Lipocalin-type prostaglandin d2 synthase production promoters

Abstract

The present application aims to provide a lipocalin-type prostaglandin D2 synthase (L-PGDS) production promoter, more specifically, a L-PGDS production promoter in pericytes or ischemia-induced pluripotent stem cells (iSCs) obtained by dedifferentiation from pericytes. In the present application, it is confirmed that the extract of a vaccinia virus-inoculated inflammatory tissue contains a substance having a L-PGDS production promoting effect. The L-PGDS production promoter is useful as a prophylactic, therapeutic or relapse-preventing drug for diseases in which an effect brought about by L-PGDS expression promotion can be expected, i.e., cerebral vascular disorders such as cerebral infarction, cognitive disorders such as Alzheimer's disease, or sleep disorders.

Description

[0001] (This application is a divisional application of application 201880084260.8, filed on December 27, 2018, entitled "Lipid-carrying protein type prostaglandin D2 synthase production promoter".) Technical Field

[0002] This invention relates to promoters of the production of lipocalin-type prostaglandin D2 synthase (hereinafter, sometimes referred to as "L-PGDS"; in addition, enzymes also referred to as "lipocalin-type prostaglandin D synthase" may be included in this invention). Background Technology

[0003] Prostaglandin D2 synthase (PGDS) is known to exist in two forms: a lipid carrier protein type (L-) PGDS distributed in the central nervous system, male reproductive organs, and the heart, and a hematopoietic type (H-) PGDS distributed in mast cells and Th2 cells. L-PGDS catalyzes the isomerization reaction from prostaglandin H2 (PGH2), a common intermediate in prostaglandin biosynthesis, to prostaglandin D2 (PGD2). On the other hand, L-PGDS structurally belongs to the lipid carrier protein family, which functions as a carrier of lipid-soluble substances. Therefore, L-PGDS is a multifunctional protein possessing both the properties of PGD2 biosynthesis enzyme and lipid-soluble carrier.

[0004] L-PGDS, distributed in the central nervous system, has also been detected in cerebrospinal fluid. Compared with other lipid transport proteins, it has a large lipophilic sac and is therefore believed to play the role of a transport protein and scavenger for various lipid-soluble molecules in the brain. Examples of the functions of such lipid transport proteins of L-PGDS include: after subarachnoid hemorrhage, the increase of L-PGDS in the brain, which binds to bilirubin, a cause of neurotoxicity, thereby preventing neurotoxicity (Inui T. et al., J. Cereb. Blood Flow Metab. 34, 1558-1567, 2014); in patients or animal models of Alzheimer's disease, L-PGDS binds firmly to regions essential for the formation of oligomers of amyloid-β protein (Aβ, senile plaques), inhibiting the formation of Aβ aggregates and cytotoxicity in cerebrospinal fluid (Kanekiyo T. et al., Proc. Natl. Acad. Sci. USA 104, 6412-6417, 2007), etc.

[0005] Furthermore, there are reports of significantly increased PGD2 production in the ischemic brain of mice, with knockout of L-PGDS and H-PGDS, or the PGD2 receptor DP1, leading to more severe cerebral edema after ischemia (Tanigichi H. et al., J. Neurosci. 27, 4303-4312, 2007); and knockout of L-PGDS resulting in increased cerebral infarction foci and cerebral edema (Saleem S. et al., Neuroscience 160, 248-254, 2009). Therefore, it can be concluded that PGD2 produced by L-PGDS or H-PGDS during cerebral ischemia exerts a neuroprotective effect through its receptor-mediated action.

[0006] Furthermore, an increase in L-PGDS gene expression was observed in axons resistant to demyelination in Twitcher mice, a mouse model of Clapham's disease caused by galactosylceramidinase deficiency. Further introduction of L-PGDS deficiency led to severe demyelination caused by oligodendrocyte loss (Taniike M. et al., J. Neurosci. 22, 4885-4896, 2002). Additionally, in L-PGDS-deficient mice, induction of peripheral nerve demyelination revealed that Gpr44, as the PGD2 receptor for Schwann cells, is essential for nerve myelination (Trimarco A. et al., Nature Neurosci. 17, 1682-1992, 2014). These findings suggest that L-PGDS and its product PGD2 are essential for the myelination and maintenance of nerve axons in oligodendrocytes (central nervous system) or Schwann cells (peripheral nerve).

[0007] Furthermore, it has been reported that the protective effect of L-PGDS is not limited to nerve cells; 15-deoxy-prostaglandin J2, a metabolite of PGD2, can also prevent cell death of gastrointestinal glial cells induced by oxidative stress (Abdo H. et al., J. Physiol. 590, 2739-2750, 2012).

[0008] Furthermore, it has been known since ancient times that PGD2 synthesized from L-PGDS in the brain plays a role in sleep regulation (Ueno R. et al., Biochem. Biophys. Res. Commun. 109, 576-582, 1982). Regarding its mechanism, Japanese researchers have conducted detailed studies, concluding that PGD2 stimulates the sleep center by secreting adenosine via the DP1 receptor in the arachnoid membrane at the fundus of the eye. L-PGDS has a high affinity for PGD2 as an enzyme product, and is therefore presumed to ensure the stability of PGD2 in cerebrospinal fluid and to transport PGD2 to nearby receptors (Urade Y. and Hayaishi O., Biochim. Biophys. Acta 1482, 259-271, 2000), and is considered to be highly involved in sleep regulation via PGD2.

[0009] As mentioned above, L-PGDS is believed to function as an enzymatic protein in the synthesis of PGD2, and also plays a role in binding, transporting, and clearing various hydrophobic low-molecular-weight molecules in the brain, possessing various functions such as brain environment regulation, neuroprotection, and sleep regulation. Therefore, it can be considered that promoting the production of L-PGDS enables these functions to be effectively performed, which is useful for the prevention and treatment of L-PGDS-related diseases. However, to date, no substances have been reported to promote the production of L-PGDS in vivo.

[0010] Although the extract of vaccinia virus inoculation inflammatory tissue (this extract) has been confirmed to have excellent L-PGDS production-promoting effects in this invention, many effects and benefits are known about this extract or preparations containing this extract. For example, as for the effects of this extract on the brain, it is known to have therapeutic effects on ischemic diseases such as cerebral infarction (Japanese Patent Application Laid-Open No. 2000-16942) and to promote the production of neurotrophic factors such as BDNF (International Patent Application Laid-Open No. WO2011 / 162317), but the L-PGDS production-promoting effect of this extract is currently unknown. Summary of the Invention

[0011] The technical problem that the invention aims to solve

[0012] This invention provides substances that promote the production of L-PGDS, which have neuroprotective and sleep-inducing effects, as well as a screening system for pharmaceutical agents with neuroprotective and sleep-inducing effects, using the L-PGDS production promotion effect as an indicator. Furthermore, it provides pharmaceuticals containing this substance as an active ingredient that are effective and safe in the prevention, treatment, or relapse prevention of diseases involving L-PGDS, such as cerebrovascular disorders like cerebral infarction, dementia like Alzheimer's disease, and insomnia. In addition, the L-PGDS production promoters involved in this invention have the effect of inhibiting and alleviating cerebral ischemia and nerve cell damage caused by cerebrovascular disorders. Furthermore, the term "treatment" used in this invention includes meanings such as "relief," "improvement," and "progression inhibition." Also, while pharmaceutical agents with sleep-inducing effects are sometimes referred to as "sleeping agents," "sleep-improving agents," "sleep-inducing agents," or "hypnotics" in this invention, substances also referred to as "sleep agents," "sleep-improving agents," "sleep-inducing agents," or "hypnotics" may be included in this invention.

[0013] Technical solutions for solving technical problems

[0014] Based on research using a mouse model of cerebral infarction (a model of permanent occlusion of the middle cerebral artery), the inventors of this invention discovered stem cells capable of differentiating into various types of neural cells, including nerve cells, astrocytes, and oligodendrocytes, within the infarcted area where mature nerve cells continuously die due to blood flow obstruction. These stem cells were named ischemia-induced multipotent stem cells (iSCs). The strong expression of the neural stem cell marker nestin in iSCs, histochemically distributed around blood vessels from the pia mater to the cerebral cortex, further demonstrates this. Additionally, iSCs express pericyte markers such as PDGFRβ (platelet-derived growth factor receptor β) and NG2 (neuron-glial antigen 2), suggesting that iSCs originate from pericytes distributed around blood vessels. Pericytes, along with neurons, astrocytes, and vascular endothelial cells, constitute the neurovascular unit (NVU), the basic functional and organic unit of the brain. They are considered to play crucial roles in the formation and maintenance of the blood-brain barrier, the regulation of neural function, and the glymphatic system. This indicates that iSCs are generated through reprogramming of pericytes during brain injuries such as ischemia, and differentiate into neurons due to the microenvironment formed by vascular endothelial cells and other cells. Therefore, iSCs can be considered stem cells that play a major role in neural repair following blood flow remodeling after stroke.

[0015] As part of their research using iSCs, the inventors of this invention investigated the effects of a vaccinia virus-inoculated rabbit inflammatory skin extract (this extract) on iSCs. Furthermore, after culturing iSCs with this extract, a comprehensive gene expression analysis using a DNA microarray of approximately 28,000 genes was performed. The results showed that this extract selectively promotes the expression of the PTGDS gene, which encodes L-PGDS. Additionally, at the protein level, the extract also confirmed its promotion of L-PGDS production. Moreover, immunohistochemical studies showed that L-PGDS expressed in ischemic brains co-distributed with pericyte markers. Therefore, it can be concluded that L-PGDS originates from pericytes or iSCs dedifferentiated from pericytes.

[0016] As described above, L-PGDS is considered to be a protein that acts as an enzyme in the synthesis of PGD2, is mainly expressed in the brain, and plays various roles such as binding, transporting, and clearing various hydrophobic low-molecular-weight molecules, as well as regulating the brain environment, neuroprotection, and sleep regulation. Therefore, L-PGDS production promoters are considered useful as drugs for the prevention, treatment, or relapse prevention of diseases involving L-PGDS, such as cerebrovascular disorders like cerebral infarction, dementia like Alzheimer's disease, and insomnia. This extract has been confirmed to have excellent L-PGDS production promoting effects, increasing the amount of L-PGDS in the brains of Alzheimer's dementia model mice and decreasing the amount of Aβ, thereby improving cognitive function. Therefore, this extract, substances containing this extract that have L-PGDS production promoting effects, or preparations containing this extract are very useful as L-PGDS production promoters.

[0017] Furthermore, the inventors of this invention have discovered that using the promoting effect of iSC on L-PGDS production as an indicator is useful for screening agents effective in treating diseases involving L-PGDS. This screening method is considered particularly helpful in the development of agents with neuroprotective or sleep-inducing effects. Additionally, this invention provides a method for testing the extract or preparations containing the extract using the promoting effect of iSC on L-PGDS production as an indicator, thereby determining or evaluating the effects and efficacy of the extract or preparations containing the extract, and thus ensuring the efficacy of the extract or preparations containing the extract. The inventors of this invention completed this invention based on the above findings.

[0018] That is, the present invention includes, for example, the following solutions.

[0019] (1) A lipid transport protein type prostaglandin D2 synthase production promoter.

[0020] (2) The lipid transport protein type prostaglandin D2 synthase production promoter as described in (1) above, which is contained in the extract of vaccinia virus-inoculated inflammatory tissue.

[0021] (3) The lipid transport protein type prostaglandin D2 synthase production promoter described in (1) above contains vaccinia virus inoculated inflammatory tissue extract.

[0022] (4) The lipid-carrier prostaglandin D2 synthase production promoter as described in any one of (1) to (3) above, wherein the lipid-carrier prostaglandin D2 synthase is produced in pericytes or ischemic induced pluripotent stem cells obtained from pericytes.

[0023] (5) The lipid transport protein type prostaglandin D2 synthase production promoter as described in any one of (1) to (4) above is a neuroprotective drug.

[0024] (6) As described in (5) above, the lipid transport protein type prostaglandin D2 synthase production promoter, wherein the neuroprotective drug works by enhancing the brain's excretion system.

[0025] (7) The lipid transport protein type prostaglandin D2 synthase production promoter as described in (5) or (6) above, wherein the neuroprotective drug is a drug for the prevention, treatment or recurrence prevention of cerebral infarction.

[0026] (8) The lipid transport protein type prostaglandin D2 synthase production promoter as described in (5) or (6) above, wherein the neuroprotective drug is a drug for the prevention and treatment of dementia.

[0027] (9) The lipid transport protein type prostaglandin D2 synthase production promoter as described in (8) above, wherein the dementia is Alzheimer's dementia.

[0028] (10) The lipid transport protein type prostaglandin D2 synthase production promoter described in (9) above has an inhibitory effect on amyloid β deposition.

[0029] (11) The lipid transport protein type prostaglandin D2 synthase production promoter as described in any one of (1) to (4) above is a hypnotic drug.

[0030] (12) The lipid transport protein type prostaglandin D2 synthase production promoter as described in any one of (2) to (11) above, wherein the inflamed tissue is inflamed skin tissue of rabbit.

[0031] (13) The lipid transport protein type prostaglandin D2 synthase production promoter as described in any one of (1) to (12) above, which is an injectable preparation.

[0032] (14) The lipid transport protein type prostaglandin D2 synthase production promoter as described in any one of (1) to (12) above, which is an oral preparation.

[0033] (15) A method for screening substances with neuroprotective or sleep-inducing effects, using the expression-promoting effect of lipid transport protein prostaglandin D2 synthase as an indicator.

[0034] (16) The screening method described in (15) above, wherein the expression-promoting effect of lipid transport protein prostaglandin D2 synthase in pericytes or ischemia-induced pluripotent stem cells dedifferentiated from pericytes is used as an indicator.

[0035] (17) The screening method described in (15) or (16) above, wherein the substance with neuroprotective effect is a drug for the prevention, treatment or recurrence prevention of cerebral infarction or a drug for the prevention and treatment of dementia.

[0036] (18) The screening method described in (17) above, wherein the dementia is Alzheimer's dementia.

[0037] (19) The screening method described in (15) or (16) above, wherein the substance with a sleep-inducing effect is a hypnotic drug.

[0038] (20) A substance with brain protection or sleep-inducing effects obtained by any one of the screening methods (15) to (19) above.

[0039] (21) The substance described in (20) above, wherein the neuroprotective effect is achieved through the excretion of amyloid β.

[0040] (22) The method for determining or evaluating the expression promotion of lipid transport protein prostaglandin D2 synthase in vaccinia virus inoculation inflammatory tissue extract or preparations containing it, with the expression promotion of lipid transport protein prostaglandin D2 synthase as an indicator.

[0041] (23) The determination or evaluation method described in (22) above uses the expression promotion effect of lipid transport protein prostaglandin D2 synthase in pericytes or ischemia-induced pluripotent stem cells obtained from pericytes as an indicator.

[0042] (24) The determination or evaluation method as described in (22) or (23) above, wherein the inflamed tissue is the inflamed skin tissue of a rabbit.

[0043] (25) A method for ensuring the quality specifications of vaccinia virus inoculation inflammatory tissue extracts or preparations containing them by performing any one of the determinations or evaluations described in (22) to (24) above.

[0044] (26) A vaccinia virus inoculation inflammatory tissue extract or a preparation containing the same, which ensures quality specifications by performing any one of the determinations or evaluations described in (22) to (24) above.

[0045] (27) A method for promoting the production of lipid-carrier prostaglandin D2 synthase, comprising administering an effective amount of vaccinia virus-inoculated inflammatory tissue extract to a patient in need of the treatment.

[0046] (28) The method for promoting the production of lipid-carrying prostaglandin D2 synthase as described in (27) above, wherein the lipid-carrying prostaglandin D2 synthase is produced in pericytes or ischemic induced pluripotent stem cells obtained from pericytes through dedifferentiation.

[0047] (29) The method for promoting the production of lipid-carrier prostaglandin D2 synthase as described in (27) or (28) above, wherein the promotion of the production of lipid-carrier prostaglandin D2 synthase plays a role in brain protection or sleep induction and improvement.

[0048] (30) The method for promoting the production of lipid transport protein type prostaglandin D2 synthase as described in (29) above, wherein brain protection is for the prevention, treatment or recurrence prevention of cerebral infarction.

[0049] (31) The method for promoting the production of lipid transport protein type prostaglandin D2 synthase as described in (29) above, wherein brain protection is for the prevention and treatment of dementia.

[0050] (32) The method for promoting the production of lipid transport protein type prostaglandin D2 synthase as described in (31) above, wherein the dementia is Alzheimer's dementia.

[0051] (33) An extract of vaccinia virus inoculated with inflammatory tissue, which promotes the production of lipid transport protein prostaglandin D2 synthase.

[0052] (34) Vaccine virus inoculation of inflammatory tissue extract as described in (33) above, wherein the lipid transport protein prostaglandin D2 synthase is produced in pericytes or ischemic induced pluripotent stem cells obtained from pericytes dedifferentiated from pericytes.

[0053] (35) Vaccine virus inoculation of inflammatory tissue extract as described in (33) or (34) above, wherein the production of lipid transport protein type prostaglandin D2 synthase promotes brain protection or sleep induction and improvement.

[0054] (36) Vaccine virus inoculation with inflammatory tissue extract as described in (35) above, wherein brain protection is for the prevention, treatment or recurrence prevention of cerebral infarction.

[0055] (37) As described in (35) above, vaccinia virus inoculation with inflammatory tissue extract, wherein brain protection is for the prevention and treatment of dementia.

[0056] (38) Vaccine virus inoculation of inflammatory tissue extract as described in (37) above, wherein the dementia is Alzheimer's dementia.

[0057] (39) Use of extracts from vaccinia virus-inoculated inflammatory tissues in the manufacture of pharmaceuticals that promote the production of lipid transport protein-type prostaglandin D2 synthase.

[0058] (40) The use as described in (39) above, wherein the lipid transport protein prostaglandin D2 synthase is produced in pericytes or ischemic induced pluripotent stem cells dedifferentiated from pericytes.

[0059] (41) As described in (39) or (40) above, the medicine that promotes the production of lipid transport protein type prostaglandin D2 synthase is a neuroprotective drug or a hypnotic drug.

[0060] (42) As described in (41) above, wherein the neuroprotective drug is a drug for the prevention, treatment or recurrence prevention of cerebral infarction.

[0061] (43) As described in (41) above, the neuroprotective drug is a drug for the prevention and treatment of dementia.

[0062] (44) The use as described in (43) above, wherein the dementia is Alzheimer's dementia.

[0063] The effects of the invention

[0064] The L-PGDS production promoter of this invention enhances the function of L-PGDS expressed in pericytes or iSCs as a lipid transport protein, acting as a transport protein to remove various hydrophobic molecules from the brain. It is strongly expected to protect the brain from damage caused by substances believed to be the cause of cerebral ischemia or dementia. Furthermore, the L-PGDS production promoter of this invention secretes intracellularly synthesized PGD2 in the cerebrospinal fluid and delivers it to PGD2 receptors in the brain, and is expected to exert sleep-regulating effects. In particular, it has been found that this extract promotes L-PGDS production in the ischemic brain of mice. Furthermore, it has been confirmed that administration of this extract to Alzheimer's disease model mice increases the amount of L-PGDS in the brain and reduces Aβ levels, thus improving cognitive function. Thus, the excellent pharmacological effects of this extract in promoting L-PGDS production have been confirmed in animal experiments. In addition, formulations containing this extract have been used for many years as safe agents with few side effects. Therefore, the usefulness of this invention is extremely high.

[0065] Furthermore, this extract is a multi-component system containing a very large number of components, making it extremely difficult to determine or evaluate its effects and ensure efficacy by measuring the content of a single or multiple components. In this respect, the method for determining or evaluating the effects of this extract or preparations containing it, using the expression-promoting effect of L-PGDS as an indicator according to this invention, allows for a simple determination or evaluation of the effects of this extract or preparations containing it, thereby ensuring the efficacy of this extract or preparations containing it. From this perspective, the invention is highly useful. Moreover, the term "determination or evaluation" in this invention encompasses all concepts related to determining effects, functions, and applicability through investigation of the object of study, such as testing and inspection. Attached Figure Description

[0066] Figure 1 This is a graph showing the results of investigating the expression level of the L-PGDS gene (PTGDS) in cultured iSCs supplemented with the test substance using real-time RT-PCR.

[0067] Figure 2 This is an electrophoresis diagram showing the results of investigating the expression level of the L-PGDS gene (PTGDS) in cultured iSCs supplemented with the test substance using the classic RT-PCR method.

[0068] Figure 3 This is an electrophoresis diagram showing the results of investigating the expression level of L-PGDS protein in cultured iSCs supplemented with the test substance using Western blotting.

[0069] Figure 4 This is a PVDF membrane representing the results of investigating the expression level of L-PGDS protein in cultured iSCs supplemented with the test substance using the dot blot method.

[0070] Figure 5 This is a graph showing the results of investigating the expression level of L-PGDS protein in cultured iSCs supplemented with the test substance using the ELISA method.

[0071] Figure 6 This is a graph showing the results of investigating the amount of prostaglandins in cell extracts from cultured iSCs with added test substances using liquid chromatography-mass spectrometry.

[0072] Figure 7 This is a graph showing the results of investigating the amount of reaction products by liquid chromatography-mass spectrometry after adding L-PGDS substrate to the culture supernatant of culture iSC containing the test substance.

[0073] Figure 8 These are images showing the distribution of L-PGDS, pericytes, and vascular endothelial cells in the brains of mice subjected to ischemic load, obtained by immunohistochemical staining.

[0074] Figure 9 This image shows the distribution of L-PGDS in the brains of mice subjected to ischemic stress, as observed by immunoelectron microscopy.

[0075] Figure 10 These are images comparing the distribution of L-PGDS and nestin, a neural stem cell marker, in cultured iSCs extracted from human cerebral infarction lesions using immunohistochemical staining.

[0076] Figure 11 This image represents the results of an immunohistochemical staining study investigating the deposition of amyloid-β protein in the brains of Alzheimer's disease model mice that had been given the test substance.

[0077] Figure 12 This image represents the results of investigating L-PGDS in the brains of Alzheimer's disease model mice that were administered the test substance using immunohistochemical staining.

[0078] Figure 13 This image shows the results of comparing the distribution of L-PGDS and vascular endothelial cells in the brains of Alzheimer's disease model mice that were administered the test substance using immunohistochemical staining.

[0079] Figure 14 This is an electrophoresis diagram showing the results of investigating the expression levels of amyloid-β and L-PGDS proteins in the brains of Alzheimer's disease model mice that were administered the test substance using Western blotting.

[0080] Figure 15 This is an electrophoresis diagram showing the results of investigating the expression level of the L-PGDS gene (PTGDS) in cultured pericytes supplemented with the test substance using the classic RT-PCR method. Detailed Implementation

[0081] This extract contains a non-protein active substance isolated from the inflamed tissue of animals inoculated with vaccinia virus and developing pox. The extract is liquid in its extracted state but can also be dried to form a solid. This preparation is very useful as a pharmaceutical product. Among the specific products manufactured and sold by the applicant in Japan as this preparation is "a preparation containing an extract of inflamed skin from rabbits inoculated with vaccinia virus" (trade name: NEUROTROPIN [registered trademark]) (hereinafter referred to as "NEUROTROPIN"). NEUROTROPIN is available in injectable and tablet forms, both of which are ethical drugs.

[0082] The indications for the injection of Neurotropin are "low back pain, cervicobrachial syndrome, symptomatic neuralgia, pruritus associated with skin diseases (eczema, dermatitis, urticaria), allergic rhinitis, and cold sensation, paresthesia, and pain as a sequela of subacute myelooptic neuropathy (SMON)". The indications for the tablet form of Neurotropin are "postherpetic neuralgia, low back pain, cervicobrachial syndrome, frozen shoulder, and osteoarthritis". This formulation was created and developed by the applicant as a pharmaceutical product. Its excellent efficacy and safety have been appreciated, and it has been sold for many years, establishing a solid position in the Japanese pharmaceutical market.

[0083] The vaccinia virus-inoculated inflammatory tissue extract of this invention can be obtained through the following steps: inoculating with vaccinia virus, breaking up the inflammatory tissue of pox, adding an extraction solvent, removing tissue fragments, performing a protein removal treatment to allow it to adsorb onto an adsorbent, and then eluting the active ingredients. That is, for example, the following steps.

[0084] (A) Collect skin tissues from rabbits, mice, etc. that have been inoculated with vaccinia virus and caused to develop pox. Break up the pox tissues and add extraction solvents such as water, phenol water, physiological saline or phenol plus glycerol water. Then, separate the extract (filtrate or supernatant) by filtration or centrifugation.

[0085] (B) Adjust the pH of the above extract to acidic and heat it to remove proteins. Then, adjust the protein-removed solution to alkaline and heat it before filtration or centrifugation.

[0086] (C) Make the obtained filtrate or supernatant acidic so that it can be adsorbed by adsorbents such as activated carbon and kaolin.

[0087] (D) Add water or other extraction solvent to the above adsorbent, adjust the pH to alkaline, and elute the adsorbed components to obtain an extract of vaccinia virus-inoculated inflammatory tissue. Then, depending on preference, evaporate and dry the eluent under reduced pressure or freeze-dry to obtain a dried solidified product.

[0088] As animals used to obtain inflammatory tissue through vaccinia virus inoculation, various animals infected with vaccinia virus, such as rabbits, cattle, horses, sheep, goats, monkeys, rats, and mice, can be used as inflammatory tissue, with rabbit inflammatory skin tissue being preferred. Any rabbit belonging to the order Lagomorpha can be used. Examples include: European rabbits (Oryctolagus cuniculus), domestic rabbits (domesticated European rabbits), wild rabbits (Japanese hares), pikas, and snowshoe hares. Among these, domestic rabbits are suitable. In Japan, there are rabbits called "domesticated rabbits" (イエウサギ), which were frequently used in the past as livestock or laboratory animals; this is also another name for domestic rabbits. Several breeds exist among domestic rabbits; the Japanese White and New Zealand White breeds are preferred.

[0089] Vaccine virus can be any strain of vaccinia virus. Examples include Lister strain, Dairen strain, Ikeda strain, EM-63 strain, and New York City Board of Health strain.

[0090] More specifically, the basic extraction steps (A) to (D) of the extract described above can be implemented as follows, for example.

[0091] Regarding process (A)

[0092] Intradermal inoculation of rabbits with vaccinia virus induces pox development, and inflamed skin tissue is collected. The collected skin tissue is cleaned and disinfected with phenol solution, etc. The inflamed skin tissue is then broken up, and 1 to 5 times its volume of extraction solvent is added. Breaking up means finely crushing it into a meat-like consistency using a grinder or similar tool. Extraction solvents can include distilled water, physiological saline, weakly acidic to weakly alkaline buffer solutions, and may also include appropriate amounts of bactericides / preservatives such as phenol, stabilizers such as glycerin, and salts such as sodium chloride, potassium chloride, and magnesium chloride. Cell tissue can also be disrupted through freeze-thaw cycles, ultrasound, cell membrane lysing enzymes, or surfactants to facilitate extraction. The resulting suspension is left to stand for 5 to 12 days. During this period, it can be heated to 30 to 45°C with or without stirring. Tissue fragments are removed by solid-liquid separation (filtration or centrifugation, etc.) to obtain a crude extract (filtrate or supernatant).

[0093] Regarding process (B)

[0094] The crude extract obtained in step (A) is subjected to protein removal treatment. Protein removal can be carried out by commonly known methods, such as heat treatment, treatment with protein denaturing agents (e.g., acids, alkalis, urea, guanidine, acetone, and other organic solvents), isoelectric point precipitation, and salting out. Next, using common methods for removing insoluble matter, such as filtration using filter paper (cellulose, nitrocellulose, etc.), glass filters, diatomaceous earth, Seitz filters, ultrafiltration, or centrifugation, a filtrate or supernatant with the precipitated insoluble proteins removed is obtained.

[0095] Regarding process (C)

[0096] The filtrate or supernatant obtained in step (B) is adjusted to acidity, preferably to pH 3.5 to 5.5, for adsorption onto an adsorbent. Examples of adsorbents that can be used include activated carbon and kaolin. Adding the adsorbent to the extract and stirring, or passing the extract through an adsorbent-packed column, allows the active ingredient to be adsorbed onto the adsorbent. When the adsorbent is added to the extract, the solution is removed by filtration or centrifugation to obtain an adsorbent containing the adsorbed active ingredient.

[0097] Regarding process (D)

[0098] To elute (de-elut) the active ingredient from the adsorbent obtained in step (C), an elution solvent is added to the adsorbent, and the pH is adjusted to alkaline, preferably to 9 to 12. Elution is carried out at room temperature or with appropriate heating, or by stirring. The adsorbent is then removed by conventional methods such as filtration or centrifugation. As the elution solvent used, an alkaline solvent can be used, such as water, methanol, ethanol, isopropanol, or suitable mixtures thereof adjusted to an alkaline pH. Water adjusted to pH 9 to 12 is preferred. The amount of elution solvent can be appropriately set. To use the eluent obtained in this manner as the drug substance, the pH can be appropriately adjusted to near neutral, ultimately yielding a rabbit inflammatory skin extract inoculated with vaccinia virus (this extract).

[0099] This extract is prepared as a liquid, and therefore can be concentrated or diluted to obtain an extract of the desired concentration. When manufacturing a formulation from this extract, heat sterilization is preferably performed. To prepare an injection, for example, a solution isotonic with physiological saline can be prepared by adding sodium chloride. Alternatively, it can be administered orally in liquid or gel form, or tablets or other oral solid dosage forms can be manufactured by appropriately concentrating, drying, or solidifying the extract. Specific methods for manufacturing such oral solid dosage forms from this extract are described in Japanese Patent Nos. 3818657 and 4883798. Injectable or oral dosage forms obtained in this manner are examples of this formulation.

[0100] There are no specific restrictions on the method of administration to patients, and it can be appropriately selected according to the therapeutic purpose. For example, in addition to oral administration, subcutaneous, intramuscular, intravenous, and transdermal administration can also be listed. The dosage can be appropriately set according to the type of vaccinia virus inoculation inflammatory tissue extract. Regarding the dosage confirmed in commercially available preparations, it is generally expressed as 16 NU per day for oral administration and 3.6 to 7.2 NU per day for injection as a medical pharmaceutical product. However, it can be appropriately increased or decreased according to the type of disease, severity of illness, individual patient differences, method of administration, and duration of administration. (NU: Neurotropin unit. Neurotropin unit is defined as the ED50 value of analgesic efficacy in SART stress mice with chronic stressors that have a lower pain threshold than normal animals, tested according to the modified Randall-Selitto method. 1 NU represents 1 mg of the analgesic active ingredient in the neurotropin preparation with an ED50 value of 100 mg / kg.)

[0101] The following describes examples of methods for manufacturing the extract, as well as pharmacological test results regarding novel pharmacological effects and L-PGDS production-promoting effects of the extract, but the present invention is not limited in any way by the description of these examples.

[0102] Example

[0103] Example 1: Preparation of the extract

[0104] Intradermal inoculation of vaccinia virus was performed on the skin of healthy, mature rabbits, and the infected skin was excised and collected. The collected skin was cleaned and disinfected with phenol solution, excess phenol was removed, the skin was crushed, and mixed with phenol solution. The mixture was left to stand for 3–7 days, then stirred for 3–4 days while being heated to 35–40°C. The extract obtained from solid-liquid separation was then adjusted to pH 4.5–5.2 using hydrochloric acid, heated at 90–100°C for 30 minutes, and filtered to remove protein. The filtrate was then adjusted to pH 9.0–9.5 using sodium hydroxide, heated at 90–100°C for 15 minutes, and then subjected to solid-liquid separation.

[0105] The obtained protein-removing solution was adjusted to pH 4.0–4.3 using hydrochloric acid. Activated carbon (2% of the protein-removing solution's mass) was added, and the mixture was stirred for 2 hours, followed by solid-liquid separation. Water was added to the collected activated carbon, and the pH was adjusted to 9.5–10 using sodium hydroxide. The mixture was stirred at 60°C for 90–100 minutes, and then centrifuged to obtain the supernatant. Water was added to the activated carbon precipitated by centrifugation, and the pH was adjusted to 10.5–11 using sodium hydroxide. The mixture was stirred at 60°C for 90–100 minutes, and then centrifuged to obtain the supernatant. The two supernatants were combined and neutralized with hydrochloric acid to obtain the extract.

[0106] Example 2 Pharmacological Test

[0107] Next, the methods and results of pharmacological tests on the promoting effect of the extract obtained in Example 1 above as the test substance are presented. Furthermore, in the pharmacological tests described below, the introduction of cerebral infarction into CB-17 mice and the isolation and culture of iSCs from the infarct foci were performed according to the methods described in Nakagomi, T. et al. Eur. J. Neurosci., 29, 1842-1852, 2009.

[0108] Experimental Example 1: Analysis of Integrated Gene Expression in iSCs Treated with Test Substances

[0109] Ischemic load was induced by middle cerebral artery occlusion in CB-17 mice (n=3), and three iSCs were isolated from the infarct nests on day 3. The iSCs were cultured in Dulbecco modified Eagle medium F12 (2% FBS DMEM / F12 F / E / N) supplemented with 2% fetal bovine serum (FBS), 20 ng / mL fibroblast proliferation factor (FBS), 20 ng / mL epidermal growth factor (EGF), and 1% N2 supplement. Test substances (50, 1000 mNU / mL) or physiological saline (control) were added. Total RNA (all 9 cultures) was recovered on day 4 using the RNeasy Mini Kit (QIAGEN). For comprehensive gene expression analysis, a SurePrint G3Mouse GE microarray 8×60K (24,321 RNAs and 4,576 non-coding RNAs, GE) was used for mouse gene chip analysis. By adding test substances, three genes showing a decrease of less than 1 / 2 or more in the same direction of expression were screened. Results were expressed as the expression ratio of each screened gene relative to the control (mean ± standard error of the three strains). An example of the results is shown in Table 1.

[0110] [Table 1]

[0111]

[0112] The results of the above analysis are shown in Table 1. A total of three genes were identified: the COCH gene, which showed decreased expression, and the GBP6 and PTGDS genes, which showed increased expression. Among them, the expression level of the PTGDS gene, which encodes L-PGDS, showed a strong dose-dependent effect on the test substance.

[0113] Experimental Example 2-1: Evaluation of L-PGDS gene expression (real-time RT-PCR method)

[0114] Similar to Experimental Example 1, the culture of iSC (DMEM / F12 F / - / - without FBS) was also performed. The test substance (50 or 500 mNU / mL) or physiological saline (control) was added (n=1). After culturing for 7 days, RNA was recovered using Isogen II (trademark) according to the manufacturer's (Nippon Gene) manual. RNA purity was tested using absorbance at 260 nm and 280 nm. Reverse transcription was then performed using SuperScript IV RTase (Invitrogen) in the presence of random primers to obtain single-stranded cDNA of total RNA. The obtained cDNA was quantified using quantitative PCR with PTGDS specific primers (Prism 7900HT, Applied Biosystems), using the housekeeping gene β-actin as a standard (threshold recycling comparison method). The primer sequences used for PTGDS and β-actin are as follows: [PTGDS: 5'-gactctgaaggacgagctgaag-3' (Sequence No. 1), 5'-tcttgaatgcacttatccggttgg-3' (Sequence No. 2); β-actin: 5'-tacagcttcaccaccacagc-3' (Sequence No. 3), 5'-aaggaaggctggaaaagagc-3' (Sequence No. 4)]. Results are expressed as the expression ratio of PTGDS to β-actin at control 1. An example of the results is shown in Table 2. Figure 1 .

[0115] [Table 2]

[0116]

[0117] From Table 2 and Figure 1 As can be seen from the results shown in Experimental Example 1, it was confirmed that the test substance promoted the expression of the L-PGDS gene (PTGDS) in an amount-dependent manner in the iSC.

[0118] Experiment 2-2: Evaluation of L-PGDS gene expression (classic RT-PCR method)

[0119] iSCs were cultured for 4 days in FGF-containing medium (DMEM / F12 F / - / -) or FGF-free medium (DMEM / F12- / - / -) at concentrations of 0 (control; physiological saline), 1, 5, 50, and 100 mNU / mL. The expression level of the L-PGDS gene (PTGDS) was semi-quantitatively determined (35 cycles) using the classic RT-PCR method (SuperScript III One-Step RT-PCR System with Platinum, Invitrogen) on total RNA recovered via the RNeasy Mini Kit (QIAGEN). PCR products were separated by 2% agarose gel electrophoresis, and the bands of PTGDS and GAPDH were stained with ethidium bromide and visually detected. The primer sequences used for PTGDS and GAPDH are as follows: [PTGDS: 5'-cctccaactcaagctggttc-3' (sequence number 5), 5'-atagttggcctccaccactg-3' (sequence number 6); GAPDH: 5'-atcactgccacccagaagac-3' (sequence number 7), 5'-cacattgggggtaggaacac-3' (sequence number 8)]. An example of the result is shown below. Figure 2 .

[0120] Regarding the promoting effect of L-PGDS gene (PTGDS) expression in iSC, a more detailed investigation was conducted using the dosage of the test substance, and the results are as follows: Figure 2 As shown, the test substance increased the expression level of the L-PGDS gene in a dose-dependent manner, regardless of whether the culture medium contained FGF.

[0121] Example 3-1: Evaluation of L-PGDS protein expression (Western blotting)

[0122] iSCs were cultured for 4 days in the absence of serum with the addition of the test substance (0, 1, 5, 50, 100 mNU / mL) (DMEM / F12F / - / -). After recovery, the sample was washed with phosphate buffer and then dissolved in RIPA buffer (4°C, 50 mM Tris hydrochloric acid buffer (pH 7.6), 150 mM sodium chloride, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate) and homogenized. After adjusting to achieve a homogenized total protein content, the homogenate was separated using SDS-PAGE (BIO-RAD Any kD, trademark), transferred to a polyvinylidene fluoride (PVDF) membrane (Immun-Blot PVDF membrane, BIO-RAD), blocked with Bloking One (Nacalai Tesque), and then detected using Western blotting with specific antibodies. L-PGDS (anti-prostaglandin D synthase (lipocarboxin) antibody [EP12357], Abcam, 1:2000) and β-actin (monoclonal anti-β-actin antibody [A1978], Sigma, 1:100000) were detected. High-sensitivity chemiluminescence immunoassay (Chemi-Lumi One L, Nacalai Tesque) was used. An example of the results is shown below. Figure 3 .

[0123] from Figure 3 It was confirmed that the test substance promoted the expression of L-PGDS protein in iSC cells.

[0124] Experimental Example 3-2: Evaluation of L-PGDS protein expression (dot blot assay)

[0125] The culture was carried out for 4 days with the test substance (0, 1, 10, 50, 100 mNU / mL) (DMEM / F12F / - / -). 500 μL of culture supernatant from iSCs was spotted onto a PVDF membrane (Immun-Blot PVDF membrane, BIO-RAD), blocked with Bloking One (Nacalai Tesque), and L-PGDS was detected by dot blot assay using an anti-L-PGDS antibody (anti-prostaglandin D synthase (lipotransferase) antibody [EP12357], Abcam, 1:2000). An example of the results is shown below. Figure 4 .

[0126] L-PGDS possesses a typical secretion signal sequence and signal peptidase recognition sequence at its N-terminus, and is therefore considered to be secreted extracellularly. Therefore, the L-PGDS protein in the culture supernatant of iSCs supplemented with the test substance was measured, and the results are as follows: Figure 4It was confirmed that the amount of L-PGDS protein in the culture supernatant (extracellular) also increased depending on the amount of the test substance added.

[0127] Experiment 3-3: Evaluation of L-PGDS protein expression (ELISA method)

[0128] iSCs treated with the test substance (0 [control], 1, 10, 50, 100, 1000 mNU / mL) for 4 days were cultured in DMEM / F12 F / - / -. The supernatant was recovered. After centrifugation (1500 rpm, 10 min, 4 °C), the amount of L-PGDS in the culture supernatant was determined using a specific ELISA method (Human L-PGDS ELISA Kit (Prostaglandin D Synthase 21 kDa (Brain), Model: SEA724Hu, Cloud-Clone Corp.)) according to the manufacturer's manual. An example of the results is shown in Table 3 and Figure 5 .

[0129] [Table 3]

[0130]

[0131] From Table 3 and Figure 5 As can be seen from Example 3-2, an increase in L-PGDS protein was confirmed in the culture supernatant (extracellular) of iSCs treated with the test substance. The results of Example 3 confirm that the test substance promotes the production of L-PGDS in iSCs not only at the gene level but also at the protein level, and further confirms that it induces the secretion of L-PGDS.

[0132] Example 4: Evaluation of the enzyme activity of L-PGDS (High-performance liquid chromatography-mass spectrometry)

[0133] L-PGDS is an enzyme that biosynthesizes PGD2 using PGH2 as a substrate (EC 5.3.99.2). To confirm that L-PGDS produced in iSCs by the test substance generates PGD2 through its enzymatic activity, a comprehensive analysis was performed on PGD2 and its metabolites, as well as prostaglandins such as PGE2, which also use PGH2 as a substrate, in both A: intracellular and B: extracellular environments of iSCs.

[0134] A: Evaluation of intracellular enzyme activity in iSC cells

[0135] Cultured for 3 days with added test substance (0, 10, 50 mNU / mL) in DMEM / F12 without FBS (F / - / -). Cell extracts were prepared by treatment with RIPA buffer (4°C, 20 min) in iSC. The concentrations of prostaglandins (PGD2, PGJ2, 15-deoxy-Δ12,14-PGJ2, 13,14-dihydro-15-one-PGD2, and PGE2) in the cell extracts were determined by HPLC-MS / MS. HPLC separation was performed using an AQUITY UPLC HSS T3 column (Waters), with an API4000 LC / MS / MS system and a TripleQuadrupole mass spectrometer (both AB Sciex) as the detector and mass spectrometer, respectively. An example of the results is shown in Table 4. Figure 6 .

[0136] [Table 4]

[0137] [n=3]

[0138]

[0139] As shown in Table 4 and Figure 6 As shown, the intracellular levels of PGD2 and its non-enzymatic metabolite PGJ2 increased in a dose-dependent manner with the addition of the test substance. Furthermore, the production of 15-deoxy-Δ12,14-PGJ2, a non-enzymatic metabolite of PGJ2, was also confirmed upon the addition of 50 mNU / mL of the test substance. In addition, the non-enzymatic metabolite 13,14-dihydro-15-one-PGD2 of 15-deoxy-Δ12,14-PGJ2 was below the detection limit. On the other hand, no dose-dependent response was observed in the production of PGE2 from PGH2, a substrate common to PGD2. B: Evaluation of extracellular enzyme activity in iSCs

[0140] The substrates PGH2 and glutathione (GSH) of L-PGDS were used to act on iSCs supplemented with the test substance (0, 50, 100, 1000 mNU / mL) and cultured for 3 days (in FBS-free DMEM / F12F / - / -). The culture supernatant after the reaction (reaction conditions: 100 mM Tris-HCl (pH 8.0), 1 mM GSH, 10 mM PGH2, 37 °C, 5 min). Similar to A above, the concentrations of prostaglandins (PGD2, PGJ2, 15-deoxy-Δ12,14-PGJ2, 13,14-dihydro-15-one-PGD2, and PGE2) in the reaction solution were determined by HPLC-MS. An example of the results is shown in Table 5. Figure 7 .

[0141] [Table 5]

[0142] [n=1]

[0143]

[0144] As shown in Table 5 and Figure 7 As shown, concentrations of the test substance above 100 mNU / mL increased the concentrations of PGD2 and its non-enzymatic metabolites PGJ2 and 15-deoxy-Δ12,14-PGJ2 in the reaction solution, thus confirming the presence of L-PGDS activity in the iSC culture supernatant. Furthermore, regarding the non-enzymatic metabolite 13,14-dihydro-15-one-PGD2 of 15-deoxy-Δ12,14-PGJ2, a dose-dependent production was observed with the addition of the test substance up to 100 mNU / mL, but the concentration was below the detection limit at 1000 mNU / mL.

[0145] L-PGDS is known to be a secretory enzyme, but the results of A and B above confirm that iSCs release enzymatically active L-PGDS into the extracellular space, and the production and release of this enzyme can be promoted by adding the test substance.

[0146] Experimental Example 5-1: Study of L-PGDS-producing cells in the mouse brain (immunohistochemical staining method)

[0147] Brain sections of CB-17 mice (3 days after ischemia) subjected to ischemic load due to middle cerebral artery occlusion were prepared and immunohistochemically studied using confocal laser microscopy using specific antibodies against L-PGDS (image (panel) B, green; anti-prostaglandin D synthase (lipotransferase) antibody [EP12357], Abcam, 1:1000), pericyte marker α-SMA (image C, red; anti-actin, smooth muscle, clone ASM-1, Millipore, 1:1000) and vascular endothelial cell marker CD31 (image D, red; anti-mouse CD31 (PECAM-1) monoclonal antibody, 550274, BD Pharmingen, 1:1000). L-PGDS (Image B) and pericyte markers (Image C) or L-PGDS (Image B) and vascular endothelial cell markers (Image D) were detected using specific antibodies labeled with different fluorescent dyes via multiple staining. The images were then overlaid to compare the distribution of L-PGDS and pericyte markers or L-PGDS and vascular endothelial cell markers (Images A1 and A2). An example of the results is shown below. Figure 8 .

[0148] exist Figure 8In the upper section, the green fluorescence in image B represents the distribution of L-PGDS, and the red fluorescence in image C represents the distribution of pericyte markers. However, in image A1, which overlays the two images, the distributions of L-PGDS (green fluorescence) and pericyte markers (red fluorescence) are locally identical (200x). Furthermore, in Figure 8 In the next section, the green fluorescence in image B represents the distribution of L-PGDS, and the red fluorescence in image D represents the distribution of vascular endothelial cell markers. In image A2, which overlays the two, although L-PGDS (green fluorescence) is distributed near vascular endothelial cell markers (red fluorescence), no co-distribution was observed (200x).

[0149] L-PGDS is a protein mainly distributed in the central nervous system, present in the arachnoid membrane, pia mater, and choroid plexus of the lateral ventricles, and is believed to be secreted into the cerebrospinal fluid (Urade Y. et al., J Lipid Mediat. Cell Signal. 14, 71-82, 1996), but the cells that produce it in the brain are not fully understood. The inventors of this invention conducted a study on the brain distribution of L-PGDS using immunohistochemistry. The results showed weak expression in the normal brain of CB-17 mice, but in the aforementioned experiment 4-1, when permanent ligation of the middle cerebral artery was introduced in these mice, significant L-PGDS expression was confirmed in the cerebral infarction lesion. Figure 8 Image B). Additionally, L-PGDS were observed around CD31, a marker of vascular endothelial cells, but their distributions did not overlap. Figure 8 Image A2). On the other hand, it was learned that L-PGDS showed a partial co-distribution with a pericyte marker α-SMA ( Figure 8 Image A1). Based on the above results, it can be concluded that the L-PGDS expressed and induced in the cerebral infarction lesion originates from pericytes (or iSCs). This can be verified by the following fact: Immunoelectron microscopy in Experiment 4-2 (described later) also confirmed that L-PGDS positive products exist as high-electron-density structures in the cytoplasm of pericytes in contact with vascular endothelial cells and the basement membrane. Figure 9 (Images A and B). These results suggest the physiological role of pericytes or iSCs in the ischemic response, including the induction of L-PGDS expression.

[0150] Experimental Example 5-2: Study of L-PGDS-producing cells in the mouse brain (immunoelectron microscopy)

[0151] Brain sections of CB-17 mice subjected to ischemic load induced by middle cerebral artery occlusion (3 days after ischemia) were prepared. The expression sites of L-PGDS were observed using immunoelectron microscopy with a specific antibody against L-PGDS (anti-prostaglandin D synthase (lipotransferase) antibody [EP12357], Abcam, 1:1000). Specifically, 2 μm thick brain sections were prepared using a vibratome, reacted with an avidin-biotin horseradish peroxidase (HRP) complex kit (Vector Laboratories) and 3,3'-diaminobenzidine tetrahydrochloride (DAB), osmium-treated, embedded in epoxy resin (EPON), and ultrathin sections were prepared for electron microscopy. One example of the results is shown below. Figure 9 .

[0152] exist Figure 9 In either image A or B, regions of high electron density were identified in the cytoplasm of pericytes (pericells) present in contact with vascular endothelial cells and the basement membrane using immunoelectron microscopy of L-PGDS.

[0153] Experimental Example 6: Study of L-PGDS-producing cells in the human brain (immunohistochemical staining method)

[0154] Immunohistochemical studies were conducted in cultured iSCs extracted from human cerebral infarction foci (necrotic tissue) using specific antibodies against L-PGDS (Image B, green; anti-prostaglandin D synthase (lipotransferase) antibody [EP12357], Abcam, 1:1000) and the neural stem cell marker nestin (Image C, red; anti-nesin, clone 10C2, Millipore, 1:1000). Cells were reacted with either Alexa Fluor 488-conjugated antibody (registered trademark, hereinafter the same) or Alexa Fluor 555-conjugated antibody (1:500; Molecular Probes, Eugene) followed by nuclear staining with 4',6-diamidino-2-phenylindole (DAPI; 1:1000; Kirkegaard & Perry Laboratories). Fluorescence was observed using a fluorescence microscope (BX60; Olympus, Japan), and the distributions of L-PGDS (Image B) and nestin (Image C) were compared by overlaying the images (Image A). An example of the results is shown below. Figure 10 .

[0155] exist Figure 10 In the images, the green fluorescence in image B represents the distribution of L-PGDS, and the red fluorescence in image C represents the distribution of nestin. However, in image A, which overlays the two images, the distribution of L-PGDS (green fluorescence) is consistent with that of nestin (red fluorescence) (200-fold). This confirms that L-PGDS is expressed in almost all iSCs that express nestin. Therefore, it can be concluded that L-PGDS is produced not only in mouse brains but also in human brains in iSCs or pericytes.

[0156] Experimental Example 7: Amyloid β Deposition and L-PGDS Expression in the Brains of Animal Models of Alzheimer's Dementia Evaluation

[0157] (1) Action analysis and sample preparation of cognitive abilities

[0158] APPswe / PS1dE9 (APP / PS1) mice (female, 3 months old) were randomly divided into a test substance administration group and a control group (n=10-11 per group) with equal body weight. For approximately 3 months, the test substance (100 NU / kg body weight) or saline was administered via tail vein injection twice weekly. Following administration, behavioral analyses of cognitive abilities (Y-maze test, novelty object recognition test, and Morris water maze test) were performed. In each test, A: alternating action rate (%) in the Y-maze test, B: frequency of exposure to novelty objects (%) in the novelty object recognition test, and C: average time (seconds) required to find the platform within 4 days (days 6-9) at the end of the learning period in the Morris water maze test were measured. A composite score (A×B÷C) was calculated for each individual. The results for each group of two individuals are presented in Table 6.

[0159] [Table 6]

[0160]

[0161] As shown in Table 6, an improvement in cognitive ability was observed in the group given the test substance, and this effect was statistically significant between the groups.

[0162] Following vena cava analysis of cognitive abilities (24 hours after final administration), in both the test substance administration group and the control group, the brains were collected after decapitation under anesthesia, and the left hemisphere was rapidly frozen in liquid nitrogen. The remaining right hemisphere was fixed using either (A) a fixative containing glutaraldehyde (0.05% glutaraldehyde, 4% paraformaldehyde, 0.1M phosphate buffer) or (B) PLP fixative (0.01M sodium periodate, 0.075M lysine, 2% paraformaldehyde) at 4°C. Coronal sections (20 μm thick) containing the cerebral cortex were prepared from the fixed tissue specimens using a cryostat (Leica CM1850).

[0163] (2) Evaluation of amyloid β deposition in the cerebral cortex (immunohistochemical staining)

[0164] Coronal sections of the test substance administration group and control group prepared in (1) above were subjected to immunohistochemical staining using anti-amyloid β antibody (anti-β-amyloid, 1-16, [SIG-39300], BioLegend, 1:1000). Amyloid β was reacted with Alexa Fluor 488 conjugate antibody (1:500; MolecularProbes, Eugene), followed by nuclear staining with 4',6-diamidinyl-2-phenylindole (DAPI; 1:1000; Kirkegaard & Perry Laboratories), and observed for fluorescence using a fluorescence microscope (BX60; Olympus, Japan). An example of the results is presented below. Figure 11 .

[0165] like Figure 11 As shown, in individuals whose cognitive abilities were confirmed to have improved by administration of the test substance, the number and surface area of ​​brain amyloid plaques were reduced compared with the control group.

[0166] (3) Evaluation of L-PGDS expression in the cerebral cortex (immunohistochemical staining)

[0167] Coronal sections of the test substance administration group and control group prepared in (1) above were subjected to immunohistochemical staining using anti-L-PGDS antibody (anti-prostaglandin D synthase (lipotransferase) antibody [EP12357], Abcam, 1:1000). L-PGDS was detected using an avidin-bionic horseradish peroxidase (HRP) complex kit (Vector Laboratories) and a reaction with 3,3'-diaminobenzidine tetrahydrochloride (DAB). An example of the results is shown below. Figure 12 .

[0168] like Figure 12 As shown, in the brain of the test substance administration group, specific staining of L-PGDS was confirmed near the blood sinus (vascular ligament) (arrow part in Figure B), in contrast, no specific staining of L-PGDS was confirmed in the brain of the control group (Figure A).

[0169] In addition, the coronal sections of the test substance administration group prepared in (1) above were subjected to immunohistochemical studies using specific antibodies against L-PGDS (anti-prostaglandin D synthase (lipotransferase) antibody [EP12357], Abcam, 1:1000) and the vascular endothelial cell marker CD31 (anti-mouse CD31 (PECAM-1) monoclonal antibody, 550274, BD Pharmingen, 1:1000). In sections containing the aforementioned primary antibodies, after reaction with Alexa Fluor 488-conjugated antibody or Alexa Fluor 555-conjugated antibody (1:500; Molecular Probes, Eugene), nuclear staining with 4',6-diamidinyl-2-phenylindole (DAPI; 1:000; Kirkegaard & Perry Laboratories) was performed, followed by fluorescence observation using a confocal laser microscope (LSM780; Carl Zeiss, Jena, Germany). Images of L-PGDS (Image B) and CD31 (Image C) were overlaid using a multiple staining method to compare the distribution of L-PGDS and CD31 (Image A). An example of the results is shown below. Figure 13 .

[0170] like Figure 13 As shown, in the brains of the test substance administration group, L-PGDS (green fluorescence) was found distributed around the vascular endothelial cell marker CD31 (red fluorescence). Therefore, it can be concluded that L-PGDS expression increases in pericytes (or iSCs) that exist in response to the administration of the test substance and cover vascular endothelial cells.

[0171] (4) Evaluation of amyloid β deposition and L-PGDS expression in the brain (Western blot analysis)

[0172] Proteins extracted from the rapidly frozen left hemisphere brain collected in (1) were homogenized using a Potter homogenizer and separated by SDS-PAGE (BIO-RAD Any kD (trademark)) and transferred to a PVDF membrane (Immun-Blot PVDF membrane, BIO-RAD). After blocking with Bloking One (nacalai tesque), amyloid β (anti-β-amyloid, 1-16, [SIG-39300], BioLegend, 1:1000) and L-PGDS (anti-prostaglandin D synthase (lipotransferase) antibody [EP12357], Abcam, 1:2000) were detected by Western blotting using specific antibodies. High-sensitivity chemiluminescence immunoassay (Chemi-Lumi One L, nacalai tesque) was used for detection. An example of the results is shown below. Figure 14 .

[0173] like Figure 14 As shown in the image marked with a *, regarding the amount of amyloid-β in the brain, it can be considered that in mice (individual numbers 4 and 29) in the test substance administration group, where cognitive improvement was confirmed by test substance administration, it was reduced compared to mice (individual numbers 14 and 30) in the control group. This result is consistent with the immunohistochemical staining method in Experiment 7(2). Figure 11 In this study, compared to the control group, the amount and surface area of ​​amyloid-β antibody-positive products in the brain were inhibited upon administration of the test substance. Additionally, as... Figure 14 As shown in the image marked with B*, the amount of L-PGDS in the brain increased in the test substance administration group compared to the control group. Therefore, it can be concluded that the test substance enhanced the production of L-PGDS in the brain. From the series of results in Experiment 7 above, the increase in the expression of L-PGDS in the brain by the test substance suggests the possibility of inhibiting the deposition of amyloid-β in the brain and thus improving cognitive function.

[0174] Experimental Example 8: Study on the promoting factors of L-PGDS expression in human pericytes (classic RT-PCR method)

[0175] As mentioned above, iSCs that have been confirmed to be responsive to the test substance are believed to originate from pericytes. Therefore, the L-PGDS expression capacity of the test substance was investigated in commercially available human brain vascular pericytes (ScienCell) under different oxygen and / or glucose concentrations. Specifically, iSCs were cultured for 4 days in the presence of 0, 50, and 500 mNU / mL of the test substance under (1) iSC responsive conditions (4.5 g / L glucose and 20% O2), (2) low glucose conditions (90 mg / L glucose and 20% O2), and (3) hypoxia-hypoglycemia conditions (90 mg / L glucose and 1% O2) (in F / - / - DMEM medium without FBS). Total RNA recovered using the RNeasy Mini Kit (QIAGEN) was used to semi-quantitatively determine the expression level of the L-PGDS gene (PTGDS) using the classic RT-PCR method with the SuperScript III One-Step RT-PCR System with Platinum (Invitrogen) (35 cycles). The PCR products were separated by 2% agarose gel electrophoresis and visualized by staining the PTGDS and β-actin bands with ethidium bromide. Furthermore, the primers used for PTGDS and β-actin were the same as those used in Example 2-1 above. An example of the results is shown below. Figure 15 .

[0176] like Figure 15 As shown, although transcription of the L-PGDS gene was detected in cells cultured under condition (1), no response to the test substance was observed. However, a significant response to the test substance was observed in cells cultured under conditions (3) of hypoxia (1%) and low glucose concentration (90 mg / L). The hypoxia-hypoglycemia condition is considered to simulate the ischemic state in the brain, and therefore, under such pathological conditions, pericytes (or iSCs) can be considered to have acquired a response to external stimuli, releasing cytoprotective proteins such as L-PGDS.

[0177] Industrial availability

[0178] L-PGDS are primarily expressed in the brain, acting as binding, transporting, and scavenging agents for various hydrophobic low-molecular-weight molecules. They are considered proteins with various functions, including brain environment regulation, neuroprotection, and sleep regulation. Therefore, the L-PGDS production promoters of this invention are useful for the prevention, treatment, or relapse prevention of L-PGDS-related diseases such as cerebrovascular disorders like cerebral infarction, dementia like Alzheimer's disease, and insomnia. In particular, this extract and preparations containing it are excellent L-PGDS production promoters and are highly effective due to their safety profile and fewer side effects. Furthermore, the screening method of this invention, using the promoting effect of L-PGDS production in pericytes or iSCs dedifferentiated from pericytes as an indicator, for substances useful for the prevention, treatment, or relapse prevention of L-PGDS-related diseases, especially those with neuroprotective or sleep-regulating effects, is a very useful method for developing new therapeutic drugs.

Claims

1. A method for manufacturing a lipid transport protein-type prostaglandin D2 synthase production promoter, characterized in that: The lipid-carrying prostaglandin D2 synthase production promoter uses extracts from vaccinia virus-inoculated inflammatory tissue as its active ingredient. The manufacturing method includes a step of determining or evaluating the quality specifications of the drug produced by the method by performing a process that uses the expression-promoting effect of lipid-carrier prostaglandin D2 synthase in pericytes or ischemia-induced pluripotent stem cells dedifferentiated from pericytes as an indicator.

2. The manufacturing method as described in claim 1, characterized in that: Lipid-carrier prostaglandin D2 synthase production promoters are neuroprotective drugs.

3. The manufacturing method as described in claim 2, characterized in that: The effects of neuroprotective drugs are achieved by enhancing the brain's excretion system.

4. The manufacturing method as described in claim 2, characterized in that: Neuroprotective drugs are used for the prevention, treatment, or recurrence prevention of cerebral infarction.

5. The manufacturing method as described in claim 2, characterized in that: Neuroprotective drugs are used for the prevention and treatment of dementia.

6. The manufacturing method as described in claim 5, characterized in that: The dementia is Alzheimer's disease.

7. The manufacturing method as described in claim 6, characterized in that: Lipid-carrier prostaglandin D2 synthase promoters have an inhibitory effect on amyloid β deposition.

8. The manufacturing method as described in claim 1, characterized in that: Lipid-carrier prostaglandin D2 synthase promoters are used as hypnotics.

9. The manufacturing method according to any one of claims 1 to 8, characterized in that: The inflamed tissue was the inflamed skin tissue of the rabbit.

10. The manufacturing method according to any one of claims 1 to 8, characterized in that: The lipid transport protein-type prostaglandin D2 synthase production promoter is an injectable formulation.

11. The manufacturing method according to any one of claims 1 to 8, characterized in that: The lipid transporter prostaglandin D2 synthase production promoter is an oral preparation.

Images