LIPOCALIN-TYPE PROSTAGLANDIN D2 SYNTHASE PRODUCTION PROMOTING AGENT

Abstract

A lipocalin-type prostaglandin D2 synthase (L-PGDS) production promoting agent, more specifically an L-PGDS production promoting agent in pericytes or ischemia-induced multipotent stem cells (iSCs) dedifferentiated from pericytes. A substance having an L-PGDS production promoting action is contained in an extract from inflamed tissues inoculated with vaccinia virus. An L-PGDS production promoting agent is highly useful as a prophylactic, therapeutic or relapse prophylactic agent for a disease in which the effect by promotion of L-PGDS expression is expected to be effective, including a cerebrovascular disorder such as cerebral infarction, dementia such as Alzheimer's disease, or a sleep disorder.

Claims

1. A method for promoting production of a lipocalin-type prostaglandin D2 synthase in a patient in need thereof, the method comprising: administering a lipocalin-type prostaglandin D2 synthase production agent to the patient.

2. The method according to claim 1, wherein the agent is present in an extract from inflamed tissues inoculated with vaccinia virus.

3. The method according to claim 1, wherein the agent contains an extract from inflamed tissues inoculated with vaccinia virus.

4. The method according to claim 3, wherein the lipocalin-type prostaglandin D2 synthase is produced in pericytes or ischemia-induced multipotent stem cells dedifferentiated from pericytes.

5. The method according to claim 3, wherein the agent is a brain-protective agent.

6. The method according to claim 5, wherein the effect of the brain-protective agent is caused by an action enhancing a glymphatic system.

7. The method according to claim 5, wherein the brain-protective agent is a prophylactic, therapeutic, or relapse prophylactic agent for cerebral infarction, or a prophylactic or therapeutic agent for dementia.

8. (canceled)

9. The method according to claim 7, wherein the dementia is Alzheimer-type.

10. The method according to claim 9, wherein the agent has an amyloid deposition-inhibitory action.

11. The method according to claim 3, wherein the agent is a hypnotic.

12. The method according to claim 3, wherein the inflamed tissues are inflamed skin tissues of rabbits.

13. The method according to claim 3, wherein the agent is an injection preparation.

14. The method according to claim 3, wherein the agent is an oral preparation.

15. A screening method for a substance having a brain-protective action or a sleep-promoting action, the method comprising: determining a lipocalin-type prostaglandin D2 synthase expression-promoting action of the substance.

16. The screening method according to claim 15, wherein the determination includes measuring the lipocalin-type prostaglandin D2 synthase expression-promoting action in pericytes or ischemia-induced multipotent stem cells dedifferentiated from pericytes.

17. The screening method according to claim 15, wherein the substance having a brain-protective action is a prophylactic, therapeutic, or relapse prophylactic agent for cerebral infarction, or a prophylactic or therapeutic agent for dementia.

18. (canceled)

19. (canceled)

20. A determining or evaluating method for an extract from inflamed tissues inoculated with vaccinia virus or a preparation containing the extract, comprising determining a lipocalin-type prostaglandin D2 synthase expression-promoting action of the extract or the preparation containing the extract.

21. The determining or evaluating method according to claim 20, wherein the determination includes measuring the lipocalin-type prostaglandin D2 synthase expression-promoting action in pericytes or ischemia-induced multipotent stem cells dedifferentiated from pericytes.

22. The determining or evaluating method according to claim 20, wherein the inflamed tissues are inflamed skin tissues of rabbits.

23. The determining or evaluating method according to claim 20, wherein the method is performed to verify that the extract or the preparation containing the extract satisfies a quality standard.

24. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0062] FIG. 1 is a graph showing L-PGDS gene (PTGDS) expression level, as examined using real-time RT-PCR, in cultured iSCs to which a test substance was added.

[0063] FIG. 2 is an electropherogram showing L-PGDS gene (PTGDS) expression level, as examined using classical RT-PCR, in cultured iSCs to which a test substance was added.

[0064] FIG. 3 is an electropherogram showing L-PGDS protein expression level, as examined using Western blotting, in cultured iSCs to which a test substance was added.

[0065] FIG. 4 is a PVDF membrane showing L-PGDS protein expression level, as examined using dot blotting, in cultured iSCs to which a test substance was added.

[0066] FIG. 5 is a graph showing L-PGDS protein expression level, as examined using an ELISA method, in cultured iSCs to which a test substance was added.

[0067] FIG. 6 is a graph showing the amounts of prostaglandins, as examined using liquid chromatography-mass spectrometry, in a cell extract of cultured iSCs to which a test substance was added.

[0068] FIG. 7 is a graph showing the amounts of reaction products, as examined using liquid chromatography-mass spectrometry, after adding substrates for L-PGDS to a culture supernatant of cultured iSCs to which a test substance was added.

[0069] FIG. 8 represents images showing distributions of L-PGDS, pericytes, and vascular endothelial cells, as compared with each other using immunohistochemical staining, in the mouse brain to which ischemic insult was applied.

[0070] FIG. 9 represents images showing distributions of L-PGDS, as observed using immunoelectron microscopy, in the mouse brain to which ischemic insult was applied.

[0071] FIG. 10 represents images showing distributions of L-PGDS and nestin, which is a neural stem cell marker, as compared using immunohistochemical staining, in cultured iSCs isolated from a human cerebral infarction area.

[0072] FIG. 11 represents images showing deposition of amyloid , as examined using immunohistochemical staining, in the brain of an Alzheimer-type dementia model mouse to which a test substance was administered.

[0073] FIG. 12 represents images showing the results of immunohistochemical staining of L-PGDS in the brain of an Alzheimer-type dementia model mouse to which a test substance was administered.

[0074] FIG. 13 represents comparative images showing distributions of L-PGDS and vascular endothelial cells, using immunohistochemical staining, in the brain of an Alzheimer-type dementia model mouse to which a test substance was administered.

[0075] FIG. 14 represents electropherograms showing expression levels of amyloid and L-PGDS protein, as examined using Western blotting, in the brain of an Alzheimer disease model mouse to which a test substance was administered.

[0076] FIG. 15 represents electropherograms showing L-PGDS gene (PTGDS) expression levels, as examined using classical RT-PCR, in cultured pericytes to which a test substance was added.

MODE FOR CARRYING OUT THE INVENTION

[0077] The present extract is an extract containing a non-protein active substance extracted and separated from inflamed tissues of an animal having developed pox by being inoculated with vaccinia virus. The present extract is in liquid when it is extracted; however, the present extract may be made solid by drying. The present preparation is very useful as pharmaceuticals. One specific product that is manufactured and sold in Japan by the applicant as the present preparation is Preparation containing an extract from inflamed skins of rabbits inoculated with vaccinia virus (trade name: NEUROTROPIN [registered trademark]) (hereinafter, referred to as NEUROTROPIN). NEUROTROPIN includes injections and tablets, both of which are ethical drugs.

[0078] Indications of NEUROTROPIN injection are low back pain, cervicobrachial syndrome, symptomatic neuralgia, itchiness accompanied by skin diseases (eczema, dermatitis, urticaria), allergic rhinitis and sequelae of subacute myelo-optico-neuropathy (SMON) such as coldness, paresthesia and pain. Indications of NEUROTROPIN tablet are postherpetic neuralgia, low back pain, cervicobrachial syndrome, periarthritis scapulohumeralis and osteoarthritis. Present preparation has been created by the applicant and developed as a drug, and has been appreciated for its excellent advantage for efficacy and safety, sold for many years and established a firm position in the Japanese pharmaceutical market.

[0079] The extract from inflamed tissues inoculated with vaccinia virus used in the present invention can be obtained by the following manner: inflamed tissues inflamed by the inoculation with vaccinia virus is crushed; an extraction solvent is added to remove the tissue fragments; then deproteinization is carried out; the deproteinized solution is adsorbed onto an adsorbent; and then the active ingredient is eluted. for example, according to the following process.

(A) Inflamed skin tissues of rabbits, mice or the like by the inoculation with vaccinia virus are collected, and the inflamed tissues are crushed. To the crushed tissue an extraction solvent such as water, phenolated water, physiological saline or phenol-added glycerin water is added. Then, the mixture is filtered or centrifuged to obtain an extraction liquid (filtrate or supernatant).
(B) The pH of the extraction liquid is adjusted to be acidic and the liquid is heated for deproteinization. Then, the deproteinized solution is adjusted to be alkaline, heated, and then filtered or centrifuged.
(C) The obtained filtrate or supernatant is made acidic and adsorbed onto an adsorbent such as activated carbon or kaolin.
(D) To the adsorbent, an extraction solvent such as water is added, the pH is adjusted to alkaline, and the adsorbed component is eluted to obtain the extract from inflamed tissues inoculated with vaccinia virus. Subsequently, as desired, the eluate may be evaporated to dryness under reduced pressure or freeze-dried to give a dried material.

[0080] As for animals in order to obtain the inflamed tissues by the inoculation of vaccinia virus, various animals that is infected with vaccinia virus such as rabbits, cows, horses, sheep, goats, monkeys, rats or mice can be used, and preferred inflamed tissues are inflamed skin tissues of rabbits. With regard to a rabbit, any rabbit may be used so far as it belongs to Lagomorpha. Examples thereof include Oryctolagus cuniculus, domestic rabbit (domesticated Oryctolagus cuniculus), hare (Japanese hare), mouse hare and snowshoe hare. Among them, it is appropriate to use domestic rabbit. In Japan, there is family rabbit called Kato which has been bred since old time and frequently used as livestock or experimental animal and it is another name of domestic rabbit. There are many breeds in domestic rabbit and the breeds being called Japanese white and New Zealand white are advantageously used.

[0081] Vaccinia virus used herein may be in any strain. Examples thereof include Lister strain, Dairen strain, Ikeda strain, EM-63 strain and New York City Board of Health strain.

[0082] As to basic extracting steps (A) to (D) of the above-described for the present extract can be carried out in more detail, the following steps are used for example.

About Step (A)

[0083] The inflamed skin tissues of rabbits by the intradermal inoculation of vaccinia virus are collected. The collected skin tissues are washed and disinfected using a phenol solution, etc. This inflamed skin tissues are crushed and an extraction solvent in 1- to 5-fold thereof by volume is added thereto. Here, the term crush means to finely break down into minces using a mincing machine or the like. As to the extraction solvent, there may be used distilled water, physiological saline, weakly acidic to weakly basic buffer, etc. and bactericidal/antiseptic agent such as phenol, stabilizer such as glycerin, salts such as sodium chloride, potassium chloride or magnesium chloride, etc. may be appropriately added thereto. At that time, it is also possible that the cell tissue is destroyed by a treatment such as freezing/melting, ultrasonic wave, cell membrane dissolving enzyme or surfactant so as to make the extraction easier. The resulting suspension is allowed to stand for 5 to 12 days. During that period, the suspension may be heated at 30 to 45 C. with or without appropriate stirring. The resulting liquid is subjected to a treatment for separating into solid and liquid (filtered or centrifuged, etc.) to remove the tissue fragments whereupon a crude extract (filtrate or supernatant) is obtained.

About Step (B)

[0084] The crude extract obtained in step (A) is subjected to a deproteinizing treatment. The deproteinization may be carried out by a known method which has been usually conducted and a method such as heating treatment, treatment with a protein denaturant (such as acid, base, urea, guanidine or an organic solvent including acetone), isoelectric precipitation or salting-out may be applied. After that, a common method for the removal of insoluble matters such as filtration using filter paper (such as cellulose or nitrocellulose), glass filter, Celite or Seitz filter, ultrafiltration or centrifugation is conducted to give a filtrate or a supernatant wherefrom the separated insoluble protein is removed.

About Step (C)

[0085] The filtrate or supernatant obtained in step (B) is adjusted to acidic or, preferably, to pH 3.5 to 5.5 to conduct an operation of adsorbing with an adsorbent. Examples of the usable adsorbent include activated carbon and kaolin. An adsorbent is added to the extract followed by stirring or the extract is passed through a column filled with an adsorbent so that the active ingredient can be adsorbed with the adsorbent. When an adsorbent is added to the extract, the adsorbent with which the active ingredient is adsorbed can be obtained by means of filtration, centrifugation, etc. to remove the solution.

About Step (D)

[0086] For elution (desorption) of the active ingredient from the adsorbent obtained in step (C), an elution solvent is added to said adsorbent and adjusted to basic or, preferably, to pH 9 to 12, elution is conducted at room temperature or with suitable heating, or with stirring, and then the adsorbent is removed by a common method such as filtration or centrifugation. As to the extraction solvent used therefore, there may be used a basic solvent such as water, methanol, ethanol, isopropanol or the like adjusted to basic pH or an appropriate mixed solvent thereof and preferably, water adjusted to pH 9 to 12 may be used. Amount of the extracting solvent may be appropriately set. In order to use the eluate obtained as such as a drug substance, the pH is appropriately adjusted to nearly neutral or the like whereby an extract from inflamed skins of rabbits inoculated with vaccinia virus (the present extract) can be finally obtained.

[0087] Since the present extract is liquid at the stage of being prepared, it is also possible that said extract is appropriately concentrated or diluted to make into a desired concentration. When a preparation is manufactured from the present extract, it is preferred to apply a sterilizing treatment with heating. For making into an injectable preparation, it is possible to add sodium chloride or the like so as to prepare a solution being isotonic to physiological saline. It is also possible that the present extract is administered in a liquid or gel state. Furthermore, the present extract may be subjected to an appropriate operation such as concentration to dryness to prepare a solid preparation for oral administration such as a tablet. Specific methods for the manufacture of solid preparation for oral administration from the present extract are disclosed in the specifications of Japanese Patent Nos. 3,818,657 and 4,883,798. The present preparation includes an injectable preparation, a solid preparation for oral administration, etc. prepared as such.

[0088] The administering method to a patient is not particularly limited and may be suitably selected depending on the purpose of treatment. Examples of the method include oral administration, subcutaneous administration, intramuscular administration, intravenous administration, and transdermal administration. The dose may be suitably determined depending on the type of the extract from inflamed tissues inoculated with vaccinia virus. The dose that is approved in the commercially available preparation is principally 16 NU per day by oral administration and 3.6 to 7.2 NU per day by injection. However, the dose may be appropriately increased or decreased depending on the type of disease, degree of seriousness, individual difference in the patients, method of administration, period of administration and the like (NU: Neurotropin unit. Neurotropin unit is defined by ED50 value of analgesic effect measured by a modified Randall-Selitto method using SART-stressed mice that are chronic stressed animals showing a lowered pain threshold than normal animals. One NU indicates the activity of 1 mg of analgesic ingredients in Neurotropin preparations when the ED50 value is 100 mg/kg of the preparation).

[0089] Hereinafter, examples of methods for producing the present extract as well as a novel pharmacological action of and results of pharmacological tests regarding a L-PGDS production promoting action and a peripheral nerve regeneration promoting action of the present extract are described. The present invention is not intended to be limited to the descriptions in Examples.

EXAMPLES

Example 1 (Manufacture of the Present Extract)

[0090] Skins of healthy adult rabbits were inoculated with vaccinia virus intradermally and the inflamed skins were cut and collected. The collected skins were washed and disinfected by a phenol solution, an excessive phenol solution was removed and the residue was crushed. A phenol solution was added thereto and mixed therewith and the mixture was allowed to stand for 3 to 7 days, and further heated at 35 to 40 C. together with stirring for 3 to 4 days. After that, an extracted solution obtained by a solid-liquid separation was adjusted to pH 4.5 to 5.2 with hydrochloric acid, heated at 90 to 100 C. for 30 minutes and filtered to remove protein. The filtrate was adjusted to pH 9.0 to 9.5 with sodium hydroxide, heated at 90 to 100 C. for 15 minutes and subjected to a solid-liquid separation.

[0091] The resulting deproteinized solution was adjusted to pH 4.0 to 4.3 with hydrochloric acid, activated carbon in an amount of 2% to the mass of the deproteinized solution was added thereto and the mixture was stirred for 2 hours and subjected to the solid-liquid separation. Water was added to the collected activated carbon followed by adjusting to pH 9.5 to 10 with sodium hydroxide and the mixture was stirred at 60 C. for 90 to 100 minutes and centrifuged to give a supernatant. Water was added again to the activated carbon precipitated upon the centrifugation followed by adjusting to pH 10.5 to 11 with sodium hydroxide and the mixture was stirred at 60 C. for 90 to 100 minutes and centrifuged to give a supernatant. Both supernatants were combined and neutralized with hydrochloric acid to give the present extract.

Example 2: Pharmacological Test

[0092] Next, test methods and test results of pharmacological tests regarding an L-PGDS production promoting action using the present extract obtained in Example 1 as a test substance. Herein, in the following pharmacological tests, introduction of cerebral infarction in a C.B-17 mouse, and isolation and culture of iSCs obtained from the cerebral infarction area were performed according to methods described in Nakagomi, T. et al. Eur. J. Neurosci., 29, 1842-1852, 2009.

Test Example 1: Comprehensive Gene Expression Analysis in iSCs Treated with Test Substance

[0093] Ischemic insult was applied to C.B-17 mice (3 animals) by middle cerebral artery occlusion, and 3 sets of cultured iSCs were isolated from infarction area at day 3. To each of the cultured iSCs (Dulbecco's Modified Eagle's Medium F12 (2% FBS DMEM/F12 F/E/N) supplemented with 2% fetal bovine serum (FBS), 20 ng/mL of fibroblast growth factor (FBS), 20 ng/mL of epidermal growth factor (EGF), and 1% of N2 Supplement, 510.sup.4 cell/3 cm dish), a test substance (50, or 1000 mNU/mL) or physiological saline (control) was added. Then, total RNA was extracted from the cultured iSCs by RNeasy [registered trademark, the same applies hereinafter] Mini Kit (QIAGEN) at day 4 of culture (9 cultures in total). For comprehensive gene expression analysis, SurePrint G3 Mouse GE Microarray 860K (24,321 kinds of RNAs and 4,576 kinds of noncoding RNAs, GE), which was a gene chip for mouse, was used. Then, a gene showing changes in expression to be or less, in all of the 3 cultures, at the both concentrations of the test substance added, and a gene showing changes in expression to be twice or more, in all of the 3 cultures, at the both concentrations of the test substance added were selected. The results are shown by the ratio of expression levels of each gene selected above relative to those in the control (meanstandard error of the three cultures). An example of the results is shown in Table 1.

TABLE-US-00001 TABLE 1 Gene Ratio of Expression Level Name Protein Name 50 mNU/mL 1000 mNU/mL COCH Coagulation factor C homolog 0.40 0.01 0.31 0.03 GBP6 EGF-like domain, multiple 6 2.25 0.07 2.82 0.24 PTGDS L-type Prostaglandin 4.57 0.83 48.42 5.58 D2 synthase

[0094] From the results of the above analysis, as shown in Table 1, 3 genes, which were COCH gene with decreased expression and GBP6 gene and PTGDS gene with increased expression, were selected in total. Among these, the expression level of PTGDS gene encoding L-PGDS was strongly dependent on dose of the test substance.

Test Example 2-1: Evaluation of L-PGDS Gene Expression (Real-Time RT-PCR Method)

[0095] As in Test Example 1, to cultured iSCs (FBS-free DMEM/F12 F/-/-, 510.sup.4 cell/3 cm dish), a test substance (50, or 500 mNU/mL) or physiological saline (control) was added (n=1), and the iSCs were cultured for 7 days. Then, RNA was extracted by Isogen II [registered trademark] (NIPPON GENE CO., LTD.) according to manufacturer's instructions. Purities of the RNA were determined on the basis of absorbance at 260 nm and 280 nm. The RNA was subjected to reverse transcription reaction using SuperScript [registered trademark, the same applies hereinafter] IV RTase (Invitrogen) in the presence of random primers to obtain single-stranded cDNAs corresponding to the total RNA. With respect to the resulting cDNAs, the amounts of transcription products were determined by quantitative PCR (Prism [registered trademark] 7900HT, Applied Biosystems) with PTGDS specific primers using -Actin, which is a housekeeping gene, as a control (comparative threshold cycle method). The nucleotide sequences of the primers used for PTGDS and -Actin were as follows: PTGDS: 5-gactctgaaggacgagctgaag-3 (SEQ ID NO: 1) and 5-tcttgaatgcacttatccggttgg-3 (SE ID NO: 2), -Actin: 5-tacagcttcaccaccacagc-3 (SEQ ID NO: 3) and 5-aaggaaggctggaaaagagc-3 (SEQ ID NO: 4). The results are shown by the ratio of expression levels of PTGDS relative to that of -Actin used as a control as 1. An example of the results is shown in Table 2 and FIG. 1.

TABLE-US-00002 TABLE 2 Additive Concentration of Ratio of Expression Level of Test Substance (mNU/mL) PTGDS to -Actin (times) 0 (Control) 1.0 50 20.4 500 84.4

[0096] As apparent from Table 2 and FIG. 1, as confirmed by the results in Test Example 1, it was confirmed that expression of L-PGDS gene (PTGDS) in iSCs was promoted by the test substance in a dose dependent manner.

Test Example 2-2: Evaluation of L-PGDS Gene Expression (Classical RT-PCR Method)

[0097] In the presence of a test substance each having a dose of 0 (control; physiological saline), 1, 5, 50, or 100 mNU/mL, iSCs were cultured in a medium containing FGF (DMEM/F12 F/-/-) or FGF-free medium (DMEM/F12 -/-/-) for 4 days. From the cultured iSCs, total RNA was extracted using RNeasy Mini Kit (QIAGEN), and L-PGDS gene (PTGDS) expression level was semiquantified by classical RT-PCR method using SuperScript III One-Step RT-PCR System with Platinum (Invitrogen) (35 cycles). The PCR products were separated by 2% agarose gel electrophoresis, and bands of PTGDS and GAPDH were stained with ethidium bromide and visually detected. The nucleotide sequences of the primers used for PTGDS and GAPDH were as follows: PTGDS: 5-cctccaactcaagctggttc-3 (SEQ ID NO: 5) and 5-atagttggcctccaccactg-3 (SEQ ID NO: 6), and GAPDH: 5-atcactgccacccagaagac-3 (SEQ ID NO: 7) and 5-cacattgggggtaggaacac-3 (SEQ ID NO: 8). An example of the results is shown in FIG. 2.

[0098] When L-PGDS gene (PTGDS) expression-promoting actions in iSCs were examined using varying doses, with smaller differences from one another, of the test substance, as shown in FIG. 2, L-PGDS gene expression level was promoted by the test substance in a dose dependent manner independent of whether the medium contains FGF or not.

Test Example 3-1: Evaluation of L-PGDS Protein Expression (Western Blotting Method)

[0099] A test substance (0, 1, 5, 50, or 100 mNU/mL) was added to iSCs, and the iSCs were cultured in the absence of serum (DMEM/F12 F/-/-, 510.sup.4 cell/3 cm dish) for 4 days. The cultured iSCs were isolated, washed with phosphate buffer, and then lysed in RIPA buffer (4 C., 50 mM Tris-HCl buffer (pH 7.6), 150 mM sodium chloride, 1% Nonidet [registered trademark] P-40 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) to obtain homogenates. Concentration of each homogenate was adjusted so that the homogenates had the same total protein content. Then, the homogenate was subjected to SDS-PAGE (BIO-RAD Any kD (trademark)) to separate proteins, and the separated proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Immun-Blot PVDF membrane, Bio-Rad) and blocked by Blocking One (NACALAI TESQUE, INC.). Thereafter, by Western blotting using specific antibody, L-PGDS (anti-Prostaglandin D Synthase (Lipocalin) antibody [EP12357], Abcam, 1:2000) and -Actin (Monoclonal anti--Actin [A1978], Sigma, 1:100000) were detected. The detection was performed by a high-sensitivity chemiluminescent assay (Chemi-Lumi One L, NACALAI TESQUE, INC.). An example of the results is shown in FIG. 3.

[0100] As apparent from FIG. 3, it was confirmed that expression of intracellular L-PGDS protein in iSCs was promoted by the test substance.

Test Example 3-2: Evaluation of L-PGDS Protein Expression (Dot Blotting Method)

[0101] In the presence of a test substance (0, 1, 10, 50 or 100 mNU/mL), iSCs were cultured for 4 days (DMEM/F12 F/-/-, 510.sup.4 cell/3 cm dish), and 500 L of the culture supernatant was spotted on a PVDF membrane (Immun-Blot PVDF membrane, Bio-Rad). The membrane was blocked by Blocking One (NACALAI TESQUE, INC.), and L-PGDS was detected by dot blotting using an anti-L-PGDS antibody (anti-Prostaglandin D Synthase (Lipocalin) antibody [EP12357], Abcam, 1:2000). An example of the results is shown in FIG. 4.

[0102] Since L-PGDS has a typical secretion signal sequence and a typical consensus signal peptidase recognition sequence at the N terminus, it is thought that L-PGDS can be secreted outside from the cell. Thus, L-PGDS protein in the culture supernatant of iSCs to which the test substance was added was examined. As a result, as apparent from FIG. 4, it was confirmed that the amount of L-PGDS protein in the culture supernatant (outside of the cells) was increased depending on dose of the test substance added.

Test Example 3-3: Evaluation of L-PGDS Protein Expression (ELISA Method)

[0103] Culture of iSCs (DMEM/F12 F/-/-, 510.sup.4 cell/3 cm dish) was treated with a test substance (0 [control], 1, 10, 50, 100, or 1000 mNU/mL) for 4 days, and the supernatant was collected. The supernatant was centrifuged (1500 rpm, 10 minutes, 4 C.), and then the amount of L-PGDS in the culture supernatant was measured by a specific ELISA method (an ELISA kit for human L-PGDS (Prostaglandin D Synthase 21 kDa (Brain), product number: SEA724Hu, Cloud-Clone Corp.) according to manufacturer's instructions. An example of the results is shown in Table 3 and FIG. 5.

TABLE-US-00003 TABLE 3 Additive Concentration of Ratio of Expression Level of Test Substance (mNU/mL) L-PGDS to Control 0 (Control) 1.00 1 0.46 10 1.16 50 1.43 100 4.72 1000 6.87

[0104] As apparent from Table 3 and FIG. 5, and in Test Example 3-2, it was confirmed that when iSCs were treated with the test substance, the amount of L-PGDS protein in the culture supernatant (outside of the cells) was increased. From the results of Test Examples 3, it was found that in iSCs, L-PGDS production was promoted by the test substance at protein level besides gene level, and the produced L-PGDS was secreted.

Test Example 4: Evaluation of Enzyme Activity of L-PGDS (High Performance Liquid Chromatography (HPLC)-Mass Spectrometry Method)

[0105] L-PGDS is an enzyme (EC 5.3.99.2) which catalyzes the biosynthesis of PGD2 from PGH2 as a substrate. In iSCs, for the purpose of confirming that L-PGDS, the production of which has been promoted by a test substance, further produces PGD2 by its enzyme activity, the following analysis was performed. Prostaglandins such as PGD2 and its metabolites, and PGE2 produced from PGH2, which is a common substrate of prostaglandin synthesis, were comprehensively analyzed concerning: (A) inside of the cells and (B) outside of the cells of iSCs.

A.: Evaluation of Enzyme Activity Concerning Inside of Cells of iSCs

[0106] A test substance (0, 10, or 50 mNU/mL) was added to iSCs, the iSCs were cultured for 3 days (FBS-free DMEM/F12 F/-/-, 510.sup.4 cell/3 cm dish), and a cell extract was prepared from the cultured iSCs by RIPA buffer treatment (4 C., 20 min). Each of the concentrations of prostaglandins (PGD2, PGJ2, 15-deoxy-12,14-PGJ2, 13,14-dihydro-15-keto-PGD2, and PGE2) in the cell extract was measured by HPLC-mass spectrometry. For HPLC separation, AQUITY UPLC HSS T3 column (Waters) was used. As a detector and a mass spectrometer, API 4000 LC/MS/MS system and Triple Quadrupole (both from AB Sciex) were used, respectively. An example of the results is shown in Table 4 and FIG. 6.

TABLE-US-00004 TABLE 4 (n = 3) Additive Concentration of prostaglandins (pg/mL) Concen- 13,14 - tration dihydro- of Test 15-deoxy- 15- Substance 12, keto- (mNU/mL) PGD2 PGJ2 14-PGJ2 PGD2 PGE2 0 5.45 1.62 3.47 0.68 n.d. n.d. 0.93 0.16 10 9.63 6.75 6.59 1.71 n.d. n.d. 2.76 3.98 1000 15.67 6.89 10.57 3.28 2.83 5.58 n.d. 1.52 2.32

[0107] As shown in Table 4 and FIG. 6, intracellular level of PGD2 and PGJ2, which is a nonenzymatic metabolite of PGD2, were increased by the addition of the test substance in a dose dependent manner. Further, when 50 mNU/mL of the test substance was added, production of 15-deoxy-12,14-PGJ2, which is a nonenzymatic metabolite of PGJ2, was also observed. The amount of 13,14-dihydro-15-keto-PGD2, which is a nonenzymatic metabolite of 15-deoxy-12,14-PGJ2, was not greater than the detection limit. On the other hand, with respect to PGE2 produced from PGH2, which is a substrate for PGE2 as well as PGD2, no production response depending on dose of the test substance added was observed.

B.: Evaluation of Enzyme Activity Concerning Outside of Cells of iSCs

[0108] A test substance (0, 50, 100, or 1000 mNU/mL) was added to iSCs, and the iSCs were cultured for 3 days (FBS-free DMEM/F12 F//, 510.sup.4 cell/3 cm dish). The culture supernatant was reacted with PGH2, which is a substrate of L-PGDS, and glutathione (GSH) (reaction condition: 100 mM Tris-HCl (pH 8.0), 1 mM GSH, 10 mM PGH2, 37 C., 5 mM). As in the above-described A., each of the concentrations of prostaglandins (PGD2, PGD2, 15-deoxy-12,14-PGJ2, 13,14-dihydro-15-keto-PGD2, and PGE2) in the reaction solution was measured by HPLC-mass spectrometry. An example of the results is shown in Table 5 and FIG. 7.

TABLE-US-00005 TABLE 5 (n = 1) Additive Concentration of prostaglandins (pg/mL) Concentration of 15-deoxy- 13,14- Test Substance 12, 14- dihydro-15- (mNU/mL) PGD2 PGJ2 PGJ2 keto -PGD2 PGE2 0 351 0.7 n.d. 0.061 83.0 50 395 0.8 n.d. 0.491 76.9 100 2928 13.3 3.1 6.002 150.5 1000 3139 18.4 4.4 n.d. 155.6

[0109] As shown in Table 5 and FIG. 7, concentrations of PGD2 and PGD2, which is a nonenzymatic metabolite of PGD2, and 15-deoxy-12,14-PGJ2 were increased by the test substance at concentrations of 100 mNU/mL or higher. Thus, it was confirmed that L-PGDS activity was present in iSC culture supernatant. On the other hand, dose-dependent production of 13,14-dihydro-15-keto-PGD2, which is a nonenzymatic metabolite of 15-deoxy-12,14-PGJ2, was observed by the addition of the test substance at concentrations up to 100 mNU/mL. However, with 1000 mNU/mL of the test substance, the amount of 13,14-dihydro-15-keto-PGD2 was lower than the detection limit.

[0110] It is known that L-PGDS is a secretory enzyme. From the results of the above-described A. and B., it was confirmed that iSCs secreted L-PGDS having an enzyme activity into the outside of the cells, and the production and secretion of L-PGDS were promoted by the addition of the test substance.

Test Example 5-1: Examination of L-PGDS-Producing Cells in Mouse Brain (Immunohistochemical Staining Method)

[0111] Ischemic insult was applied to a C.B-17 mouse by middle cerebral artery occlusion. At three days after the application of ischemic insult, brain slices were prepared, and immunohistochemical examination was performed with a confocal laser microscope using specific antibodies against L-PGDS (panel B, green; anti-prostaglandin D synthase (lipocalin) antibody [EP12357], Abcam, 1:1000), a pericyte marker a-SMA (panel C, red; anti-actin, smooth muscle, clone ASM-1, Millipore, 1:1000), and a vascular endothelial cell marker CD31 (panel D, red; anti-mouse CD31 (PECAM-1) monoclonal antibody, 550274, BD Pharmingen, 1:1000). By multiple staining, L-PGDS (panel B) and the pericyte marker (panel C), or L-PGDS (panel B) and the vascular endothelial cell marker (panel D) were detected using specific antibodies each labeled with different fluorescent dyes, and images were obtained. The images were merged, and distribution was compared between L-PGDS and the pericyte marker, or L-PGDS and the vascular endothelial cell marker (panel A1, panel A2). An example of the results is shown in FIG. 8.

[0112] In images in the upper half of FIG. 8, green fluorescence in panel B represents distribution of L-PGDS, and red fluorescence in panel C represents distribution of the pericyte marker. In panel A1 in which these two images are merged, L-PGDS (green fluorescence) partially overlaps with the distribution of the pericyte marker (red fluorescence) (magnification of 200). On the other hand, in images in the lower half of FIG. 8, green fluorescence in panel B represents distribution of L-PGDS, and red fluorescence in panel D represents distribution of the vascular endothelial cell marker. In panel A2 in which these two images are merged, although distribution of L-PGDS (green fluorescence) is close to that of the vascular endothelial cell marker (red fluorescence), no image showing co-distribution was obtained (magnification of 200).

[0113] L-PGDS is a protein mainly distributed in the central nervous system, exists in the arachnoid membrane and the pia mater, the choroid plexus of the lateral ventricles of the brain, and the like, and it is thought that L-PGDS is secreted into cerebrospinal fluid (Urade Y. et al., J Lipid Mediat. Cell Signal. 14, 71-82, 1996). However, L-PGDS-producing cells in the brain have not yet sufficiently been elucidated. The present inventors have made investigations of distribution of L-PGDS in the brain using an immunohistochemical method. As a result, it was found that the expression of L-PGDS in the normal brain of a C.B-17 mouse was weak. In the above-described Test Example 4-1, it was also found that when permanent ligation was performed on a middle cerebral artery of the mouse, a significant L-PGDS expression was observed in the cerebral infarction area (panel B of FIG. 8). In addition, although distribution of L-PGDS was observed around the vascular endothelial cell marker CD31, the distribution of L-PGDS and the distribution of CD31 did not overlap with each other (panel A2 of FIG. 8). On the other hand, it was found that L-PGDS was partially co-localized with a pericyte marker -SMA (panel A1 of FIG. 8). From the above results, it was thought that L-PGDS, the expression of which was promoted in a cerebral infarction area, was derived from pericytes (or iSCs). This was also confirmed from the following results, which were obtained by immunoelectron microscopy in Test Example 4-2, that L-PGDS-positive product was observed as an electron-dense structure in the cytoplasm of pericytes, which were present in contact with vascular endothelial cells and the basal membrane (panels A and B of FIG. 9). From these results, it was supposed that the promotion of L-PGDS expression was included in physiological roles of pericytes or iSCs in ischemic response.

Test Example 5-2: Examination of L-PGDS-Producing Cells in Mouse Brain (Immunoelectron Microscopy)

[0114] Ischemic insult was applied to a C.B-17 mouse by middle cerebral artery occlusion. At three days after the application of ischemic insult, brain slices were prepared, and L-PGDS expression sites were observed by immunoelectron microscopy using a specific antibody against L-PGDS (anti-prostaglandin D synthase (lipocalin) antibody [EP12357], Abcam, 1:1000). Specifically, the observation was carried out as follows. Brain slices each having a thickness of 2 m were prepared using a vibratome. The slices were subjected to a reaction using an avidin-biotin horseradish peroxidase (HRP) complex kit (Vector Laboratories) and 3,3-diaminobenzidinetetrahydrochloride (DAB), treated with osmium, and embedded in epon. Then, ultrathin slices were prepared from the epon-embedded slice, and the ultrathin slices were observed by electronmicroscopy. An example of the results is shown in FIG. 9.

[0115] In both panels A and B of FIG. 9, by immunoelectron microscopic observation about L-PGDS, electron-dense regions were observed in the cytoplasm of vascular perivascular cells (pericytes), which were present in contact with vascular endothelial cells and the basal membrane.

Test Example 6: Examination of L-PGDS-Producing Cells in Human Brain (Immunohistochemical Staining Method)

[0116] According to a method described in Tatebayashi K. et al., Stem Cells and Develop. 26, 787-797, 2017, immunohistochemical investigation of cultured iSCs isolated from a human cerebral infarction area (necrotic tissue) was performed using specific antibodies against L-PGDS (panel B, green; anti-prostaglandin D synthase (lipocalin) antibody [EP12357], Abcam, 1:1000) and nestin, which is a neural stem cell marker, (panel C, red; anti-nestin, clone 10C2, Millipore, 1:1000). The cells were reacted with an Alexa Fluor [registered trademark, the same applies hereinafter] 488-conjugated antibody or an Alexa Fluor 555-conjugated antibody (1:500; Molecular Probes, Eugene), and thereafter subjected to nuclear staining with 4,6-diamidino-2-phenylindole (DAPI; 1:1000; Kirkegaard & Perry Laboratories). Fluorescence imaging of the stained cells were performed using a fluorescent microscope (BX60; Olympus, Japan), images of L-PGDS (panel B) and nestin (panel C) were merged, and distributions of L-PGDS and nestin were compared with each other (panel A). An example of the results is shown in FIG. 10.

[0117] In FIG. 10, green fluorescence in panel B represents distribution of L-PGDS, and red fluorescence in panel C represents distribution of nestin. In panel A in which these two images are merged, L-PGDS (green fluorescence) overlaps with the distribution of nestin (red fluorescence) (magnification of 200). That is, it was confirmed that L-PGDS was expressed in almost all nestin-expressing iSCs. From these results, it was thought that L-PGDS was produced in iSCs or pericytes not only in the mouse brain but also in the human brain.

Test Example 7: Evaluation of Cerebral Amyloid Deposition and L-PGDS Expression in Alzheimer-Type Dementia Model Animal

(1) Behavioral Analysis of Cognitive Ability and Sample Preparation

[0118] APPswe/PS1dE9 (APP/PS1) mice (female, 3-month old) were divided into a test substance-administration group and a control group (10 to 11 animals/group) such that each group had the same body weight. The test substance (100 NU/kg body weight) and physiological saline were administered to the animals of the test substance-administration group and the animals of the control group, respectively, twice a week for about 3 months by tail vein injection. After the final administration, behavioral analyses of cognitive ability (a Y-maze test, a novel object recognition test, and the Morris water maze test) were performed. In each of the tests, each of the following A to C was measured. In the Y-maze test, (A) a rate of alternation behavior (%) was measured. In the novel object recognition test, (B) a frequency of access to a novel object (%) was measured. In the Morris water maze test, (C) an average time (seconds) required for finding the platform during 4 days until the end of the learning period was measured (from day 6 to day 9). Then, a composite score (ABC) was calculated for each individual animal. The evaluation results in two individuals of each group are shown in Table 6.

TABLE-US-00006 TABLE 6 Novel Morris Object Water Y-maze Recognition Maze Composite Individual Test Test Test C Score Number A (%) B (%) (Sec.) (A B C) Control Group #14 36.8 57.1 84.4 25.0 #30 40.0 47.1 90.0 20.9 Test Substance- #4 52.6 66.7 48.9 71.8 Administration #29 61.5 60.0 40.0 92.3 Group

[0119] As apparent from Table 6, an improvement action on cognitive ability was observed in the test substance-administration group, and the difference in this action was statistically significant between the two groups.

[0120] After the behavioral analysis of cognitive ability (24 hours after the final administration), brains were harvested from animals of the test substance-administration group and the control group by decapitation under anesthesia, and the left hemisphere of the brain was quickly frozen in a liquid nitrogen bath. On the other hand, the remaining right hemisphere was fixed (at 4 C.) with (A) a fixative solution containing glutaraldehyde (0.05% .sub.glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer) or (B) a PLP fixative solution (0.01 M sodium metaperiodate, 0.075 M lysine, and 2% paraformaldehyde). From the fixed tissue sample, a coronal plane slices (thickness: 20 m) including the cerebral cortex was prepared using a cryostat (Leica CM1850).

(2) Evaluation of Amyloid Deposition in Cerebral Cortex (Immunohistochemical Staining Method)

[0121] The coronal plane slices of the test substance-administration group and the control group prepared in the above-described (1) were immunohistochemically stained with an anti-amyloid antibody (anti--amyloid, 1-16, [SIG-39300], BioLegend, 1:1000). The anti-amyloid antibody-stained slices were reacted with an Alexa Fluor 488-conjugated antibody (1:500; Molecular Probes, Eugene), thereafter nuclear staining was performed with 4,6-diamidino-2-phenylindole (DAPI; 1:1000; Kirkegaard & Perry Laboratories), and fluorescence imaging of amyloid was performed using a fluorescent microscope (BX60; Olympus, Japan). An example of the results is shown in FIG. 11.

[0122] As shown in FIG. 11, in individuals that were improved in cognitive ability by the administration of the test substance, the number and the area of amyloid plaques in the brain were decreased as compared to the control group.

(3) Evaluation of L-PGDS Expression in Cerebral Cortex (Immunohistochemical Staining Method)

[0123] The coronal plane slices of the test substance-administration group and the control group prepared in the above-described (1) were immunohistochemically stained with an anti-L-PGDS antibody (anti-prostaglandin D synthase (lipocalin) antibody [EP12357], Abcam, 1:1000). L-PGDS was detected by the 3,3-diaminobenzidine tetrahydrochloride (DAB) reaction using an avidin-biotin horseradish peroxidase (HRP) complex kit (Vector Laboratories). An example of the results is shown in FIG. 12.

[0124] As shown in FIG. 12, in the brain of the test substance-administration group, specific staining of L-PGDS was observed in the vicinity of vascular cavities (indicated by the arrow in panel B). On the other hand, in the brain of the control group, no specific staining of L-PGDS was observed (panel A).

[0125] With respect to the coronal plane slices of the test substance-administration group prepared in the above-described (1), immunohistochemical examination was performed using specific antibodies against L-PGDS (anti-prostaglandin D synthase (lipocalin) antibody [EP12357], Abcam, 1:1000) and a vascular endothelial cell marker CD31 (anti-mouse CD31 (PECAM-1) monoclonal antibody, 550274, BD Pharmingen, 1:1000). A slice to which the above-described primary antibodies were bound was reacted with an Alexa Fluor 488-conjugated antibody or an Alexa Fluor 555-conjugated antibody (1:500; Molecular Probes, Eugene), and thereafter subjected to nuclear staining with 4,6-diamidino-2-phenylindole (DAPI; 1:000; Kirkegaard & Perry Laboratories). Fluorescence imaging of the stained slice was performed using a confocal laser microscope (LSM780; Carl Zeiss Jena, Germany). By the multiple staining, images of L-PGDS (panel B) and CD31 (panel C) were merged, and distribution was compared between L-PGDS and CD31 (panel A). An example of the results is shown in FIG. 13.

[0126] As shown in FIG. 13, in the brain of the test substance-administration group, it was observed that L-PGDS (green fluorescence) was distributed around the vascular endothelial cell marker CD31 (red fluorescence). From the results, it was thought that in response to the administration of the test substance, the expression of L-PGDS was increased in pericytes (or iSCs) which were present around vascular endothelial cells.

(4) Evaluation of Cerebral Amyloid Deposition and L-PGDS Expression (Western Blotting Method)

[0127] The left hemisphere of the brain harvested and quickly frozen in the above-described (1) was homogenized using Potter-Elvehjem homogenizer to extract proteins. The extracted proteins were separated by SDS-PAGE (BIO-RAD Any kD (trademark)), and transferred to a PVDF membrane (Immun-Blot PVDF membrane, Bio-Rad). The PVDF membrane was blocked with Blocking One (NACALAI TESQUE, INC.). Then, amyloid (anti--amyloid, 1-16, [SIG-39300], BioLegend, 1:1000) and L-PGDS (anti-Prostaglandin D Synthase (Lipocalin) antibody [EP12357], Abcam, 1:2000) were detected by Western blotting using specific antibodies. The detection was performed by a high-sensitivity chemiluminescent assay (Chemi-Lumi One L, NACALAI TESQUE, INC.). An example of the results is shown in FIG. 14.

[0128] As shown in the position indicated by the symbol * in panel A of FIG. 14, in the mice in the test substance-administration group (individual numbers: 4 and 29) which were improved in cognitive ability by the administration of the test substance, the amount of amyloid was reduced as compared to the mice in the control group (individual numbers: 14 and 30). The results were correlated with the results of the immunohistochemical staining in Test Example 7 (2) (FIG. 11) that the number and the area of cerebral amyloid antibody-positive product were reduced by test substance administration as compared to the control group. In addition, as shown in the position indicated by the symbol * in panel B of FIG. 14, in the test substance-administration group, the amount of intracerebral L-PGDS was increased as compared to the control group, and therefore it was confirmed that intracerebral L-PGDS production was promoted by the test substance. From the above-described results in Test Example 7, it is thought that it may be possible that the test substance promotes the expression of L-PGDS in the brain, leading to prophylaxis of cerebral amyloid 8 deposition, which in turn improve cognitive function.

Test Example 8: Examination of Contributing Factor for Promotion of L-PGDS Expression in Human-Derived Pericytes (Classical RT-PCR Method)

[0129] As described above, it is thought that iSCs that respond to the test substance are derived from brain pericytes. Thus, using commercially available Human Brain Vascular Pericytes (ScienCell), which is a human pericyte cell line, under different oxygen and/or glucose concentrations, L-PGDS expression activity of the test substance was examined. Specifically, in the presence of different doses, which were 0, 50, or 500 mNU/mL, of the test substances, iSCs were cultured for 4 days (FBS-free DMEM medium F/-/-) under the following conditions: (1) a condition in which iSCs show reactivity (4.5 g/L of glucose and 20% of O.sub.2), (2) a low glucose condition (90 mg/L of glucose and 20% of O.sub.2), and (3) a low oxygen and low glucose condition (90 mg/L of glucose and 1% of O.sub.2). From the cultured iSCs, total RNA was extracted using RNeasy Mini Kit (QIAGEN), and L-PGDS gene (PTGDS) expression level was semiquantified by classical RT-PCR method using SuperScript III One-Step RT-PCR System with Platinum (Invitrogen) (35 cycles). The PCR products were separated by 2% agarose gel electrophoresis, and bands of PTGDS and -Actin were stained with ethidium bromide and visually detected. The primers used for PTGDS and -Actin were same as used in Test Example 2-1. An example of the results is shown in FIG. 15.

[0130] As shown in FIG. 15, although the transferred L-PGDS gene of the cells cultured under condition (1) was capable of being detected, no responsivity to the test substance was observed. On the other hand, in the cells cultured under condition (3), which is a low oxygen (1%) and low glucose concentration (90 mg/L) condition, a remarkable responsivity to the test substance was observed. It is thought that the low oxygen and low glucose condition mimics the ischemic condition in the brain. Thus, it is thought that under conditions of ischemic diseases, pericytes (or iSCs) acquire responsivity to an external stimulus, and the pericytes (or iSCs) release cyto-protective proteins such as L-PGDS.

INDUSTRIAL APPLICABILITY

[0131] It is said that L-PGDS is a protein that is mainly expressed in the brain, plays roles as a binder and transporter or a scavenger for various hydrophobic low-molecular weight compounds, and has various functions such as an intracerebral environment-controlling and brain-protective function, and a sleep-controlling function. Thus, the L-PGDS production promoting agent of the present invention is useful as a prophylactic, therapeutic or relapse prophylactic agent for L-PGDS associated diseases including a cerebrovascular disorder such as cerebral infarction, dementia such as Alzheimer's disease, and insomnia. In particular, the present extract and a preparation containing the present extract are highly useful as excellent L-PGDS production promoting agents and highly safe drugs with little problem such as side effects. In addition, a screening method of the present invention for a substance useful for prophylaxis/treatment or relapse prophylaxis of L-PGDS associated diseases, in particular, a substance having a brain-protective action or a sleep-promoting action, using an L-PGDS production promoting action in pericytes or iSCs dedifferentiated from pericytes as an index is an extremely useful method that contributes to the development of a new therapeutic agent.

SEQUENCE LISTING

[0132]