JPWO2006051987A1 - Estrogen-dependent diseases, prostaglandin D (PGD) -dependent gynecological diseases, immune diseases, cancer, and novel pharmaceuticals for inhibiting angiogenesis - Google Patents

Abstract

Provided are pharmaceuticals for inhibiting estrogen-dependent diseases, prostaglandin D (PGD) -dependent gynecological diseases, immune diseases, cancer and angiogenesis. The medicament of the present invention is a medicament for at least one selected from the group consisting of estrogen-dependent diseases, PGD-dependent gynecological diseases, immune diseases, cancer, and angiogenesis inhibition, and suppresses histamine release factor (HRF) activity It is characterized by including an ingredient. By suppressing the HRF activity, for example, the expression of genes such as IL-1β receptor and 17β hydroxyl steroid dehydrogenase (HSD) is suppressed, so that the estrogen activity is suppressed, and estrogen-dependent diseases are suppressed. Can prevent, treat or improve. It is preferable to use artemisinin as the HRF activity suppressing component.

Description

  The present invention relates to novel pharmaceuticals for suppressing estrogen-dependent diseases, prostaglandin D (PGD) -dependent gynecological diseases, immune diseases, cancer and angiogenesis.

  With the human genome decoding project started in the 1980s, human genetic decoding has progressed, and accordingly, the relationship between diseases and genes is becoming clear. In response, in the medical field, elucidation of molecular mechanisms of diseases and establishment of preventive and therapeutic methods based on them are rapidly progressing. When a certain molecular mechanism (molecular cascade) is clarified in a living body, the prevention and treatment of a disease can be achieved by inhibiting or enhancing the cascade.

  On the other hand, estrogen-dependent diseases such as endometriosis, infertility, immune diseases and cancer are considered to be complex diseases involving various genes. Therefore, if the molecular cascade of these diseases is clarified, it becomes possible to develop pharmaceuticals for their prevention and treatment. In addition, angiogenesis is related to various diseases, and development of suppression technology and enhancement technology is required.

  Therefore, an object of the present invention is to provide pharmaceuticals for suppressing estrogen-dependent diseases, prostaglandin D (PGD) -dependent gynecological diseases, immune diseases, cancer, and angiogenesis.

  To achieve the above object, the medicament of the present invention is a medicament for at least one selected from the group consisting of estrogen-dependent diseases, PGD-dependent gynecological diseases, immune diseases, cancer and angiogenesis suppression, It is characterized by containing a histamine releasing factor (HRF: IgE-dependent histamine-releasing factor, also known as TCTP: Translationally controlled tumor protein) activity inhibitory component.

The present inventors have elucidated a molecular cascade starting from HRF such as the following (A) to (I). According to this cascade, malignant tumors such as estrogen-dependent diseases, PGD-dependent gynecological diseases, immune diseases and certain cancers are found to originate from HRF and are related at any stage in this cascade. If the expression of the molecule is enhanced or suppressed, the above-mentioned diseases can be prevented, improved and treated.
(A) The expression of at least one of interleukin-1β receptor (IL-1β receptor, IL-1βR) and 17β-hydroxysteroid dehydrogenase (17βHSD) is enhanced by enhancing the activity of HRF, whereby estrogen Cascade with enhanced activity.
(B) Cascade in which the expression of the Wnt gene is suppressed by enhancing the activity of HRF, thereby enhancing the aromatase activity and enhancing the estrogen activity.
(C) Cascade in which the expression of SMAD gene or transforming growth factor β (TGF-β) gene is enhanced by enhancing the activity of HRF.
(D) Cascade that suppresses the expression of lipocalin-type PGD synthase gene by enhancing the activity of HRF.
(E) Cascade in which the expression of Ras gene is enhanced by enhancing the activity of HRF.
(F) Cascade in which the expression of MEK (MAP kinase kinase) gene is enhanced by enhancing the activity of HRF.
(G) Cascade in which the expression of PLC (phospholipase C) gene is enhanced by enhancing the activity of HRF.
(H) A cascade in which the expression of a high affinity IgE receptor (FcεRI) gene is suppressed by enhancing the activity of HRF.
(I) A cascade in which the expression of a KDR (kinase insert domain receptor) gene is enhanced by enhancing the activity of HRF.

  That is, since the pharmaceutical of the present invention contains an HRF activity suppressing component, the expression of the gene is suppressed or enhanced, thereby enabling prevention, improvement, treatment, etc. of the above-mentioned diseases. Moreover, since the expression of genes related to angiogenesis such as KDR, TGF-β and IL-1β is suppressed or enhanced by the HRF activity suppressing component, angiogenesis can be suppressed.

FIG. 1 is an explanatory diagram of a molecular cascade starting from HRF. FIG. 2 is an LC-MS total ion chromatogram of the artemisinin preparation used in the examples of the present invention. FIG. 3 is an MS pattern of the relative retention time of 3.42 minutes for the artemisinin preparation. FIG. 4 is a graph of a calibration curve created in the example of the present invention. FIG. 5 is a graph showing that the activity of HRF was suppressed by administering artemisinin in the examples of the present invention.

  The molecular cascade starting from the HRF is shown in FIG. As shown, dioxin (TCDD) was known to target HRF, but downstream it is involved in estrogen-dependent diseases, PGD-dependent gynecological diseases, immune diseases and angiogenesis The present inventors are the first to clarify the above.

  As shown in the figure, when HRF activity is promoted, the expression of IL-1βR gene and 17βHSD gene is promoted, and estrogen activity is improved accordingly. By promoting the HRF activity, the expression of the Wnt gene is suppressed, and accordingly, the aromatase activity is improved and the estrogen activity is improved. This improvement in estrogenic activity causes estrogen-dependent diseases. By promoting HRF activity, the expression of SMAD gene or TGF-β gene is promoted. Furthermore, when HRF activity is promoted, the expression of the lipocalin-type PGD synthase gene is suppressed (attenuated), resulting in PGD-dependent gynecological diseases such as infertility, implantation disorders and miscarriages. On the other hand, the promotion of HRF activity generates immune diseases by suppressing the expression of FcεRI gene and promoting the expression of PLC gene, Ras gene and MEK gene.

  In addition, it has been obtained separately that HRF is associated with the development of estrogen-dependent diseases. That is, when morbidity of an estrogen-dependent disease was determined based on the HRF concentration in menstrual blood, all 15 patients judged positive (HRF concentration of 0.10 μg / mL or more) were afflicted with an estrogen-dependent disease, and negative Of the 3 persons judged to have an HRF concentration of less than 0.10 μg / mL, 2 persons did not suffer from estrogen-dependent diseases.

  The present invention will be described in detail below.

  Since the pharmaceutical of the present invention includes an HRF inhibitory component as described above, the HRF inhibitory component can suppress HRF activity and suppress the expression of IL-1β gene, thereby suppressing estrogen activity. Estrogen-dependent diseases such as membrane disease can be prevented, treated or ameliorated. It should be noted that IL-1β is a factor associated with estrogen-dependent diseases such as endometriosis, see, for example, J Clin Endocrinol Metab. 2002 Dec; 87 (12): 5785-92. And Ann N Y Acad Sci. 2002 Jun; 966: 166-86. Review. It is described in. Similarly, the pharmaceutical of the present invention can suppress the estrogen activity such as endometriosis, because it can suppress the expression of 17βHSD gene by suppressing the HRF activity by the HRF inhibitory component, thereby suppressing the estrogen activity. Can be treated or improved. The fact that 17βHSD is a related factor of estrogen-dependent diseases such as endometriosis is described in, for example, J Clin Endocrinol Metab. 2000 Sep; 85 (9): 3292-6. And J Clin Endocrinol Metab. 1998 Dec; 83 (12): 4474-80. It is described in. The pharmaceutical product of the present invention suppresses HRF activity by the HRF inhibitory component and promotes the expression of Wnt gene, thereby suppressing aromatase activity and estrogen activity. For example, it depends on estrogen such as endometriosis Sexual diseases can be prevented, treated or ameliorated. The inhibition of aromatase expression by Wnt is described in, for example, J Clin Endocrinol Metab. 2002 Sep; 87 (9): 4369-77. It is described in. Since the pharmaceutical of the present invention can suppress the estrogen activity by suppressing the expression of SMAD gene or TGF-β gene by suppressing the HRF activity by the HRF inhibitory component, for example, estrogen-dependent diseases such as endometriosis Can prevent, treat or improve. SMAD and TGF-β are related factors for estrogen-dependent diseases such as endometriosis, see, for example, J Clin Endocrinol Metab. 2003 Oct; 88 (10): 4967-76. It is described in.

  In addition, since the pharmaceutical of the present invention can suppress the expression of the KDR gene by suppressing the HRF activity by the HRF inhibitory component, for example, it can prevent, treat or ameliorate endometriosis or the like, or can suppress angiogenesis. . The fact that KDR is a related factor of diseases such as endometriosis is described in, for example, Ann NY Acad Sci. 2002 Mar; 955: 60-74; discussion 86-8, 396-406. It is described in. Since the pharmaceutical of the present invention can suppress the HRF activity by the HRF inhibitory component and promote the expression of the lipocalin type PGD synthase gene, this can prevent, treat or improve PGD-dependent diseases. The pharmaceutical agent of the present invention suppresses the expression of Ras gene by suppressing the HRF activity by the HRF inhibitory component, thereby preventing, treating or improving diseases such as endometriosis and ovarian cancer. Ras is a relevant factor for diseases such as endometriosis and ovarian cancer, see, for example, Mol Hum Reprod. 2004 Oct; 10 (10): 719-28. It is described in. The pharmaceutical product of the present invention suppresses the expression of the MAPK gene by suppressing the expression of the MEK gene by suppressing the HRF activity by the HRF inhibitory component, thereby preventing, treating or improving diseases such as endometriosis. .

  Furthermore, since the pharmaceutical of the present invention can suppress the expression of Ras gene and MEK gene by suppressing HRF activity by the HRF inhibitory component, and can promote the expression of PLC gene and FcεRI gene. Inhibiting the cascade involved, thereby preventing, treating or ameliorating immune diseases.

  In the present invention, “HRF activity suppression” not only means inactivating HRF, but also means, for example, inhibition or suppression of expression of HRF gene, suppression of synthesis or secretion of HRF, and the like.

  In the present invention, the “medicine for the disease” means a drug for treating, preventing or ameliorating the disease.

In the pharmaceutical product of the present invention, examples of the HRF activity-suppressing component include artemisinin and derivatives or synthetic products thereof, herbal medicines such as Seiko, Artemisia apiaceance, and the like. This is because artemisinin acts on HRF directly or indirectly and inactivates HRF. Examples of the artemisinin derivative include artesunate, dihydroartemisinin, artemeta and the like. The chemical formula of artemisinin is shown in the following chemical formula (1). Examples of the synthetic product of artemisinin include RBx11160 represented by the following chemical formula (2). As said artemisinin etc., what was isolated and refined from the crude drug etc. which are mentioned later may be used, for example, and a commercial item may be used. Artemisinin and the like have been conventionally used as a therapeutic agent for malaria, and its safety has been proven.

  In addition to the above-mentioned HRF activity-suppressing component, the pharmaceutical product of the present invention includes, for example, 17βHSD gene, IL-1β gene, IL-1β receptor gene, SMAD gene, Ras gene, MEK gene, MAPK gene, PLC gene, KDR Components and inhibitors that suppress the expression of genes and TGF-β genes, components that promote the expression of Wnt gene, lipocalin-type PGD synthase gene, PLC gene, FcεRI gene, and various additives may be included. . Specifically, for example, methotrexate (PubMed ID: 8292668), NO-releasing nonsteroidal anti-inflammatory analgesics (NO-NSAIDs, PubMed ID: 11046058), caspase drug (PubMed ID: 8603722) and hyaluronic acid (PubMed ID) : 12213700) and 17-β HSD inhibitors such as 16-ketoestrone (PubMed ID: 6572146). The hyaluronic acid can promote or suppress cytokines such as IL-1β depending on its concentration.

  In the pharmaceutical product of the present invention, the estrogen-dependent disease is, for example, dysmenorrhea, infertility, miscarriage, endometriosis, uterine fibroid, uterine adenomyosis, ovarian chocolate cyst, ovarian cancer, breast cancer, endometrial cancer, It includes cervical cancer, premenstrual syndrome (PMS: Syndrome that changes in mind and body with menstrual cycle), coronary heart disease, lipid metabolism abnormality and thyroid function abnormality. The PGD-dependent gynecological diseases include, for example, infertility, implantation disorders and miscarriages. Examples of the immune disease include food allergy, drug allergy, pollen allergy, insect allergy, asthma, rhinitis, urticaria, anaphylaxis, allergic bronchiectasis, hay fever, cold urticaria and atopic dermatitis.

  Next, the treatment, prevention, amelioration or suppression method of the disease of the present invention (hereinafter referred to as the treatment method of the present invention) is selected from the group consisting of estrogen-dependent diseases, PGD-dependent gynecological diseases, immune diseases and cancer. A method for treating, preventing or ameliorating a disease or inhibiting angiogenesis, comprising inhibiting HRF activity.

  In the treatment method of the present invention, in order to suppress HRF activity, for example, it is preferable to include administration of artemisinin and its derivatives or synthetic compounds, herbal medicines such as blue candy. Moreover, in order to suppress HRF activity, the pharmaceutical product of the present invention may be administered. The derivatives and synthetic products are the same as the pharmaceutical product of the present invention. The artemisinin or the like may be a commercially available product such as a commercially available artemisinin preparation, or may be produced by a known method.

  In the treatment method of the present invention, the disease is an estrogen-dependent disease, for example, IL-1β gene, IL-1β receptor gene, 17βHSD gene, Wnt gene, SMAD gene, TGF-β gene, Ras gene, It is preferable to include selecting a treatment method according to the measurement result by measuring the expression level and / or aromatase activity of the MEK gene and the KDR gene. The measurement result may be the expression level of any one gene or the expression levels of a plurality of genes. Since it can be judged from various aspects, it is more preferable to use the expression levels of a plurality of genes. Among the genes, it is preferable to use the expression levels of IL-1β gene, IL-1β receptor gene, 17βHSD gene and Wnt gene. Further preferred. Examples of the selection of the treatment method include changing the dose, administration interval, administration method and the like of artemisinin and the pharmaceutical of the present invention. The expression level of the gene can be measured using, for example, a microarray, and the method for measuring the aromatase activity is, for example, an in vitro measurement method using animal cells or yeast, a tritium water release assay method, a fluorescence method , Liquid chromatography methods and measurement methods using mRNA.

The treatment method of the present invention preferably further comprises inhibiting at least one of the following (a) to (d).
(A) Expression of a gene selected from the group consisting of IL-1β gene, IL-1β receptor gene and 17βHSD gene.
(B) Suppression of Wnt gene expression.
(C) Expression of at least one of SMAD gene and TGF-β gene.
(D) Aromatase activity.

  In the treatment method of the present invention, the (a) is preferably the expression of at least one of the IL-1β gene and the IL-1β receptor gene and the expression of the 17βHSD gene.

  In the treatment method of the present invention, the (c) is preferably both a SMAD gene and a TGF-β gene.

  In the treatment method of the present invention, the disease is preferably a PGD-dependent gynecological disease, and preferably comprises measuring the expression level of a lipocalin-type PGD synthase gene and selecting a treatment method according to the expression level. Furthermore, it is more preferable to include administering a prostaglandin preparation and promoting PGD activity. Examples of the promotion of PGD activity include promotion of expression of a lipocalin-type PGD synthase gene, activation of PGD, promotion of PGD synthesis or secretion, and the like. The treatment method can be selected in the same manner as in the case of the estrogen-dependent disease.

  In the treatment method of the present invention, the disease is an immune disease. For example, the expression level of a gene such as FcεRI gene, Ras gene, MEK gene and PLC gene is measured, and the treatment method is selected according to the expression level. It is preferable to further include, for example, and it is more preferable to include suppression of expression of, for example, Ras gene, MEK gene, FcεRI gene and the like. The measurement result may be the expression level of any one gene or the expression levels of a plurality of genes. Since it can be judged from many aspects, it is more preferable to use the expression level of a plurality of genes. The treatment method can be selected in the same manner as in the case of the estrogen-dependent disease.

  In the treatment method of the present invention, when it is a method of suppressing angiogenesis, it further includes, for example, suppressing the expression of genes related to angiogenesis such as KDR gene, IL-1β gene and TGF-β gene. Is preferred. In addition, for example, the expression level of genes such as KDR gene, IL-1β gene, and TGF-β gene may be measured, and a treatment method may be selected based on the measurement result. The measurement result may be the expression level of any one gene or the expression levels of a plurality of genes. Since it can be judged from many aspects, it is more preferable to use the expression level of a plurality of genes. The treatment method can be selected in the same manner as in the case of the estrogen-dependent disease.

  Next, the screening method of the present invention is a method for screening for an HRF activity suppressing component or an HRF activity enhancing component, which comprises a 17βHSD gene, an IL-1β gene, an IL-1β receptor gene, a Wnt gene, a SMAD gene, a lipocalin type. Select an HRF activity-suppressing component or an HRF activity-enhancing component by measuring the expression level of a gene selected from the group consisting of PGD synthase gene, Ras gene, MEK gene, PLC gene, FcεRI gene, KDR gene and TGF-β gene The process of carrying out is included. The method for measuring the expression level of the gene is not particularly limited, and a conventionally known method can be used. For example, a method capable of rapid measurement such as a method using a microarray and a method using PCR such as a real-time PCR method. Can be given.

  As a method of using the microarray, a microarray in which probes corresponding to the marker transcripts are arranged is prepared, and a labeled target prepared using RNA in a sample as a template is hybridized to the microarray and bound to the probe. And a method of quantifying the RNA strand by measuring the labeled signal of the target. The hybridization method and conditions are not particularly limited and can be performed by a conventionally known method. Those skilled in the art can appropriately adjust depending on the type of probe, target, polynucleotide and the like. The method for detecting and measuring the labeled signal of the target hybridized with the probe is not particularly limited, and can be measured by a conventionally known method or apparatus according to the type of label.

  The screening method of the present invention includes, for example, a pharmaceutical for at least one selected from the group consisting of estrogen-dependent diseases, PGD-dependent gynecological diseases, immune diseases, cancer and angiogenesis inhibition, food for specified health use, food for nutritional function Or it can be used for the screening of general food. Examples of the general food include natural foods, health foods supplemented with health functional ingredients, and supplements.

  Next, the microarray of the present invention is a microarray used in the screening method of the present invention, and includes a 17βHSD gene, an IL-1β gene, an IL-1β receptor gene, a Wnt gene, a SMAD gene, and a lipocalin type PGD synthase gene. A polynucleotide spot that can be used as a probe for detecting the expression of a gene selected from the group consisting of a Ras gene, MEK gene, PLC gene, FcεRI gene, KDR gene, and TGF-β gene is arranged.

  The production method of the microarray is not particularly limited. For example, a conventionally known method such as a method of directly synthesizing a probe polynucleotide on the substrate surface (on-chip method) or a method of immobilizing a previously prepared probe on the substrate surface. Is mentioned. As the on-chip method, selection is made in a predetermined region of a minute matrix by combining the use of a protective group that is selectively removed by light irradiation, and photolithography technology and solid phase synthesis technology used in semiconductor manufacturing. The method of performing a synthetic synthesis is mentioned. Examples of the method using the probe prepared in advance include a method in which a probe solution is finely dropped onto a substrate by an ink jet method and chemically or physically fixed. The substrate is not particularly limited, and examples thereof include glass, metal, and plastic.

  Next, the screening kit of the present invention is a screening kit used in the screening method of the present invention, and includes the microarray of the present invention. The kit may further contain, for example, a primer or reagent for preparing total RNA from a sample, a labeled primer or reagent of the present invention for preparing a target, and the like. As the reagent, a conventionally known reagent can be used, and examples thereof include a polymerase, a nucleotide, a labeled compound, and a buffer.

  Next, the production method of the present invention includes a pharmaceutical for at least one selected from the group consisting of estrogen-dependent diseases, PGD-dependent gynecological diseases, immune diseases, cancer and angiogenesis inhibition, food for specified health use, nutritional function A method for producing a food or general food, comprising the step of selecting an HRF activity-suppressing component by the screening method of the present invention. The general food is as described above.

  Next, the determination method of the present invention is a method for determining the susceptibility of a disease selected from the group consisting of an estrogen-dependent disease, a PGD-dependent gynecological disease, an immune disease, and cancer, in a biological sample of a subject Selected from the group consisting of 17βHSD gene, IL-1β gene, IL-1β receptor gene, Wnt gene, SMAD gene, lipocalin-type PGD synthase gene, Ras gene, MEK gene, PLC gene, FcεRI gene and TGF-β gene A measurement step for measuring the expression level of the gene to be compared, a determination step for comparing the measurement value with that of a normal biological sample, thereby determining the HRF dependence, and determining the susceptibility of the disease based on the HRF dependence Including a determination step. The determination includes, for example, determination of whether or not the subject suffers from the disease, determination whether or not there is a risk of suffering from the disease in the future, and risk of recurrence of the disease after treatment Determination of whether or not a sex exists, and determination of the degree of risk and the risk of the disease.

  Next, the second microarray of the present invention is a microarray used in the determination method of the present invention, and includes a 17βHSD gene, an IL-1β gene, an IL-1β receptor gene, a Wnt gene, a SMAD gene, and a lipocalin type PGD. A polynucleotide spot that can be used as a probe for detecting the expression of a gene selected from the group consisting of a synthetic enzyme gene, Ras gene, MEK gene, PLC gene, FcεRI gene, and TGF-β gene is arranged. The manufacturing method of the microarray is the same as that of the first microarray.

  Next, the second kit of the present invention is a kit used in the determination method of the present invention, includes the second microarray of the present invention, and is otherwise the same as the first kit of the present invention. Things can be included.

  Next, the method for determining angiogenesis according to the present invention includes measuring the HRF activity in a biological sample and comparing the measured value with that of a normal biological sample to determine the presence or absence of angiogenesis. HRF activity can be measured, for example, by enzyme immunoassay (EIA) such as solid phase enzyme immunoassay (ELISA) or sandwich ELISA (RIA) or radioimmunoassay (RIA). In addition, the method for determining angiogenesis according to the present invention may include, for example, a measurement step of measuring the expression level of a KDR gene, a TGF-β gene, an IL-1β gene, and the like. It can also be judged.

  Next, the use of the present invention comprises artemisinin and its derivatives for the manufacture of a medicament for at least one selected from the group consisting of estrogen dependent diseases, PGD dependent gynecological diseases, immune diseases, cancer and angiogenesis inhibition or At least one use selected from the group consisting of composites. Another aspect of the use of the present invention is artemisinin and its derivatives or compounds for at least one selected from the group consisting of estrogen-dependent diseases, PGD-dependent gynecological diseases, immune diseases, cancer and angiogenesis inhibition Is the use of. In the use of the present invention, it is preferable that the artemisinin is derived from a herbal medicine such as green candy. The derivatives and synthetic products are the same as the pharmaceutical product of the present invention.

  Moreover, this invention contains the food for specific health containing the component which suppresses HRF activity, a nutritional functional food, a general food, etc. as another aspect. The said general food and the said HRF activity suppression component are the same as that of the said pharmaceutical of this invention.

  Furthermore, since angiogenesis is promoted by promoting HRF activity as described above, the present invention further promotes an angiogenesis promoting agent containing an HRF activity enhancing component, and HRF activity as another aspect. A method of promoting angiogenesis by the method. Such an angiogenesis promoter is useful for cardiovascular diseases, for example.

  In still another aspect, the present invention relates to a drug for idiopathic nephrotic syndrome, a drug containing an HRF activity-suppressing component, an idiopathic nephrotic syndrome by administering an HRF activity-suppressing component or suppressing HRF activity. Method for treating, pharmaceutical for idiopathic nephrotic syndrome including a step of measuring HRF activity, screening method for food for specified health use, nutritional functional food or general food, and idiopathic nephrotic syndrome including step of measuring HRF activity Including diagnostic methods. HRF is considered to be a disease-specific gene in idiopathic nephrotic syndrome in children and is specifically expressed not only in the state of nephrosis but also at the protein level in peripheral blood mononuclear cells activated with concanavalin A. This is because HRF is thought to be related to the pathogenesis of nephrotic syndrome because of increased HRF secretion in the same cell (Toyabe, S., et al. Clin Exp Immunol. 2005 Oct). 142 (1): 162-6.).

  In still another aspect, the present invention provides a drug for Alzheimer's disease or Down's syndrome, which contains a component that enhances HRF activity, a method for treating Alzheimer's disease or Down's syndrome by activating HRF, and measuring HRF activity. And a method for screening Alzheimer's disease or Down's syndrome, a pharmaceutical product for Alzheimer's disease or Down's syndrome, a method for screening for food for specified health use, a nutritional functional food or a general food, and a diagnosis method for Alzheimer's disease or Down's syndrome using HRF as a marker. HRF is significantly reduced in patients with Down's syndrome, for example, in the temporal cortex, thalamus and supranuclear nucleus, while in Alzheimer's disease patients, for example, in the temporal cortex, it is recognized in neurodegeneration. Since the cause of the decrease in function is thought to be a decrease in the activity of HRF (Kim, SH., Et al. Neurosci Lett. 2001 Mar 2; 300 (1): 41-4.), It activates HRF. Therefore, it is considered possible to treat Alzheimer's disease or Down syndrome. The method for activating HRF is not particularly limited, and examples thereof include a method of administering HRF itself, and in addition, a range that covers a sufficient part to function in an appropriate expression vector such as a retrovirus. For example, the above-mentioned cDNA is inserted into a region where HRF is decreased by developing Alzheimer's disease and Down's syndrome, such as temporal cortex, and then cells in the region are transformed.

  Hereinafter, the present invention will be described in more detail based on examples and comparative examples. However, the present invention is not limited by the following examples.

  Using microarray, HRF was added to enhance or suppress the expression of various genes. The results are shown in Table 1, Table 2 and Table 3 below. In addition, the evaluation of gene expression by a microarray was performed by a conventional method.

Specifically, the evaluation of gene expression by the microarray was performed as follows. That is, first, HL-60 cells and 10% FBS-RPMI1640 as a medium were prepared. Next, HRF was added to the medium at a concentration of 10 μg / mL, and the HL-60 cells were cultured at 37 ° C. for 24 hours. The cells were washed with PBS, and then trade name ISOGEN (manufactured by Nippon Gene) was added to the RNA. Extracted. 2 μg of the extracted RNA was used, and microarraying was performed using a trade name CodeLink Human Whole Genome Bioarray (manufactured by Amersham Bioscience). Then, trade name Agilent G2565 microarray scanner and scanned using (Agilent Ltd. Technologies), the resulting signal (image), and quantified by trade name CodeLink Expression2 (manufactured by ^{Amersham Bioscience), GeneSpring (R)} (Silicon Genetics Manufactured).

  As shown in Tables 1 to 3, the expression levels of various genes varied, and among them, the increase in the expression levels of MEK, 17βHSD, IL-1 receptor and the like was large.

  This example is an example showing that administration of artemisinin inactivates HRF and improves symptoms of estrogen-dependent diseases.

(1) Analysis of artemisinin in artemisinin preparation First, 82 g of artemisinin preparation was suspended in 1.5 mL of purified water, extracted twice with 4 mL of a hexane: ether (2: 1) mixture, shaken for 10 minutes, Centrifugation was performed and the solvent was distilled off. Next, 2 mL of acetonitrile was added, and after refrigerated storage to precipitate impurities, the mixture was centrifuged, the supernatant was distilled off, dissolved in 100 μL of the developing solution, and analyzed by LC-MS under the following conditions. It was. The results are shown in FIGS.

(Conditions for liquid chromatography)
Column: Product name XTerra MS C ₁₈ (3.5 μm, inner diameter 2.1 mm × length 100 mm, manufactured by Waters)
Eluent: Acetonitrile: 2.5 mM ammonia acetate aqueous solution (60:40)
Flow rate: 0.2 mL / min Injection volume: 10 μL

The masses of the artemisinin molecule-related ion and dihydroartemisinin molecule-related ion are as follows.
Artemisinin molecule-related ion Molecular ion (M): 282
Hydrogen addition ion: 283
Ammonia adduct ion: 300
Na addition ion: 305
Trimolecular Na addition ion: 351
Dihydroartemisinin molecule-related ion Molecular ion (M): 284
Hydrogenation ion: 285
Ammonia adduct ion: 302
Na addition ion: 307

  FIG. 2 is an LC-MS total ion chromatogram of the artemisinin preparation used in the examples of the present invention, and FIG. 3 is an MS pattern of the relative retention time of the artemisinin preparation of 3.42 minutes. From these results, the artemisinin preparation was composed of artemisinin and a small amount of dihydroartemisinin.

(2) Preparation of calibration curve First, HRF producing cells that specifically react with HRF were established, and these producing cells were administered intraperitoneally to mice. The obtained mouse ascites was purified with a protein G column to obtain a highly pure monoclonal IgG antibody. This monoclonal antibody specifically recognizing HRF was dissolved in a sodium bicarbonate buffer (0.1 M, pH 9.5) in advance so as to have a concentration of 10 μl / ml, and this was dissolved in 96 wells for ELISA. 100 μL each was added to the immunoplate and allowed to stand overnight at 4 ° C. to allow the antibody to bind to the plate.

  Next, the solution is discarded from the plate, and each well is washed three times with PBS (phosphate buffered saline) containing 0.05% Tween 20 (trade name) and then Tris-HCl buffer containing 2% animal serum. 200 μL of the solution was added, and the mixture was allowed to stand at room temperature for 1 to 2 hours, and the antibody-free site was blocked with animal serum. The plate was washed 3 times with PBS containing 0.05% Tween 20 (trade name), and then HRF standard antigen (baculovirus-derived recombinant antigen: 2000, 1000, 500, 250, 125, 63, 32) adjusted to a predetermined concentration. 16, 8, 4, 2, 1, 0 ng / mL) was added at 100 μL per well, left at room temperature for 2 hours, and washed 3 times with PBS containing 0.05% Tween 20 (trade name). Then, 100 μL per well of anti-rabbit HRF antibody with different antigen recognition sites (polyclonal antibody obtained by immunizing subcutaneously with a synthetic peptide antigen (15 amino acid residues having an HRF antigen active site) 4 times) was added. The mixture was allowed to stand at room temperature for 1 hour and washed 3 times with PBS containing 0.05% Tween 20 (trade name). Horseradish peroxidase-labeled goat anti-rabbit IgG antibody (Bio-Rad) was added at 100 μL per well, left at room temperature for 1 hour, and washed 3 times with PBS containing 0.05% Tween 20 (trade name). Then, add 100 μL of enzyme substrate solution (TMB: 3-3-5-5′tetramethylbenzidine) per well, let it stand for 30 minutes at room temperature, develop color, add 100 μL of 1M phosphoric acid per well, and react. Stopped. Absorbance was measured using an immunoleader (BIO-RAD, Model 680) at a wavelength of 490 nm and a sub-wavelength of 680 nm to prepare a calibration curve. The obtained calibration curve is shown in FIG.

(3) Determination of dysmenorrhea The degree of dysmenorrhea was determined using the score table shown in Table 4 below based on subjective symptoms and use of analgesics. This score table is obtained by modifying the Andersch method with reference to the method of Bileroglu.

(4) Administration of artemisinin The degree of patients with dysmenorrhea (including endometriosis) was evaluated using the score. From the results of the evaluation, two patients considered to have severe symptoms were taken as the administration group, and the above-mentioned artemisinin preparation was administered to these patients in an amount of 100 mg per day for 5 months. On the other hand, two patients with mild symptoms compared with the administration group were designated as the non-administration group, and the artemisinin preparation was not administered to these patients. About the said patient, HRF activity was measured every month and the symptom was confirmed. The measurement result of HRF activity is shown in FIG. 5, and the change in score is shown in Table 5 below. The measurement of the HRF activity was performed in the same manner as in the preparation of the calibration curve (1) except that serum or menstrual blood from a subject was used instead of the HRF standard antigen, and antibody was obtained using the calibration curve. The amount of HRF bound to was determined as HRF activity.

  As shown in FIG. 5 and Table 5, administration of artemisinin inactivated HRF, and accordingly, symptoms of dysmenorrhea were also improved. Therefore, it can be said that estrogen-dependent diseases can be improved by suppressing the activity of HRF.

  The pharmaceutical agent of the present invention is useful as a pharmaceutical agent for preventing, treating or ameliorating estrogen-dependent diseases, infertility and immune diseases, and a pharmaceutical agent for suppressing angiogenesis.

Claims (32)

  A pharmaceutical product for at least one selected from the group consisting of estrogen-dependent disease, prostaglandin D (PGD) -dependent gynecological disease, immune disease, cancer and angiogenesis inhibition, and inhibiting histamine release factor (HRF) activity Medicinal product containing ingredients.   The pharmaceutical product according to claim 1, wherein the HRF activity-suppressing component is selected from the group consisting of artemisinin and its derivatives or synthetic products.   The pharmaceutical product according to claim 1, wherein the HRF activity-suppressing component is green candy.   The estrogen-dependent diseases include dysmenorrhea, infertility, miscarriage, endometriosis, adenomyosis, uterine fibroid, ovarian chocolate cyst, ovarian cancer, breast cancer, endometrial cancer, cervical cancer, premenstrual syndrome ( The pharmaceutical product according to claim 1, selected from the group consisting of PMS), coronary heart disease, lipid metabolism abnormality and thyroid function abnormality.   The pharmaceutical product according to claim 1, wherein the PGD-dependent gynecological disease is selected from the group consisting of infertility, implantation disorders and miscarriage.   The immune disease is selected from the group consisting of food allergy, drug allergy, pollen allergy, insect allergy, asthma, rhinitis, urticaria, anaphylaxis, allergic bronchiectasis, hay fever, cold urticaria and atopic dermatitis The pharmaceutical product according to claim 1.   A method for screening an HRF activity inhibitory component or an HRF activity enhancing component, comprising a 17β hydroxyl steroid dehydrogenase (HSD) gene, an IL-1β gene, an IL-1β receptor gene, a Wnt gene, a SMAD gene, a lipocalin type PGD A gene selected from the group consisting of a synthetic enzyme gene, Ras gene, MEK gene, phospholipase C (PLC) gene, high affinity IgE receptor (FcεRI) gene, KDR gene and transforming growth factor β (TGF-β) gene A screening method comprising a step of selecting an HRF activity suppressing component or an HRF activity enhancing component by measuring the expression level of.   Claim for use in screening for pharmaceuticals, foods for specified health use, nutritional functional foods or general foods for at least one selected from the group consisting of estrogen-dependent diseases, PGD-dependent gynecological diseases, immune diseases, cancer and angiogenesis suppression The screening method according to claim 7.   A microarray used in the screening method according to claim 8, comprising a 17βHSD gene, an IL-1β gene, an IL-1β receptor gene, a Wnt gene, a SMAD gene, a lipocalin-type PGD synthase gene, a Ras gene, a MEK gene, A microarray in which spots of polynucleotides that can be used as probes for detecting the expression of a gene selected from the group consisting of a PLC gene, an FcεRI gene, a KDR gene, and a TGF-β gene are arranged.   A screening kit for use in the screening method according to claim 8, comprising the microarray according to claim 9.   A method for producing a medicinal product, food for specified health use, nutritional functional food or general food for at least one selected from the group consisting of estrogen-dependent diseases, PGD-dependent gynecological diseases, immune diseases, cancer and angiogenesis suppression, The manufacturing method including the process of selecting the HRF activity suppression component by the screening method of Claim 7.   A method for determining the susceptibility of a disease selected from the group consisting of an estrogen-dependent disease, a PGD-dependent gynecological disease, an immune disease and a cancer, comprising: a 17βHSD gene, an IL-1β gene, an IL- Measurement step of measuring the expression level of a gene selected from the group consisting of 1β receptor gene, Wnt gene, SMAD gene, lipocalin-type PGD synthase gene, Ras gene, MEK gene, PLC gene, FcεRI gene and TGF-β gene A determination method comprising a determination step of comparing the measured value with that of a normal biological sample, thereby determining an HRF dependency, and a determination step of determining the susceptibility of the disease based on the HRF dependency.   A microarray for use in the determination method according to claim 12, comprising a 17βHSD gene, an IL-1β gene, an IL-1β receptor gene, a Wnt gene, a SMAD gene, a lipocalin-type PGD synthase gene, a Ras gene, a MEK gene, A microarray in which spots of polynucleotides that can be used as probes for detecting the expression of a gene selected from the group consisting of a PLC gene, an FcεRI gene, and a TGF-β gene are arranged.   A kit for use in the determination method according to claim 12, which comprises the microarray according to claim 13.   A method for determining angiogenesis, comprising determining HRF activity in a biological sample and determining the presence or absence of angiogenesis by comparing the measured value with that of a normal biological sample.   A method for treating, preventing or ameliorating a disease selected from the group consisting of estrogen-dependent diseases, PGD-dependent gynecological diseases, immune diseases and cancer, or inhibiting angiogenesis, comprising inhibiting HRF activity .   The method according to claim 16, comprising administering at least one selected from the group consisting of artemisinin and a derivative or composite thereof in order to suppress HRF activity.   17. A method according to claim 16, comprising administering blue cocoon to inhibit HRF activity.   The disease is an estrogen-dependent disease, and is from the group consisting of IL-1β gene, IL-1β receptor gene, 17βHSD gene, Wnt gene, SMAD gene, TGF-β gene, Ras gene, MEK gene and KDR gene The method according to claim 16, comprising selecting a treatment method according to the measurement result by measuring the expression level and / or aromatase activity of the selected gene. The method according to claim 19, further comprising inhibiting at least one of the following (a) to (d):
(A) Expression of a gene selected from the group consisting of IL-1β gene, IL-1β receptor gene and 17βHSD gene.
(B) Suppression of Wnt gene expression.
(C) Expression of at least one of SMAD gene and TGF-β gene.
(D) Aromatase activity.   21. The method according to claim 20, wherein (a) is expression of at least one of IL-1β gene and IL-1β receptor gene and expression of 17βHSD gene.   The method according to claim 20, wherein (c) is both a SMAD gene and a TGF-β gene.   The method according to claim 16, wherein the disease is PGD-dependent gynecological disease, and comprises measuring the expression level of a lipocalin type PGD synthase gene and selecting a treatment method according to the expression level.   24. The method of claim 23, further comprising administering a prostaglandin formulation.   24. The method of claim 23, further comprising promoting PGD activity.   The disease is an immune disease, and comprises measuring the expression level of a gene selected from the group consisting of FcεRI gene, Ras gene, MEK gene and PLC gene, and selecting a treatment method according to the expression level The method of claim 16.   27. The method according to claim 26, further comprising suppressing the expression of a gene selected from the group consisting of Ras gene, MEK gene and FcεRI gene.   The method according to claim 16, further comprising suppressing the expression of a gene selected from the group consisting of a KDR gene, an IL-1β gene, and a TGF-β gene.   Selected from the group consisting of artemisinin and its derivatives or compounds for the manufacture of a medicament for at least one selected from the group consisting of estrogen dependent diseases, PGD dependent gynecological diseases, immune diseases, cancer and angiogenesis inhibition At least one use.   30. The use according to claim 29, wherein the artemisinin is derived from blue cocoon.   Use of artemisinin and its derivatives or compounds for at least one selected from the group consisting of estrogen-dependent diseases, PGD-dependent gynecological diseases, immune diseases, cancer and angiogenesis inhibition.   32. Use according to claim 31, wherein the artemisinin is derived from blue cocoon.

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