibody (Biovision).

(7) Genomic DNA fragmentation assay
RAW264 cells (3 × 10 ⁵ ) were seeded in 12-well plates. After 24 hours, dehydrocorridin (24 μM) and LPS (1 μg / ml) were added. On the other hand, the apoptosis positive group was irradiated with ultraviolet rays in the same manner as described above (30 seconds). After 24 hours, genomic DNA was extracted and subjected to agarose electrophoresis.

(8) Mitochondrial membrane potential assay
RAW264 cells (1 × 10 ⁵ ) or peritoneal macrophages (2 × 10 ⁵ ) were seeded in 24-well plates. After 24 hours, cells were incubated with 24 μM dehydrocorridin, 1 μg / ml LPS, or 24 μM dehydrocorridin and 1 μg / ml LPS for 4-16 hours. Thereafter, staining was performed with rhodamine 123 (Molecular Probes, USA). A total of 1 × 10 ⁴ cells were analyzed with a flow cytometer (Beckman, USA) and used as an indicator of mitochondrial membrane potential (Johnson LV, Walsh ML, Chen LB (1980). Localization of mitochondria in living cells with rhodamine 123. See Proc Natl Acad Sci USA 77: 990-994.) The amount of rhodamine 123 stored in mitochondria was measured.

(9) Intracellular ATP content assay
RAW264 cells (2 × 10 ⁴ ) or peritoneal macrophages (8 × 10 ⁴ ) were seeded in 96-well plates. After 24 hours, cells were incubated for 16 hours with 24 μM dehydrocorridin, 1 μg / ml LPS, or 24 μM dehydrocorridin and 1 μg / ml LPS. Then, the amount of ATP in the cell is determined using the luciferin-luciferase reaction (see Lyman GE, DeVincenzo JP (1967). Determination of picogram amounts of ATP using the luciferin-luciferase enzyme system. Anal Biochem 21: 435-443.). It was measured.

(10) Interleukin (IL) production assay
RAW264 cells (1 × 10 ⁵ ) or peritoneal macrophages (2 × 10 ⁵ ) were seeded in 24-well plates. After 24 hours, cells were incubated with 1 μg / ml LPS for an additional 24 hours in the presence of 0-24 μM dehydrocorridin. Then, measure the IL-1β and IL-6 concentrations in the culture using the IL-1β ELISA kit (Pierce, USA) and IL-6 ELISA kit (R & D Systems, USA) did.

(11) Animal experiments
Eight week old female ICR mice were purchased from Japan SLC. Mice were injected subcutaneously with saline containing 1 mg / ml LPS (dose 10 mg / kg) and 0-480 μM dehydrocorridin (dose 0-4.8 μmol / kg). After administration, body weight was measured daily. To compare IL-6 concentrations in plasma, LPS (10 mg / kg) and dehydrocorridin (0 or 4.8 μmol / kg) were injected subcutaneously into mice and blood samples were collected 24 hours later. Alternatively, mice were prepared by subcutaneous injection of only dehydrocorridin (9.6 μmol / kg). After 24 hours, body weight is measured and a blood sample is collected, and alanine amino acid in plasma using Determiner ALT II (Kyowa Medex Co., Ltd.) and Urea NB Test Wako (Wako Pure Chemical Industries, Ltd., Japan) Transferase (ALT) concentration and blood urea nitrogen (BUN) concentration were measured. Under the approval of the Ethics Committee of the Nagoya University School of Medicine, animal experiments were conducted in accordance with the guidelines for laboratory animal facilities attached to the Nagoya University School of Medicine.

2. Results (1) Effect of dehydrocorridin on cell viability of RAW264 cells activated with LPS
Cultivate RAW264 cells in the presence of 1 μg / ml LPS and 24 μM herbal components (Albiflorin, Aucubin, Barbaloin, Benzoylmesaconine, Catalpol, Dehydrocorydaline, Epihesperidin, Gentiopicroside, 6-Gingerol, Liquiritin, Paeoniflorin, Swertiamarin) Performance was evaluated (FIG. 1A). As a result, it was found that dehydrocorydaline significantly reduces cell viability. When cultured in a dehydrocorridin-containing medium, it can be seen that cell viability was greatly reduced in the presence of LPS compared to the absence of LPS, and significant cell death was induced (FIG. 1B). Similar results were obtained with mouse-derived peritoneal macrophages (FIG. 6A). In the Annexin-V / 7-AAD assay, significant cell death induction was observed in the presence of LPS (FIG. 1C).

(2) Effect of dehydrocorridin on apoptosis induction
In the presence of LPS, dehydrocorridin decreased the cell viability of RAW264 cells, but caspase 9 and caspase 3/7 activation were not observed (FIGS. 2A and 2B). As a positive control for caspase activation, RAW264 cells were irradiated with UV light for 30 seconds (FIGS. 2A and 2B). This treatment reduced the cell viability of RAW264 cells to the same extent as when cultured in the presence of 24 μM dehydrocorridin and 1 μg / ml LPS (FIGS. 1B and 2C). Irradiation with UV light induces cytochrome c release from mitochondria and genomic DNA fragmentation, but such a phenomenon was not observed in the presence of dehydrocorridin and LPS (FIG. 2D, E).

(3) Effects of dehydrocorridin on mitochondrial membrane potential and intracellular ATP level
After RAW264 cells were cultured in a medium containing dehydrocorridin (Deh) for 4 to 16 hours, the mitochondrial membrane potential was evaluated using the fluorescent dye rhodamine 123 that accumulates in the mitochondria depending on the mitochondrial membrane potential. When mitochondrial membrane potential rises but dehydrocorridin is also present after 8-16 hours in the presence of LPS and in the absence of dehydrocorridalin compared to control (in the absence of LPS and in the absence of dehydrocorridin) The increase was inhibited in the presence of LPS and dehydrocorridin (FIG. 3A). When only dehydrocorridin is present (in the absence of LPS and in the presence of dehydrocorridin), there is no effect on the mitochondrial membrane potential compared to the control (FIG. 3A). On the other hand, after culturing RAW264 cells for 16 hours, the amount of intracellular ATP was evaluated. In the absence of dehydrocorridin, the amount of intracellular ATP was maintained even when activated with LPS, but when activated with LPS in the presence of dehydrocorridin, the amount of intracellular ATP was markedly reduced (FIG. 3B). Similar results were obtained with mouse-derived peritoneal macrophages (FIGS. 6B and 6C).
(3) Effect of dehydrocorridin on cytokine production
RAW264 cells were cultured in a medium containing LPS and dehydrocorridin for 24 hours, and then the IL-1β and IL-6 concentrations of the medium were evaluated. Since IL-1β and IL-6 are produced by LPS stimulation, these concentrations are significantly increased, but dehydrocorridin suppresses the increase in the concentration (FIG. 4). Similar results were obtained with mouse-derived peritoneal macrophages (FIG. 6D).

(4) In vivo effect of dehydrocorridin
When LPS was administered, the mice were weakened and the body weight was significantly reduced, but when dehydrocorridin was administered simultaneously, the weight loss could be improved (FIG. 5A). The plasma IL-6 concentration increased with LPS administration, but when dehydrocorridin was administered simultaneously, the increase in IL-6 concentration was suppressed (FIG. 5B). When dehydrocorridin was administered alone, no change in body weight was observed, and neither ALT concentration nor BUN concentration in plasma was observed (FIG. 5C).

3. Summary As a result of screening, it was found that dehydrocorridin, one of the components of Engosaku, induces significant cell death on activated macrophages. In activated macrophages, an increase in mitochondrial membrane potential was observed, and it was found that dehydrocorridin inhibits this increase in mitochondrial membrane potential, thereby depleting intracellular ATP and inducing cell death (FIG. 7). The above results mean that inhibiting the increase in mitochondrial membrane potential of activated macrophages is an effective means for inducing cell death specifically for activated macrophages. If cell death of activated macrophages is induced, excessive cytokine production from activated macrophages can be suppressed. Excess cytokine production from activated macrophages is involved in the pathogenesis of refractory inflammatory diseases such as sepsis and inflammatory bowel disease (ulcerative colitis, Crohn's disease). Therefore, dehydrocorridin is useful for improving the pathology of these diseases.

  According to the cell death inducer of the present invention, cell death can be induced specifically for activated macrophages. Therefore, the present invention is useful as a means for treating various diseases associated with activated macrophages represented by sepsis.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (12)

  An activated macrophage-specific cell death inducer comprising dehydrocorridin or a salt thereof as an active ingredient.   The activated macrophage-specific cell death inducer according to claim 1, wherein the activated macrophages are macrophages activated by LPS.   The activated macrophage-specific cell death inducer according to claim 1 or 2, which is a therapeutic agent for a disease in which macrophages activated by LPS are involved in pathogenesis.   The activated macrophage-specific cell death inducer according to claim 3, wherein the disease is sepsis, ulcerative colitis or Crohn's disease.   Use of dehydrocorridin or a salt thereof for producing an activated macrophage-specific cell death inducer.   A therapeutic method comprising the step of administering a therapeutically effective amount of dehydrocorridin or a salt thereof to a patient having a disease in which macrophages activated by LPS are involved in pathogenesis.   The method according to claim 6, wherein the disease is sepsis, ulcerative colitis or Crohn's disease.   A screening method for an activated macrophage-specific cell death inducer, comprising examining whether or not a test substance exhibits an activity of inhibiting an increase in mitochondrial membrane potential of macrophages by LPS. The screening method according to claim 8, comprising the following steps (1) to (3):
(1) culturing macrophages in the presence of LPS and in the presence of a test substance;
(2) a step of measuring the mitochondrial membrane potential of macrophages; and (3) a step of determining the effectiveness of the test substance based on the measurement results, wherein inhibition of increase in mitochondrial membrane potential by LPS is recognized. Indicative step.   Prepare macrophages (control group 1) cultured under the same conditions as in step (1) except in the presence of LPS and in the absence of test substance, and compare the mitochondrial membrane potential measurement results of control group 1 The screening method according to claim 9, wherein efficacy is determined in step (3).   Steps other than macrophages cultured under the same conditions as in step (1) except in the absence of LPS and in the absence of test substance (control group 2), and in the absence of LPS and in the presence of test substance Prepare macrophages cultured under the same conditions as in (1) (control group 3), and compare the measurement results of mitochondrial membrane potential between the control group 2 and the control group 3 to determine nonspecific toxicity of the test substance. The screening method according to claim 9 or 10, wherein determination is performed.   The test substance is determined to have nonspecific cytotoxicity when the mitochondrial membrane potential in the measurement result of the control group 3 is lower than the mitochondrial membrane potential in the measurement result of the control group 2. Screening method.

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