WO2002067950A1 - Promoteurs de régénération vasculaire - Google Patents

Promoteurs de régénération vasculaire Download PDF

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Publication number
WO2002067950A1
WO2002067950A1 PCT/JP2002/000368 JP0200368W WO02067950A1 WO 2002067950 A1 WO2002067950 A1 WO 2002067950A1 JP 0200368 W JP0200368 W JP 0200368W WO 02067950 A1 WO02067950 A1 WO 02067950A1
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ginsenoside
pharmaceutical composition
expression
cells
rbi
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PCT/JP2002/000368
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Japanese (ja)
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Masahiro Sakanaka
Koji Hashimoto
Junya Tanaka
Ryuji Hata
Hidemitsu Uno
Makoto Kuramoto
Noriaki Mitsuta
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Japan Science And Technology Corporation
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Publication of WO2002067950A1 publication Critical patent/WO2002067950A1/fr

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    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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    • A61P19/00—Drugs for skeletal disorders
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    • A61P25/00—Drugs for disorders of the nervous system
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
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Definitions

  • the present invention relates to a pharmaceutical composition for inducing the expression of a major factor that promotes the formation, regeneration or remodeling of blood vessels, that is, VEGF (vascular endothelial growth factor). More specifically, the present invention relates to a ginsenoside derivative such as dihydrozincenoside Rbt, which prevents, treats, or treats a disease or condition that causes impaired blood flow such as cerebrovascular disorder, cerebral infarction, spinal cord injury, and wound.
  • the present invention relates to a pharmaceutical composition for intravenous administration, a preparation for intravenous administration, an external preparation for skin, or an agent for promoting regeneration and reconstruction of blood vessels including cerebral blood vessels.
  • the present invention relates to a disease (including a pathological condition) or apoptosis or apoptosis-like cell death that causes a blood flow disorder through enhanced expression of Be1—X and suppression of expression of Z or caspase-3.
  • the present invention also relates to the aforementioned pharmaceutical composition for preventing, treating or treating a resulting disease.
  • the present invention relates to a pharmaceutical composition comprising ginsenosides for inducing Be1-1XL expression or VEGF expression in cells by activating the transcription factor STAT5 or HIF-1.
  • the present invention relates to a method for preventing, treating or treating diseases or conditions which cause impaired blood flow, which comprises administering a test substance to cultured cells and measuring the expression regulating action of the Bc1-2 protein group. It also relates to a method of searching for a pharmaceutical composition.
  • ischemic tissue In many organic diseases, for example, diseases that cause impaired blood flow, blood flow insufficiency is improved while cells in the ischemic tissue undergo apoptosis or apoptotic cell death. Furthermore, apoptosis or apoptosis-like cell death in ischemic tissue progresses, and irreversible higher-order dysfunction appears in the living body. This will be described in more detail below, taking as an example a cerebral infarction disease in which the cortical branch of the middle cerebral artery (MCA) is permanently obstructed in cerebrovascular disorders.
  • MCA middle cerebral artery
  • apoptosis of nerve cells or "apoptotic-like nerve cell death.”
  • ACA anterior cerebral artery
  • PCA posterior cerebral artery
  • B c 1 — 2 proteins have an inhibitory effect on apoptosis or apoptotic cell death (B el — 2, Be 1 -x L, B el — w, M cl — 1, B fl — l ZB od , Nr-13, BRAG-1, BooZDiva, Galectin-3) and those that promote apoptosis or apoptotic cell death (Bax, Bak, Bel — xs , Bad, B ik / N bk, B id, B i m / BodHrk / DP 5 BNIP / NixBokZM tdBlk EGL-1) (Apoptosis proofing method by organ; Editing, Katsunori Otsuki, Takehiko Oji, Kei Watanabe Nanedo, 2000p 16-24).
  • apoptosis inhibitor Antiapoptotic factors
  • apoptosis-promoting factors proapoptoic factors
  • apoptosis or apoptosis-like cell death can be suppressed by simultaneously regulating the expression of a plurality of apoptosis-related factors described above.
  • Bc1-1x> J is particularly abundantly expressed in all living tissues such as mature brain / nervous tissue, immune tissue, hematopoietic tissue, skin tissue, and cardiac muscle tissue. It plays an essential role in the survival of cells.
  • the mitochondria-related protein Be 1 — inhibits the binding of A paf 1 to procaspase 9 by binding to A pafl (Adams JM and Cory S.
  • a paf 1 protein dissociates from the B c 1 — X protein and binds to procaspase 9 to release mitochondria. It is believed that procaspase 9 is activated in conjunction with cytochrome C leakage (Adams JM and Cory S. Science, 281, 1322-1326, 1998).
  • procaspase 9 in the cytoplasm was activated, caspase 9 and caspase 3, the final cell excecutioner, were subsequently activated, These proteolytic enzymes accelerate the process by which cells self-lyse and die (apoptosis). Perhaps activation stage of procaspase 9, there is a high possibility that the cells are destined death is determined, B e 1 - x L be further stop the activation of pro-caspase 9 increased expression agent, cell It is considered a good measure to prevent death.
  • VEGF vascular endothelial growth factor
  • VEGF is used for the proliferation of vascular endothelial cells, migration or migration of endothelial cells, luminal formation, coagulation and fibrinolysis of endothelial cells such as tissue factor and plasminogen activator. It is involved in the renewal, regeneration or remodeling of blood vessels by promoting production of protein, expression of cell adhesion molecules in endothelial cells, and expression of Bc1-2 in endothelial cells. Therefore, if a compound that promotes the expression of VEGF is found, it can be said that it can be used as a pharmaceutical composition for promoting the regeneration and / or reconstruction of blood vessels.
  • HIF-1 hyperoxia inducible fact or-1
  • VEGF vascular endothelial growth factor 1
  • the outline of the regeneration and Z or remodeling of blood vessels is described in Japanese Patent Application No. 2000-248584 and PCT / JP0Z055554. Naka, Tanaka) describes the wound healing phenomenon as an example.
  • dihydroginsenoside R bi one of the ginsenoside derivatives, has the following structural formula
  • This compound is represented by the following structural formula as described in PCTZ JP 00/04102 and PCT / JP00 / 055554.
  • ginsenoside R bi Can be prepared by using the ginsenoside R bi shown in the above as a raw material.
  • ginsenoside derivatives such as dihydrozincenoside Rb showed excellent stroke treatment effects and skin tissue. It has been shown to show regeneration / remodeling promoting effects, open wound healing effects, etc., but whether ginsenoside derivatives such as dihydroginsenoside R bi promotes the neovascularization, regeneration or reconstruction of blood vessels No specific test examples based on the molecular mechanism are disclosed.
  • ginsenoside derivatives such as dihydroginsenoside Rbi are not capable of apoptosis of cells, especially neurons. Alternatively, it is disclosed to suppress apoptosis-like neuronal cell death, but its molecular mechanism is not disclosed.
  • the present inventors have found that a pharmaceutical composition comprising a ginsenoside derivative such as dihydrozincenoside R b increases the expression of VEGF and Bc 1-: _ in a low optimal extracellular solution concentration range, In addition, they found that the expression of caspase 3 was suppressed, and completed the present invention. That is, Jinsenosai earth induction of such dihydric Dorojinsenosai de R bi is, VEGF expression enhancing action, B c 1 -.
  • X L onset current potentiation and Roh or caspase 3 shows expression suppressive action but because cerebrovascular disorders or wound like It has been found that it can be used as a pharmaceutical composition for preventing, treating or treating diseases or conditions causing blood flow disorders, and as an agent for promoting the regeneration and reconstruction of blood vessels. Further, in the present invention, a disease or condition causing blood flow disorder is provided.
  • Ginsenoside derivatives such as dihydrozinenoside Rbi are found to be effective in vivo (in ViVo) against cerebrovascular disorders, spinal cord injury, cerebral infarction, wounds, etc. Was done.
  • the present inventors have found that ginsenosides or ginsenoside derivatives induce the expression of Bc1- ⁇ _ or VEGF through activation of the cellular transcription factor STAT5 or HIF-1. Was completed.
  • ginsenoside Rb Described above is taken as a representative of natural ginsenosides.
  • dihydroginsenoside R bi will be described.
  • Natural ginsenosides and ginsenoside derivatives are collectively referred to herein as ginsenosides. Disclosure of the invention
  • An object of the present invention is to provide a pharmaceutical composition for inducing the expression of a major factor that promotes vascular neogenesis, regeneration or remodeling, ie, VEGF. More specifically, the present invention comprises a ginsenoside derivative such as dihydrozincenoside Rb, which prevents, treats, or treats a disease or condition that causes impaired blood flow, such as cerebrovascular disorder, cerebral infarction, spinal cord injury, and wound. It is intended to provide a pharmaceutical composition for intravenous administration, an external preparation for skin, or an agent for promoting the regeneration and reconstruction of blood vessels including cerebral blood vessels.
  • the present invention through the B e 1-X L expression enhancement and Z or caspase 3 expression inhibited, the diseases causing the disease (including conditions) or Apoptosis one cis or apoptotic-like cell death leading to impaired blood flow
  • the pharmaceutical composition for prevention, treatment or treatment is also provided.
  • Et al is, the present invention is B e 1 by activating the transcription factor STAT 5 or transcription factor HIF one first cell - provides a pharmaceutical composition comprising Jinsenosa Lee earth to induce the expression of X L or VEGF .
  • the present invention relates to a medicament for preventing, treating or treating a disease or condition causing blood flow disorder, which comprises administering to a test substance cultured cell and measuring the expression regulating action of the Bc1-2 protein group. Also provided is a method of searching for a composition. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows the NMR chart of dihydrozincenoside Rb.
  • FIG. 2 is a photograph of a MAP2 immunoblot instead of a drawing, showing the protective effect of dihydrozincenoside Rbi on apoptosis or apoptosis-like neuronal cell death of cultured neurons by SNP.
  • FIG. 3 is a graph showing the protective effect of dihydrozincenoside Rbi on apoptosis or apoptosis-like neuronal cell death of cultured neurons by SNP.
  • Saline solution after disability onset Shows the TTC staining results of the pulse in the administered rats brain (2 cases)
  • t FIG. 5 is a photograph replacing in the drawings, dihydric Dorojinsenosa after MCA permanent occlusion (i.e. after onset of cerebral vascular disorders)
  • Lee de R bi 2 is a photograph instead of a drawing, showing the results of TTC staining of rat brain (2 cases) administered intravenously (day).
  • FIG. 6 is a graph showing the effect of intravenous administration of dihydrozincenoside Rb (6 / ig / day, 0.6 zg / day) on cerebrovascular disorder (cerebral infarction) rats.
  • FIG. 7 is a photograph replacing a drawing showing a rat on the second day after spinal cord injury.
  • the photograph on the left shows an example of intravenous administration of saline, and the photograph on the right shows an example of intravenous administration of dihydrozincenoside R b.
  • FIG. 8 is a photograph replacing a drawing showing another rat on the second day after spinal cord injury.
  • the photograph on the left is an example of intravenous administration of saline, and the photograph on the right is an example of intravenous administration of dihydrozincenoside Rb.
  • FIG. 9 is a photograph as a drawing which shows the effect of topical administration of 1 0 4 -1 0 7% by weight of the dihydric Dorojinseno rhino de R b for open wounds of rats.
  • the first 0 Figure is a Darafu showing the effect of topical administration of dihydric Dorojinse Nosai de R b 1 0 4 -1 0 7% by weight with respect to open wounds in rats Bok.
  • FIG. 11 is a photograph of RT-PCR instead of a drawing, showing the effect of dihydrozincenoside R t on the expression of VEGF mRNA if the expression of Be 1 —x L mRNA in neurons.
  • FIG. 12 is a photograph of RT-PCR instead of a drawing, showing the effect of dihydroginsenoside Rbi on caspase-3 mRNA expression in nerve cells.
  • FIG. 13 is a diagram showing a part of chemical derivatives that can be prepared by using ginsenoside R b as a lead compound.
  • the first 4 figures Jinsenosai de R bi of by neuronal B e 1 - shows the outgoing current enhancing effect of x L mRNA, a photograph of the place of RT- PCR in the drawings.
  • FIG. 15 is a western blot photograph instead of a drawing, showing the effect of ginsenoside R b on enhancing the expression of Bc 1 -X ⁇ protein in neurons.
  • FIG. 16 is a graph quantifying the results of Western blotting, which shows the action of ginsenoside R b to enhance the expression of Bc 1 -protein in neurons.
  • FIG. 17 is a schematic diagram showing an outline of preparation of Be 1 —x Promo overnight / luciferase plasmid.
  • the first 8 figures Jinsenosai de B e 1 by R bi - is a graph showing changes in x L promoter first active Sunawa Chi STAT 5 activation.
  • FIG. 19 is a photograph of a western plot instead of a drawing, showing the effect of dihydrozincenoside Rb on the expression of Bc 1 -protein in nerve cells.
  • FIG. 20 is a graph quantifying the results of Western blotting, which shows the effect of dihydrozincenoside Rbi on enhancing the expression of Bc1-XL protein in neurons.
  • FIG. 21 is a photograph of RT-PCR instead of a drawing, showing the action of ginsenoside Rbi to enhance VEGF mRNA expression of astrocytes.
  • FIG. 22 is a photograph of RT-PCR instead of a drawing, showing the action of ginsenoside Rbi to enhance the expression of human skin keratinocyte VEGFmRNA.
  • FIG. 23 is a graph showing the effect of ginsenoside Rbi on VEGF protein secretion of human skin keratinocytes.
  • FIG. 24 is a graph showing activation of STA TA5 in ⁇ neurons and the site of the first mouth by ginsenoside R bi.
  • FIG. 25 is a graph showing HIF-1 activation of skin keratinocytes by ginsenoside R b.
  • FIG. 26 is a photograph, instead of a drawing, of RT-PCR showing the effect of dihydrozine cenoside Rt on enhancing the expression of VEGFmRNA by astrocyte.
  • FIG. 27 is a photograph of a western plot, instead of a drawing, showing the action of dihydrozincenoside Rbi to enhance VEGF protein expression in nerve cells.
  • FIG. 28 is a graph quantifying the results of Western blotting, which shows the effect of dihydrozincenoside Rbt to enhance VEGF protein expression ′ in neurons.
  • the present invention relates to a pharmaceutical composition for inducing the expression of a major factor that promotes the formation, regeneration or remodeling of blood vessels, ie, VEGF. More specifically, the present invention relates to ginsenoside derivatives such as dihydro ginsenoside Rbi, Prevention, treatment or treatment of diseases or conditions that cause impaired blood flow, such as occlusion, wounds, or spinal cord injury. For accelerators.
  • the present invention relates to a disease (including a pathological condition) or apoptosis or apoptosis-like cell death that causes blood flow disorder through enhancement of Be1 expression and / or suppression of caspase 3 expression. It also relates to said pharmaceutical composition for the prevention, treatment or treatment.
  • Et al is, the present invention relates to a pharmaceutical composition comprising Jinsenosai earth to induce the expression of B e 1 one x L or VEGF by activating the transcription factor S TAT 5 or transcription factor HIF 1 cells.
  • the present invention relates to a pharmaceutical composition for preventing, treating or treating a disease causing blood flow disorder, which comprises administering a test substance to cultured cells and measuring the expression regulating action of the Be1-2 protein group. It also relates to how to search for things.
  • the dihydrozincenoside R bi of the present invention is represented by the above-mentioned structural formula.
  • the dihydrozincenoside R bi is PCT / JP00 / 04201 or PCTZJP0 / 05555.
  • Ginsenoside R b can be prepared as a raw material according to the method described in No. 4.
  • a ginsenoside derivative such as dihydrozincenoside Rb of the present invention can be used in a free form, but it can also be used with an appropriate salt. They can also be used as solvates such as hydrates thereof.
  • the concentration of ginsenoside derivatives or metabolites thereof such as dihydroginsenoside Rbi of the present invention is preferably low, and more specifically, the concentration of extracellular fluid in the affected tissue is 100 g / m 1.
  • (About 90 ⁇ M) or less preferably 100 ng Zm1 (about 90 nM) or less, more preferably I ng Zml (about 0.9 nM) or less, and still more preferably 10 ng Zml (about 0.9 nM) or less.
  • the density is less than 0 fg / m1 (about 90 fM).
  • a ginsenoside derivative such as dihydrozincenoside Rbi of the present invention
  • the preparation should be prepared so that the concentration of extracellular fluid in the affected area of the patient becomes the above-mentioned concentration. Adjustment is preferred.
  • the pharmaceutical composition and preparation of the present invention can provide a sufficient effect even when the concentration of extracellular fluid in the affected area is about 0.001 to 100 fg / m 1.
  • concentration of natural ginsenosides in the extracellular solution is described in PCT / JP 00/04102 or PCT / JP 00/055 It is described in No. 54.
  • the preparation for intravenous administration containing the ginsenosides of the present invention may be any one which can be directly administered intravascularly, preferably intravenously, and even if it is a single intravenous injection preparation, it may be intravenously administered. It may be a preparation for continuous administration. It may also be a dosage form that can be used by adding to an intravenous formulation such as a point application composition.
  • the pharmaceutical composition of the present invention is preferably a preparation for intravenous administration, an external preparation for skin, or an external preparation for mucosa, preferably a local external preparation for lesions, a local injection for lesions, an oral administration preparation, an intraarticular injection preparation, an intrathecal injection preparation, Intra-arterial preparations, nasal drops, eye drops, eye ointments, ear drops, suppositories (including vaginal suppositories), subcutaneous injections, intradermal injections, intramuscular injections, inhalants, sublinguals, transdermal drugs Any or known administration route can be selected. It may also be used as a sustained release agent.
  • dihydrozincenoside R bi was administered intravenously to reduce cerebral vascular injury, especially the area of cerebral infarction lesions, to about 1Z3 in the non-administration group, and furthermore, the cell death suppressing gene product B It has a unique mechanism of action that enhances c11- ⁇ expression and suppresses caspase-3 expression, and protects all types of cells, especially brain and nerve cells, as well as acute and chronic cerebral infarction. It can also be used as a neuroprotective drug for cerebral hemorrhage, subarachnoid hemorrhage, acute and chronic phases of cerebral embolism, and cerebrovascular disorders such as transient ischemic attacks.
  • ginsenoside derivatives such as dihydroginsenoside Rb, metabolites thereof, and salts thereof are substances that can be intravenously drip-injected into ambulances for patients suspected of having cerebrovascular disorders such as stroke.
  • Natural ginsenosides such as ginsenoside Rbi also have the same 'efficacy' applications as those of ginsenoside derivatives such as dihydrozincenoside Rb, as described in WO 00/374841.
  • the intravenous administration of the dihydrozincenoside Rb of the present invention not only reduces the area of cerebral vascular injury, particularly the cerebral infarction lesion area to about 1Z3, but also reduces the area around the ischemic lesion (is chemi c penumb ra) by intravenous administration. Disruption 'It is said that the reduced vascular network can be returned to almost normal state through enhanced VEGF expression. Therefore, intravenous administration of ginsenoside derivatives such as dihydrozincenoside R bi promotes regeneration or remodeling of the broken or reduced cerebral vascular network after a stroke, and thus, after the end of intravenous administration of the pharmaceutical composition. To make the rescued brain tissue function normally over time Can be.
  • the dihydrozine senoside R bi of the present invention not only has a direct protective effect on nerve cells such as enhancement of Bc 1 _ protein expression, suppression of caspase 3 expression and suppression of apoptosis-like nerve cell death, but also cerebral vascular Regeneration of the blood vessels, including the net-Protecting living tissues such as the brain that has become ischemic through indirect and long-lasting defense mechanisms rather than remodeling.
  • the pharmaceutical composition comprising ginsenosides of the present invention induces the expression of BeI- or VEGF by activating the cell transcription factor STAT5 or transcription factor HIF-1.
  • the pharmaceutical composition of the present invention comprises cytokines capable of activating the transcription factor STAT 5 (interleukin 2, interleukin 3, interleukin 5, interleukin 7, interleukin 9, interleukin 9, inleukin leukin). It can be said that it has the same action as granulocytes, macrophages, colonies (stimulators of erythropoietin), growth hormone, prolactin, etc.
  • the pharmaceutical composition of the present invention is thought to induce the transcription of 0-casein, oncostatin M, and the like, through activation of STAT5.
  • the pharmaceutical composition of the present invention provides not only VEGF but also erythropoietin, transferrin, transferrin receptor, VEGF receptor (FLT-1), glycolysis through activation of the transcription factor HIF-1. It can also be said to induce the expression of various system enzymes, type 1 and type 3 glucose transporters, adenylate kinase 3, hemoxygenase or tyrosine dehydrogenase.
  • ginsenoside derivatives such as dihydrozincenoside Rbi of the present invention have a novel effect of regenerating, regenerating and remodeling blood vessels through enhancement of VEGF expression.
  • Disease, disease or condition eg, transient cerebral ischemic attack, diabetes, heart failure, cardiomyopathy, thrombophlebitis, fracture, neurodegenerative disease, glue Primary disease, cerebrovascular disorder, cerebral hemorrhage, subarachnoid hemorrhage, cerebral infarction, arteriosclerosis, peripheral circulatory failure, immunodeficiency disease, AIDS, myelodysplastic syndrome, osteoporosis, knee osteoarthritis, osteoporosis, Angina pectoris, myocardial infarction, vasculitis, central retinal arteriovenous obstruction, liver, kidney, heart, cerebral ischemia reperfusion injury, vascular injury, poor blood color, coldness, physical fatigue, anorexia, frail constitution, stomach and intestinal weakness , After illness, Behcet's disease,
  • ginsenoside derivatives such as dihydrozincenoside R bi promotes retinal vascular regeneration and reconstruction after photocoagulation.
  • blood flow disorder is the main symptom
  • dihydrozincenoside R b it is an indispensable indication of dihydrozincenoside R b. Therefore, ginsenoside derivatives such as dihydroginsenoside Rb are expected to reduce tissue damage through at least two mechanisms of action even in peripheral blood flow disorders.
  • ginsenosides such as ginsenoside Rbi have similar effects, uses and effects. Furthermore, as described in PCT / JP 00/055554, ginsenosides have an excellent effect in preventing, treating or treating the above-mentioned diseases through the action of promoting the regeneration and reconstruction of living tissues. Is shown.
  • compositions comprising ginsenoside derivatives such as dihydrozincenoside Rbi are used to enhance the expression of cells, particularly neurons, through the enhancement of Be1-X expression and / or suppression of caspase-3 expression.
  • God for one cis or apoptosis Since it suppresses transcellular death, it can be used for prevention, treatment or treatment of any disease (including pathological condition) that causes apoptosis, cell death or apoptosis-like cell death.
  • Polyglutamine disease amyotrophic lateral sclerosis, glaucoma, senile macular degeneration, diabetic retinopathy, central retinal arteriovenous occlusion, retinal detachment, retinitis pigmentosa, AIDS encephalopathy, hepatic encephalopathy, encephalitis, encephalopathy Paralysis, head trauma, spinal cord injury, carbon monoxide poisoning, neonatal asphyxia, peripheral neuropathy, spastic paraplegia, brain tumors, encephalitis, alcoholism, toxic neurological disorders, sphingoribideosis, progressive supranuclear palsy, spinal cord Vascular disorders, mitochondrial encephalomyopathy, meningitis, etc.), stroke, neurotrauma, head trauma, transient ischemic attacks, spinal cord injury, ⁇ Liver ⁇ renal ischemia-reperfusion injury, cardiomyopathy, heart failure, myocardial infarction, angina, peripheral circulatory failure, pressure sores, wound
  • Natural ginsenosides such as ginsenoside Rbi, also have the same 'use' effect.
  • Many of the above-mentioned diseases that cause apoptosis, cell death, or apoptosis-like cell death include blood flow impairment, although the degree of the disease varies to some extent, and therefore, they are included in the category of diseases that cause blood flow impairment. in addition Is also good.
  • dihydrozincenoside Rb was administered intravenously at a daily dose of 6 ig or 0.6 g in a permanent occlusion rat (body weight of about 300 g) in the middle cerebral artery cortical branch (MCA). Reduces the volume of cerebral infarction to about one third of the non-administration group.
  • the dihydrozine senoside Rbi of the present invention shows excellent effects and efficacy on spinal cord injury rats even at a dose of 1.2 ⁇ g Z-day as shown in the Examples below.
  • ginsenoside derivatives such as dihydroginsenoside Rbi, as well as natural ginsenosides such as ginsenoside Rbi
  • spinal cord injury or nerve trauma such as neurology.
  • Secondary tissue degeneration, edema, cerebral edema, edema of neural tissue, apoptosis or apoptotic cell death of oligodendrocyte, demyelination, vascular damage, neurogenic bladder, autonomic dysfunction, sensory disturbance, urination It is useful for the prevention, treatment, and treatment of disorders, defecation disorders, sexual dysfunction, skin ulcers, pressure sores, nerve palsy, and peripheral circulatory failure.
  • the optimal intravenous dose of ginsenoside derivatives for patients with a blood flow disorder of 60 kg or a patient with cell death is estimated Calculated from 0.12 to 1.2 mg per day. Therefore, the daily systemic dose (including oral dose) of the pharmaceutical composition of the present invention to a human or a vertebrate weighing 60 kg depends on the individual differences and the medical condition of the patient. lmg or more, preferably lmg or more, more preferably 10 mg or more.
  • the pharmaceutical composition of the present invention has few side effects, and the upper limit of a systemic dose (including an oral dose) for the prevention, treatment or treatment of the above-mentioned disease or condition can be considerably large.
  • the lower limit of the dose is around 0.01 fg / day, as judged from the effective extracellular fluid concentration. It is.
  • the pharmaceutical composition of the present invention is externally administered locally to a lesion, the dose may be about 1/10 to 1/100 of the above systemic dose.
  • the dose of natural ginsenosides such as ginsenoside Rb is considered to be the same as or about 10 times higher than the dose of ginsenosides.
  • intravenous administration is preferable.
  • the dose can be administered intermittently or continuously.
  • the ginsenoside derivatives described in PCT / JP00Z04102 or PCT / JP0Z0555554 are dihydrozinsenosai HRb which is an active ingredient of the present invention. It can be formulated in the same manner as described above.
  • the water-soluble pharmaceutical composition of the present invention is obtained by dissolving a lyophilized crystal in a biologically (pharmaceutically) acceptable carrier such as physiological saline, distilled water, phosphate buffer and glucose solution. It can be a preparation for intravenous administration.
  • Fat emulsions and ribosome preparations may be used as biologically or pharmaceutically acceptable carriers.
  • concentration of the preparation for intravenous administration can be adjusted to any concentration as long as the concentration is not so high, for example, 0.00000;
  • the dose can be about ⁇ lmg Zml.
  • the dose is less than or equal to lmg, preferably less than 1 / g, more preferably less than 1 ng per day for a human or vertebrate weighing 60 kg. is there.
  • the upper limit of the local dose of a lesion site for the prevention, treatment or treatment of the above-mentioned disease or condition is about 100 mg, preferably about 10 mg per day.
  • Natural ginsenosides such as ginsenoside Rbi can be administered in the same manner.
  • the preparation can be a solid or liquid preparation.
  • Solid preparations include, for example, tablets, pills, powders or granules.
  • the active substance is mixed with a pharmaceutically acceptable carrier, such as sodium bicarbonate, calcium carbonate, potato starch, sucrose, mannitol, carboxymethyl cellulose and the like.
  • a pharmaceutically acceptable carrier such as sodium bicarbonate, calcium carbonate, potato starch, sucrose, mannitol, carboxymethyl cellulose and the like.
  • the preparation operation is carried out according to a conventional method, but may contain additives other than the above-mentioned carriers for preparation, for example, lubricants such as calcium stearate and magnesium stearate.
  • Enteric substances such as cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, polyvinyl alcohol phthalate, styrene maleic anhydride copolymer or methacrylic acid, methyl methacrylate copolymer may be added to the above solid preparations.
  • An enteric preparation can be obtained by spraying a solution or an aqueous solution of the above with an organic solvent to apply an enteric coating.
  • Solid preparations such as powders and granules can be enclosed in enteric capsules.
  • Liquid preparations for oral administration include, for example, emulsions, solutions, suspensions, syrups or elixirs. These preparations contain commonly used pharmaceutically acceptable carriers, such as water or liquid paraffin.
  • An oily base such as coconut oil, fractionated coconut oil, soybean oil, and corn oil can also be used as a carrier.
  • Pharmaceutically acceptable carriers include other adjuvants, flavoring agents, stabilizing agents, or preservatives that are commonly used as needed.
  • Liquid preparations may also be administered in capsules made of a substance that can be absorbed, such as gelatin.
  • Solid preparations for vaginal or rectal administration include suppositories containing the active substance and produced by known methods.
  • a suppository containing such ginsenosides may be used as an external preparation for mucosa or skin for prevention, treatment or treatment of diseases causing blood flow disorders such as hemorrhoids in the rectum or anus, It may be used as a systemic agent for the prevention, treatment or treatment of diseases or conditions that cause impaired blood flow.
  • Preparations for parenteral administration containing ginsenoside derivatives are administered as sterile aqueous or non-aqueous solutions, suspensions or emulsions.
  • Non-aqueous solutions or suspending agents include, for example, propyl glycol, polyethylene glycol, vegetable oils such as olive oil or soybean oil, and injectable organic esters such as ethyl ethyl oleate. I do.
  • Such formulations may also contain adjuvants such as preserving, wetting, emulsifying, dispersing, and stabilizing agents.
  • adjuvants such as preserving, wetting, emulsifying, dispersing, and stabilizing agents.
  • These solutions, suspensions, and emulsions can be sterilized by appropriate treatment, such as filtration through a bacteria-retaining filter, heating, blending of a bactericide, or irradiation with ultraviolet light.
  • a sterile solid preparation can be produced and dissolved in sterile water or a sterile injectable solvent immediately before use.
  • water is added to a homogeneous solution of a vegetable oil such as soybean oil, a phospholipid such as citylene, and the ginsenoside derivative used in the present invention.
  • a homogenized fat emulsion can also be used as an injection.
  • the natural ginsenosides, particularly ginsenoside Rbi described in PCT / JP 00/055554 and PCTZ
  • Ginsenoside derivatives such as dihydrozincenoside Rbi are used in the same manner as for ginsenoside Rt described in WO 00/37848, as in keratinocyte culture for skin transplantation. It is also effective in protecting, preserving, and maintaining nutrition sheets.
  • the preservation and maintenance of cells for the preparation of cultured skin the preservation and maintenance of stem cells for the preparation of artificial organs, and organs for transplantation ⁇ tissues or cells (liver, kidney, heart, It is considered to be useful for preservation and maintenance of lungs, lungs, meninges, bones, joints, ligaments, digestive tract, cornea, skin, blood vessels, peripheral nerves, etc.
  • ginsenoside derivatives such as dihydrozincenoside Rbi are disclosed in Japanese Patent Application No. 2000-004 / 203 or Japanese Patent Application No. 2000-014969.
  • blood cell components for blood transfusion, platelet preservation and maintenance, and frozen cells sperm, eggs, skin keratinocytes, cord blood, ES cells, fertilized eggs, stem cells, etc.
  • frozen cells sperm, eggs, skin keratinocytes, cord blood, ES cells, fertilized eggs, stem cells, etc.
  • the present inventors have proposed that dihydrozincenoside Rbi or a metabolite thereof or a salt thereof, which is newly obtained by chemically modifying ginsenoside Rb, regenerates and regenerates blood vessels through a VEGF expression enhancing action.
  • the present invention has been found for the first time to exhibit a construction promoting action, and therefore, the present invention seeks to use ginsenoside Rb or a metabolite thereof for other active ingredients for preventing, treating or treating diseases that cause impaired blood flow.
  • a ginsenoside derivative such as dihydrozincenoside Rbi
  • an arbitrary or known administration route can be selected.
  • examples of such a prodrug include, for example, those obtained by esterifying the hydroxyl group of a ginsenoside derivative, but are not limited thereto.
  • the blood flow disorder (including the pathological condition) in this specification may be caused by, for example, damage to a living tissue, a wound, a trauma, a burn or a defect, or an external physical or chemical force.
  • the disorder may be caused by any cause such as a pathological one.
  • poor blood color, coldness, hypotension, and physical fatigue are also included in the pathological conditions caused by impaired blood flow.
  • the “ginsenosides” of the present invention are compounds called natural ginsenosides, such as ginsenoside R bi, which is a component of ginseng, and contain ginsenosides. Ginseng or other natural products or extracts, extracts, fractionation components, or purified fractions thereof. (Ginsenosides) may be a compound derived by chemically modifying ginsenosides by chemical means (ie, a ginsenoside derivative). As the “ginsenosides” or naturally occurring ginsenoside compounds in the present invention, ⁇ includes, for example, the following.
  • Jinsenosai de R o (ginsenoside Ro; tick set saponin V; chik use t susaponin V; it ⁇ n A; saponin A), Jinsenosai de R ai (ginsenos i de Rai) , Jinsenosai de R a 2 (ginsenoside Ra 2 ), Jinsenosai de R bi (g insenoside Rbi; saponin D (saponin D), Jinsenosai de R b 2 (ginsenosid e Rb 2 ), Jinsenosai de R b 3 (ginsenoside Rb 3) , Jinsenosai de R c (gin senoside Rc) Ginsenoside Rd, ginsenoside Re
  • ginsenoside Re i ⁇ FR a 3 (ginsenoside Ra3); Bruno Tojinseno Sai de R 4 (notoginsenos ide R 4) ; Kinkenosai de (kinkenoside Ri); di Nsenosai de R s 1 (ginsenos ide Rs i ); Jinsenosai de R s 2 (ginsenoside R & ⁇ ); (20 s) — ginsenoside R g 3 (20 s- ginsenoside Rg 3 ); 20 — dalco ginsenoside R f (20-glucoginsenoside Ri); ginsenoside Rf (ginsenoside Rf); (2 OR) — ginsenoside R g 2 (20R-ginsenoside Rg 2 ); (20 R) ginsenoside R hi (20R-ginsenoside Rhi); ginsenoside Gf (ginsenoside Rf); ginsenoside R gi (ginsenos ide Rgi
  • the natural product such as ginseng containing the ginsenoside compound or an extract, extract, fraction component, or purified fraction thereof may be the ginsenoside described above.
  • Any natural product containing a relatively large amount of the compound may be used.
  • the natural product may be used as it is, or an extract obtained by extracting and concentrating a component containing a ginsenoside compound may be used. It may be an extract or tablet prepared by formulating the extract in liquid or solid form, and may be a fraction containing a ginsenoside compound obtained by purifying and separating the extract, such as a saponin fraction.
  • the ginsenoside compound-containing fraction may be purified to give a ginsenoside compound as a main component.
  • Preferred natural products such as ginseng containing ginsenoside compounds or extracts, extracts, fractionation components or purified fractions thereof include, for example, ginseng, ginseng extract, and medicinal products.
  • the crude saponin fraction of ginseng can be mentioned.
  • the ginsenosides of the present invention can be used not only as a pharmaceutical composition, but also as a cosmetic composition, a composition for chemical peeling, a composition for hair growth and hair growth, a composition for external mucosa, and a composition for regulating the growth of animals and plants. It has been found in PCT / JP 00/055554 that it can also be used.
  • ginsenosides are also used as a composition for physical pigmenting, a composition for food for specified health use, a composition for OTC preparations, or a composition for dietary supplements.
  • the pharmaceutical composition of the present invention is used as a composition of an OTC preparation, it is effective for weak constitution, physical fatigue, illness, gastrointestinal weakness, anorexia, poor blood color, and coldness.
  • the composition of the OTC preparation is also included in the pharmaceutical composition.
  • a chemical peeling composition or a physical peeling composition composed of ginsenosides may be added to the peeling site immediately after the peeling, as described in PCTZ JP 0/0555554.
  • the topical administration of a particularly low concentration promotes regeneration of skin tissue at the peeling site.
  • the composition for chemical peeling or the composition for physical peeling of the present invention may be administered externally before or during peeling.
  • ginsenoside derivatives As a compound derived by chemically modifying ginsenoside compounds such as naturally occurring ginsenoside R bi by chemical means, the chemical structure of the natural ginsenosides described above was modified in the following manner. (In the present specification, these compounds are referred to as “ginsenoside derivatives”.)
  • a double bond in the side chain is cleaved to give an aldehyde end
  • Acylation or acetylation An arbitrary functional group such as an alkyl group or a aryl group bonded to the end of the side chain in addition to cetylation.
  • a double bond in the side chain is cleaved to form a carboxyl group.
  • a double bond in the side chain is cleaved to bind to an arbitrary functional group such as a carboxyl group or an aldehyde group.
  • One methyl group at the side chain terminal is A hydrogen atom, and the other methyl group substituted with an arbitrary functional group such as an alkyl group or a aryl group.
  • a double bond in the side chain is converted into a single bond, and the arbitrary functional group For example, one having one or more hydroxyl groups bonded thereto, or one obtained by dehydrating and epoxidizing two molecular hydroxyl groups, (10) a Diels-Alder using a diene compound such as cyclopentene at the double bond of the side chain.
  • Te is PCT / JP 0 0/0 4 1 0 2
  • dihydric Dorojinsenosai de R b 1 neuronal protective effect which is one of the Jinsenosai earth derivatives, which describes the preparation method as well as NMR chart, etc. I have.
  • the above-mentioned ginsenoside derivatives are described in PCT / JP 00/04102, PCT / JP 00/0555554, and Japanese Patent Application No. 2004-17034509. It is considered that all of the effects, indications, and uses of ginsenoside Rb or dihydrozinesenoside Rbi described in Japanese Patent Application No. 2000-40032 are combined. It should be noted that an attempt to create a compound having a new medicinal effect by using a natural ginsenoside, which is difficult to synthesize at present, as a lead compound was first performed by the present inventors.
  • oleanolic acid having a slightly different chemical structure for example, ginsenoside Ro (chixessaponin V)
  • ginsenoside Ro chixessaponin V
  • a steroid-like skeleton of ginsenosides oleanoic acid
  • a substance obtained by reducing one double bond existing in the chemical structure of aglycone so-called dihydrozinenosides
  • a hydrogen atom at the reduction site substituted with an arbitrary functional group (for example, a hydroxyl group, an alkyl group, an aryl group, etc.), (3) an esterified propyloxyl group, and (4) an acylated or acetylated hydroxyl group.
  • the ginsenoside derivatives described above or their stereoisomers are considered to have a common effect 'efficacy' because they have similar chemical structures to each other, and therefore can be used alone in the present invention. Alternatively, it can be used simultaneously in combination with a plurality of different ginsenoside derivatives or ginsenosides.
  • the metabolite of the ginsenoside derivative of the present invention is a compound produced as a result of metabolism of the ginsenoside derivative of the present invention in a living body, in particular, a product in which a sugar chain is cleaved.
  • the components are not limited to the above-mentioned ginsenoside derivatives, but are metabolites of these in vivo, and have the object of the present invention.
  • the pharmaceutical composition of the present invention can of course be used as a veterinary pharmaceutical composition for vertebrates, but in this specification, the expression "veterinary pharmaceutical composition" will be omitted.
  • the pharmaceutical composition of the present invention may be used as a veterinary pharmaceutical composition for invertebrates such as fish, shrimp, oysters and shellfish.
  • the pharmaceutical compositions of the present invention include veterinary pharmaceutical compositions.
  • the low-dose and low-concentration ginsenosides of the present invention have a therapeutic effect on cerebrovascular disorders, a blood vessel regeneration / reconstruction promoting effect, a wound therapeutic effect, a spinal cord injury therapeutic effect, a VEGF expression enhancing effect, Bc1—X.
  • the expression enhancing effect, caspase 3 expression suppressing effect, transcription factor STAT5 • HIF-1 activating effect, etc. will be described in detail based on specific examples. For this reason, description will be made based on experimental results using dihydrozincenoside R, which is a typical ginsenoside derivative of the present invention, and ginsenoside Rbi, which is a typical natural ginsenoside.
  • the present inventors first prepared dihydrozincenoside Rbi.
  • dihydrozincenoside Rbi The production examples and NMR data of dihydrozincenoside Rbi are shown below.
  • 10 mg of 10% Pd / C palladium charcoal
  • 1 ml of methanol (special grade) is added and suspended.
  • the present inventors first examined the concentration at which the dihydrozincenoside R bi obtained by the above-mentioned method exerts a favorable effect on cells. For this reason, the present inventors have developed apoptosis or apoptosis-like neurons in cultured neurons. The concentration at which death was suppressed by dihydrozincenoside R b was examined.
  • the present inventors (Sakanaka, Tanaka) show that short-term exposure of cultured neurons to the nitric oxide donor, nitropurs sodium (SNP), can result in neuronal apoptosis or apoptotic neuronal death. (Toku K. et al., J. Neurosci. Res., 53, 415-425, 1998).
  • ginsenoside Rb has an apoptosis-like or apoptosis-like neuron at an optimal extracellular solution concentration of 110 fg / m1. It has been found that deterrence can be suppressed (WO 0 Z 37481). Therefore, the neuroprotective effect of dihydrozine senoside Rb was examined using a similar experimental system.
  • Nerve cells were isolated from the fetal cerebral cortex of a 17-day-old rat fetal cerebral cortex using trypsin EDTA and plated on a polyellidine-coated 24-well plate. After culturing for 16 hours in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, the culture solution is subjected to neurons containing insulin, transferrin, etc. The medium was replaced with a serum-free medium for culture and cultured for 3 to 4 days. On day 3 or 4 of culture, sodium nitroprusside (SNP) was added at a concentration of 300 M and incubated for 10 minutes.
  • DMEM Dulbecco's modified Eagle's medium
  • SNP sodium nitroprusside
  • FIG. 2 is a photograph instead of a drawing, showing the result of the immunoblot of microtuble-associated protein 2 ( ⁇ ⁇ AP 2).
  • the first lane from the left is the control cultured neurons, with a clear MAP 2 band. (Ie, a marker band of nerve cells). SNP treatment caused many neurons to undergo apoptosis or apoptosis-like neuronal death, so the MAP2 band was clearly weakened, as in the second lane from the left.
  • dihydrozincenoside Rb in the optimal extracellular fluid concentration range of 0.01 fg / ml to lng / m1 significantly suppressed apoptosis of neurons or apoptosis-like neuronal death It turned out to be. That is, dihydrozincenoside R bi has a favorable anti-cellular activity, particularly against nerve cells, in an optimal extracellular solution concentration range which is considerably wider than that of ginsenoside R b described in WO 00/37481. It is thought to exert an apoptotic effect.
  • ginsenoside derivatives such as dihydrozincenoside Rb also promote nervous tissue regeneration and / or remodeling by extending neurites.
  • ginsenoside derivatives such as dihydrozincenoside Rbi have an extracellular solution concentration of 100 g / m1 (about 90 M) or less, preferably 100 ng / m1 (about 90 nM) or less, more preferably I n gZm l (about 0.9 n M) or less, and even more preferably 0.OOOO li gZm l (about 0.0 0 0 0 0 9 f M) to 100 It is considered that by suppressing the apoptosis or apoptosis-like cell death of the cells at the rate of 0.000 fg / m 1 (about 900 fM), an excellent cytoprotective action is exhibited.
  • Ginsenoside derivatives such as dihydrozincenoside Rbi are thought to protect cells of living tissues exposed to impaired blood flow through anti-apoptotic action.
  • Ginsenoside derivatives such as dihydrozincenoside Rbi are used for transplantation tissues ⁇ organ cells (stem cells, fertilized eggs, embryos, ES cells, skin keratinocytes, etc.), cells derived from any tissue, frozen cells for transplantation, transplantation It is also considered useful for protecting or preserving frozen tissues for use, frozen organs for transplantation, blood cell components for blood transfusion, platelets, and germ cells (frozen ovum, frozen sperm, frozen fertilized egg).
  • * In Fig. 3 indicates P ⁇ 0. 001 indicates **, and ** indicates P ⁇ 0.0001.
  • Statistical analysis is based on the post hoc test of 8 1 ⁇ 0 + 3 (; 116 ⁇ 6.
  • ginsenoside derivatives such as dihydrozincenoside Rb can be used for cells, particularly neuronal cells, in an optimal extracellular concentration range that is considerably wider than ginsenoside Rbi described in WO 00/37481. Inhibition of apoptosis or apoptosis-like neuronal cell death has been shown in an in vitro experimental system, but dihydrozine cenoside R has also been demonstrated in an in vivo animal experimental system that actually impairs blood flow. The present inventor next examined whether or not ginsenoside derivatives such as b show excellent effects similarly to the ginsenoside R bi described in WO 00/37481.
  • cerebrovascular disorder is selected as a disease or condition causing blood flow disorder as follows, and intravenous administration of dihydrozincenoside Rbt, which is one of the ginsenoside derivatives, is effective for cerebrovascular disorder, especially for cerebral infarction. I checked whether it was effective for treatment.
  • SH spontaneously hypertensive rats
  • MCA left middle cerebral artery cortical branch
  • FIG. 4 shows two cases of administration of saline
  • Fig. 5 shows dihydrozine cenoside R bi S Two cases in which g / day was administered intravenously are shown.
  • the results are shown in FIG.
  • Fig. 6 compared with the cerebral infarction area in the cerebral infarction group given vehicle (saline), dihydrogen senoside R b!
  • the cerebral infarction area (infarct area) of the treated cerebral infarction group was reduced to about one third.
  • the ** mark indicates 0.01 according to the Mann-Whitney test.
  • the therapeutic effect of dihydrozincenoside Rb on cerebrovascular disorders is superior to the effect of ginsenoside Rbi disclosed in WO 00/37481.
  • ginsenoside derivatives such as dihydroginsenoside Rbi are effective and efficacious in diseases or conditions that cause impaired blood flow such as cerebrovascular disorder and cerebral infarction.
  • ginsenoside derivatives such as dihydrozincenoside R bi promote regeneration and / or remodeling of cerebral blood vessels, similarly to ginsenoside R bi described in WO 0 Z4 686. .
  • the present inventor further doubled the intravenous dose of dihydrozine cenoside Rb (single dose of 12 ig and then dosed on the following day) to determine whether a similar effect for treating cerebral infarction could be obtained.
  • ginsenoside R bi showed an excellent therapeutic effect on cerebral infarction even in a 6-day dose to SH-SP rat with a body weight of about 300 g in WO 00/374814.
  • ginsenoside derivatives such as dihydrozincenoside Rb do not necessarily exert a cerebral infarction treatment effect and / or a cerebral blood vessel regeneration / remodeling promotion effect at such a high dose.
  • dihydrozincenoside R for cerebral infarction rats weighing about 300 g is lower than the optimal dose of ginsenoside Rb. It was considered to be less than 0 z gZ days, preferably less than / day.
  • dihydroginsenoside Rb inhibits neuronal apoptosis or apoptosis-like neuronal death in a broader concentration range than ginsenoside Rb, but in vivo (in vivo).
  • Dihydrozincenoside Rb has excellent cerebral infarction treatment effect and cerebral blood vessel regeneration / reconstruction in the same dosage amount as ginsenoside Rbt or as low as 1/10 to 1/100 It can be said that it exerts a promoting effect.
  • other ginsenoside derivatives such as dihydroxyxenosenoside R bi or epoxy ginsenoside R bi may be used at a dose and concentration that is equal to or approximately 100 times higher than ginsenoside Rb. Effect similar to b ⁇ Efficacy is expected.
  • the present inventors have further investigated whether low doses of dihydrozine cenoside Rb also have a favorable effect on neurological trauma, such as spinal cord injury, which causes impaired blood flow.
  • neurological trauma such as spinal cord injury and head trauma also results in impaired blood flow due to rupture of blood vessels and edema of brain and spinal cord tissues, resulting in irreversible higher-order neuropathy. Therefore, the present inventors have taken spinal cord injury as one of the diseases causing blood flow disorder and decided to examine the effect of dihydrozine cenoside Rbi on spinal cord injury. For this reason, the following is an experimental example in which dihydrozine cenoside Rb was infused intravenously into a spinal cord injury rat (body weight: about 300 g) for 7 days at a dose of 1.2 gZ days.
  • Figs. 7 and 8 show the saline administration rats on the second day after spinal cord injury
  • the right photographs in Figs. 7 and 8 show dihydrozincenoside Rb1.2 at the same time. g / day) dosing rate is indicated.
  • the saline administration rat with 20 g of pressure applied to the lower thoracic spinal cord for 20 minutes was used not only on the day of spinal cord injury but also after spinal cord injury. He also exhibited paraplegia on both legs during the day.
  • dihydrozincenoside Rt administered intravenously at a dose of 1.2 ⁇ g / day
  • the paraplegia of the lower limb was reduced on the day of spinal cord injury.
  • paraplegia of both lower limbs improved remarkably, and the rat was able to stand up while holding on to an object, as shown in the right photographs in Figs. 7 and 8.
  • Dihydrozincenoside Rbt at a dose of 8 g / day, 16 iZg / day or 60 / _t g / day administered intravenously to rats with spinal cord injury did not show any significant effect. .
  • ginsenoside derivatives such as dihydrozincenoside R bi are significantly superior to spinal cord injuries even when compared to ginsenoside R b described in WO 00/46808. ⁇ It has been found to be effective in treating nerve trauma. Furthermore, the optimal dose of dihydrozincenoside Rb, for a spinal cord injury rat weighing 300 g, was considered to be around 1.2 ig to 2.4 ⁇ g / day or less.
  • dihydrozincenoside Rb when dihydrozincenoside Rb is used as a pharmaceutical composition for treating nerve trauma, head trauma, and spinal cord injury, the optimal dose is WO 0 Z 4 680 or PC TZ JP 0 Optimal dosage of ginsenoside Rbi described in 0/04201
  • dihydrozinc senoside R bi can be produced from high-purity ginsenoside R bi at a 97% yield, so that dihydrozin senoside R b is more efficient than ginsenoside R bi. It can be used to prevent, treat, and treat neurological trauma, stroke, and other brain disorders that cause blood flow disorders.
  • ginsenoside derivatives such as dihydrozincenoside Rbi indicate that blood flow disorders are caused by diseases such as cerebrovascular disorders and spinal cord injuries. It has been invented to be a pharmaceutical composition for prevention, treatment or therapy. Possibly, Jihi Drosin Senoside R b! Derivatives such as ginsenoside derivatives, which inhibit cell apoptosis or apoptotic cell death and promote vascular regeneration / remodeling, can cause blood flow disorders (eg, cerebrovascular disorders, head trauma) , Nerve trauma, spinal cord injury, etc.).
  • ginsenoside derivatives such as dihydridine senoside Rb will be added.
  • a spinal cord injury occurs due to a pressure load on a segment of the spinal cord, for example, the lower thoracic cord, not only the gray matter nerve cells in the area but also the white matter pathway in the area are damaged.
  • the white matter pathway consists of the nerve cell processes (ie, axons or dendrites) and the myelin from oligodendrocytes that insulates them (myelin).
  • Impairment of the white matter pathway further extends to the distal (caudal) side, and secondary degeneration or apoptosis of the cell originating the pathway, ie, the upper nerve cell body that projects fibers into the pathway. causes cis-like cell death.
  • impairment of the white matter pathways of the lower thoracic spine under pressure overload is caused by the origin of the pathways and the pathways below the lower thoracic spinal cord (ie lumbar spinal cord, sacral medulla).
  • spinal cord injury in addition to the above-mentioned disorders specific to nerve tissue, blood flow disorders, and blood vessel injuries, neuropathic bladder, cerebral edema, nerve tissue edema, edema, dysuria, Defecation disorders, sexual dysfunction, skin ulcers, pressure sores, etc. occur. Many of these diseases, symptoms, or conditions are caused by spinal cord injuries that cause damage to not only motor nerves, but also autonomic nerves and sensory nerves, resulting in dysfunction and impaired blood flow in the area. Conceivable. It is also known that vascular damage and edema easily occur when nerve tissue (spinal tissue) is subjected to excessive mechanical or physical pressure. The above-mentioned symptoms, diseases, lesions or conditions associated with spinal cord injury are also found in head trauma to varying degrees.
  • ginsenoside derivatives such as dihydrozinosenoside Rb are WO 0 Spinal cord injury at doses / concentrations equivalent to or wider than the ginsenoside R bi described in 0/4866 08 It is considered to be useful for the prevention, treatment and treatment of the above-mentioned conditions, symptoms and diseases caused by wounds and nerve trauma (including head trauma).
  • Ginsenoside derivatives such as dihydroginsenoside Rb ⁇ are expected to be indicated for the following conditions, symptoms, and diseases: secondary degeneration of nerve tissue, edema, brain edema, edema of nerve tissue, oligodendrocyte Apoptosis or apoptosis-like cell death, demyelination, vascular damage, neurogenic bladder, autonomic nervous disorder, sensory disturbance, dysuria, defecation dysfunction, sexual dysfunction, skin ulcer, pressure sore, palsy, Peripheral circulatory failure and the like.
  • ginsenoside derivatives such as dihydrozincenoside Rbi, as well as natural ginsenosides such as ginsenoside Rbi, regenerate and regenerate central nervous tissue and regenerate and remodel cerebral spinal cord blood vessels. It is thought that it exerts its effects and effects on the above-mentioned conditions, symptoms or diseases through its anti-apoptotic action.
  • intravenous administration of a ginsenoside derivative such as dihydrozincenoside Rb of the present invention shows a novel effect of regenerating and reconstructing a blood vessel or a nerve tissue.
  • Diseases that cause alterations, damage to blood vessels, or diseases or conditions that are primarily caused by impaired blood flow e.g., transient cerebral ischemia, aortitis syndrome, diabetes, cancer, malignancy, sarcoma, Malignant neoplasm, heart failure, cardiomyopathy, acute peripheral arterial occlusion, DIC, thrombosis, thrombophlebitis, collagen disease, cerebrovascular disease, cerebral bleeding, subarachnoid hemorrhage, cerebral infarction, atherosclerosis, peripheral Circulatory insufficiency, chills, poor blood color, physical fatigue, menopause, cyanosis, gastrointestinal weakness, anorexia, alopecia, subdural hematoma, epidural hematoma, obstructive thromboang
  • ginsenoside derivatives such as dihydrozincenoside Rbt reduce tissue cell damage in peripheral blood flow disorders, injuries, trauma or wounds through at least two mechanisms of action.
  • Natural ginsenosides such as ginsenoside Rbi can also be used as a pharmaceutical composition for preventing, treating or treating the above-mentioned diseases and conditions.
  • a pharmaceutical composition comprising a ginsenoside derivative, such as ginsenoside derivative, suppresses secondary nerve lesions and secondary lesions in a region of the brain having synaptic communication, so Degenerative disease, peripheral neuropathy, demyelinating disease, inflammatory brain neurological disease, toxic brain neurological disease, cerebrospinal vascular disease (e.g.
  • Alzheimer's disease Pick's disease, progressive supranuclear palsy, spinocerebellar degeneration, Parkinson's disease, chorea, polyglutamine disease, carbon monoxide poisoning, cerebral palsy, neonatal asphyxia, hypoxic encephalopathy, AIDS encephalopathy, encephalitis, acute sporadic encephalomyelitis, acute cerebellar encephalitis, transverse myelitis, amyotrophic side It is also effective for secondary lesions such as chordosis sclerosis and multiple sclerosis, and slows the progression of higher nervous dysfunction due to these diseases. of Life) can be increased.
  • these brain neurological diseases include those described in a compendium (Handbook of Neurology, Differential Diagnosis and Treatment, Second Edition, Edited by Mikuni Mikuni, Medical Shoin 1999).
  • P As described in CT / JP 0 0 Z 0 410 102 (a brain cell or ginseng protective agent consisting of ginseng), these brain-neurotic diseases are mediated through an apoptotic-like nerve cell death inhibitory effect. It is thought that ginsenoside derivatives such as dihydroginsenoside Rbt also exert an effect on lesions.
  • a ginsenoside derivative such as dihydrozincenoside Rbi of the present invention is considered to significantly improve paralysis in spinal cord-injured animals.
  • nervous tissue is the most vulnerable to trauma compared to other peripheral tissues, so dihydrozincenoside R b!
  • Pharmaceutical compositions consisting of ginsenoside derivatives such as dihydrozinoseside Rbi indicate that pharmaceutical compositions comprising ginsenoside derivatives such as dihydrozinoseside Rbi are injured in peripheral tissues other than central nervous tissue. It is also effective on wounds (including burns, frostbite, electrolysis, radiation damage, laser damage, ultraviolet light damage, and internal organ damage).
  • wounds including burns, frostbite, electrolysis, radiation damage, laser damage, ultraviolet light damage, and internal organ damage.
  • these peripheral tissue traumas are also included in diseases and conditions that cause impaired blood flow.
  • ginsenoside derivatives such as dihydrozincenoside Rt also have an effect on peripheral tissue diseases that cause impaired blood flow due to disruption or cutting of blood vessels.
  • wounds particularly open wounds, were selected as diseases in which blood vessels were ruptured or severed, and the therapeutic effect of dihydrozincenoside Rbi on open wounds was examined.
  • each open wounds, dihydric Doroji Nsenosai de R bi respectively 0.0 0 0 1 wt% (1 0 4% by weight), 0.0 0 0 0 1 wt% (I t) - 5 wt%) , 0.0 0 0 0 0 1 wt% (1 0 6 wt%), 0.0 0 0 0 0 1 wt% (1 0-7 wt%), Purobe bets (ophthalmic containing a concentration of White petrolatum) was applied once a day 0.1 g for 9 days. Only the same amount of the plot was externally applied to the control. Immediately after the animal was euthanized by anesthesia, the wound skin was collected and photographed.
  • FIG. Figure 9 is a photograph replacing the drawing.
  • four examples are shown, and a first example, a second example, a third example, and a fourth example are shown from above.
  • the concentration of the composition in the external preparation is 0.01% by weight or less, preferably less than 0.01% by weight.
  • 0 0 0 1 wt% or less, more preferably 0. 0 0 0 0 0 0 1 wt% (1 0-7 wt%) were considered to be preferable to set the longitudinal or less. Therefore, for the prevention, treatment or treatment of diseases or conditions that cause impaired blood flow (for example, wounds, burns, pressure sores, skin ulcers, hemorrhoids, etc.), it consists of ginsenoside derivatives such as dihydroginsenoside R bi.
  • the concentration in the external preparation for skin or the topical preparation is less than or equal to 0.001% by weight, preferably less than 0.01% by weight. It is preferably set to not more than 0.001% by weight (100 to 15 % by weight), more preferably not more than 0.001% by weight (10 to 7 % by weight).
  • the upper limit of the concentration of ginsenoside derivatives in external skin preparations and topical administration (including external mucosal preparations and suppositories) for the prevention, treatment or treatment of the above-mentioned diseases is 5% by weight or less, preferably 1% by weight or less. And more preferably 0.1% by weight or less.
  • ginsenoside derivatives such as dihydrozincenoside Rb have an extracellular fluid concentration of the affected tissue of less than 100 ⁇ g / m1, preferably 100 ng / m 1 or less, more preferably 1 ng / m 1 or less, and still more preferably 0.0000.
  • compositions of the present invention are believed to be able to accomplish these complex events reliably and quickly.
  • ginsenoside derivatives such as dihydroginsenoside Rbi may be useful for the prevention, treatment, and treatment of malignant neoplasms (including cancer and sarcoma), if the action of promoting the remodeling of blood vessels is selectively exerted.
  • malignant neoplasms including cancer and sarcoma
  • natural ginsenosides such as ginsenoside Rbi have similar effects, efficacy and uses.
  • Any organ or tissue that has caused damage or histopathological changes (cornea, oral mucosa, outer ear, gastrointestinal mucosa, nasal mucosa, eardrum, vagina, bladder, uterus, urethra, airway mucosa, rectum, rectal mucosa, anus, etc. ) Can promote the regeneration and reconstruction of vascular and other diseased tissues.
  • suppositories containing ginsenosides at low concentrations may be used for the prevention, treatment or treatment of hemorrhoids. Or, if applied externally to the rectum (mucosa), excellent effects can be obtained. From the results of this experiment, it was found that the amount of ginsenoside derivatives, especially dihydrogin genoside Rbi, per 10 g of the probe was 0.1 mg or less, and preferably 0.001 mg or less. . That is, the optimal skin external dose of ginsenoside derivatives, particularly dihydrozinsenoside Rbi, to humans or vertebrates having skin diseases is quite small.
  • ginsenoside derivatives such as dihydrozincenoside Rbi are mixed per 10 g of the protolyte for the purpose of prevention, treatment or treatment of the above-mentioned disease
  • the upper limit is 1 g or less, preferably 0.1 g or less, Preferably it is less than 0.000 lg.
  • Concentrations when natural ginsenosides such as ginsenoside Rb are used as an external preparation for skin and a mucosal preparation are described in PCT / JP00 / 0555554.
  • ginsenoside derivatives such as dihydrozincenoside Rbi to the skin promotes regeneration and reconstruction of cut blood vessels, as well as epidermal tissues of the skin, connective tissues of the dermis, papillae of the dermis, and subcutaneous
  • tissue, blood vessels, pilates, sebaceous glands, sweat glands, dermal papilla, hair follicles, etc. are promoted to be regenerated and reconstructed is, of course, the topical application of ginsenoside derivatives such as dihydrozine senoside R bi to skin.
  • Achieving blood vessel and skin tissue regeneration / reconstruction or wound healing in a vivid way alone means that cytokines involved in blood vessel regeneration / reconstruction and skin tissue wound healing / regeneration / reconstruction / reconstruction Growth factors or growth factors and their receptors and transcription factors (eg, EGF, TGF-j31, SCF, TGF-a, FGF, VEGF, angiopoietin, Tie_l, Tie — 2, ephrin— B2, Eph4B, CXCR4, PDGF_BB, TGF—] 31, PDGF-AB, IGF, She, SCL, ets—10, NDF, transcription Factor HIF-1, FKB, VEGFR, transcription factor STATs, EGFR, HGFR, TGFR, IGFR, KGF, HGF, erythropoietin, PDGF, PDGFR, TGF—32 , TGF-03, FGF-2, U-PA, t-PA, etc.) and low-dose gins
  • vascular regeneration and remodeling phenomena can be achieved by dividing, proliferating, migrating, migrating, differentiating, adhering, and forming lumens of vascular endothelial cells, regenerating and remodeling extracellular matrix, intima, Complicated processes such as regeneration and reconstruction of the basal, medial, adventitia, and autonomic nerves must be performed, but ginsenoside derivatives such as dihydroginsenoside Rb cause these vital phenomena. It is thought that it can be adjusted systematically. That is, it is considered that ginsenoside derivatives such as dihydrozincenoside Rbt promote regeneration and reconstruction of all cells constituting blood vessels and skin tissues and secretions thereof.
  • ginsenoside derivatives such as dihydroginsenoside Rbi are useful for treating biological tissue, especially blood vessel or skin tissue, against organic diseases that cause histopathological changes in living tissues such as skin and diseases that cause impaired blood flow. It can be said that it exerts its effects and effects through the action of promoting regeneration and reconstruction.
  • the diseases that cause such impaired blood flow of the skin or histopathological changes include the following.
  • ginsenoside derivatives such as dihydrozincenoside Rb are particularly suitable for ginsenosides.
  • natural ginsenosides such as ginsenoside Rbi or natural products containing ginsenoside Rbi are used in the present invention. It may be used instead of a pharmaceutical composition (ie, a ginsenoside derivative such as dihydrozincenoside R b).
  • a natural ginsenoside such as ginsenoside R b, or a natural product containing ginsenoside R bt or an extract thereof is used as a pharmaceutical composition for preventing, treating or treating diseases or conditions that cause impaired blood flow.
  • the concentration is preferably set to less than 0.001% by weight.
  • the concentration of ginsenosides such as ginsenoside Rbi is less than 0.0002% by weight. It is preferable to set Of course, the concentration of ginsenoside derivatives such as dihydrozincenoside Rb is also 0. It may be set to less than 0.002% by weight.
  • Natural ginsenoside compounds such as ginsenoside Rt or ginsenosides have an extracellular fluid concentration of 10 ng / m1 (about 9 nM) or less, preferably lng / ml (about 0.9 n M) Below, more preferably from 0.01 to 100 fg / ml or from 1 to L: 0.000 fg / ml to promote regeneration and reconstruction of blood vessels.
  • ginsenoside R b It is said that natural ginsenosides such as and the like promote the regeneration and remodeling of tissues such as blood vessels by increasing, decreasing or regulating the molecular groups involved in tissue regeneration and remodeling as described above.
  • the pharmaceutical composition of the present invention is considered to prevent, treat, or treat a disease or condition causing blood flow disorder through an anti-apoptosis effect and a blood vessel regeneration / remodeling promotion effect. Therefore, the present inventors next investigated whether ginsenoside derivatives such as dihydrozinosenoside Rb regulate the expression of major factors (groups of molecules) involved in cell apoptosis or vascular regeneration / remodeling.
  • Be 1 — X and caspase 3 are typical molecules involved in apoptosis or apoptosis-like cell death
  • VEGF is a typical molecule involved in revascularization and Z or remodeling. We examined whether the expression of these molecules was altered by dihydrozincenoside Rbi.
  • the cerebral cortical neurons isolated from the embryonic day 17 rat were cultured in a medium containing 10% fetal calf serum. On day 2 of the culture, the medium was replaced with a serum-free medium. On day 3 of the culture, dihydroginsenoside Rbi was added at a concentration of 0, 1, 100 ⁇ g / m 1, and the cells were cultured for 6 hours. Thereafter, total RNA was extracted from the cultured neurons.
  • cDNA was prepared from total RNA using oligo dT primer and reverse transcriptase.
  • the PCR reaction was performed using Taq [polymerase].
  • the PCR-primers and PCR reaction conditions used are as follows. I3-actin is an internal standard.
  • Sense primer AGA AGA GCT ATG AGC TGC CTG ACG Antisense primer- TAG TTG CGC TCA GGA GGA GCA ATG
  • Sense primer AAG CGT AGA CAA GGA GAT GCA
  • Antisense primer GGA GCT GAT CTG AGG AAA AAC C
  • Sense primer CCA TGA ACT TTC TGC TCT CTT G
  • Antisense primer GGT GAG AGG TCT AGT TCC CG
  • Sense primer GCT AAC CTC AGA GAG ACA TTC ATG
  • Antisense primer TTA GTG ATA AAA GTA CAG TTC TTT
  • FIG. 11 and FIG. Figures 11 and 12 are photographs replacing the drawings.
  • VEGF mRNA expression was enhanced.
  • the strongest expression of VEGF mRNA was observed in the group to which lf gZml was added.
  • the VEGF of the rat is divided into three subtypes, VEGF120, VEGF164, and VEGF188, corresponding to the number of constituent amino acids. At least two are allowed as shown in the figure.
  • dihydrogin genoside Rb ⁇ The expression of caspase-3 mRNA was suppressed in neurons added at a concentration of 1,100 fg / 1. In particular, the strongest suppression of caspase 3 mRNA expression was observed in the 1 fg / m 1 addition group.
  • ginsenoside derivatives are books such as Be1 — or caspase 3 (Experimental Medicine, Vol. 17, No. 13, Planning, Shigeichi Nagata, 1999, Yodo It can be said that apoptosis or apoptosis-like cell death is inhibited by increasing, decreasing, or regulating the expression of the apoptosis-related molecules described in Jpn.
  • Ginsenoside derivatives, especially dihydroginsenoside Rbi are described in VEGF and other books (Experimental Medicine, Vol. 17, No. 6, Planning, Masashi Shibuya, 1999, Yodosha). It is thought that increasing, decreasing or regulating the expression of the assembly-related molecules promotes the regeneration and remodeling of blood vessels.
  • a pharmaceutical composition comprising a ginsenoside derivative such as dihydrozincenoside Rb can promote apoptosis or apoptosis through promotion of Be1-X expression and / or suppression of caspase-3 expression. It is used for the prevention, treatment, and treatment of diseases (including pathological conditions) that inhibit cis-like cell death and cause cell death.
  • ginsenoside derivatives such as dihydro ginsenoside R bi may also enhance, attenuate or regulate the action of Bel — and / or caspase 3 transcription factors (eg, NF ⁇ B, STATs, etc.). .
  • ginsenoside derivatives other than dihydroginsenoside Rbi will be briefly described by taking the ginsenoside Rb derivative in Fig. 13 as an example.
  • (1) at the upper left of FIG. 13 is an example of a derivative in which a hydroxyl group is acylated or acetylated.
  • a double bond in a side chain may be reduced.
  • (2) is an acylation or case This is an example in which a double bond in the side chain is converted to a single bond and an arbitrary functional group (for example, one or more hydroxyl groups) is bonded to the same part in addition to the chilling. It is also possible to convert.
  • (3) in FIG. 13 is an example of a derivative in which the terminal is converted to an aldehyde group by cleaving the double bond in the side chain in addition to acylation or acetylation.
  • (4) is an example in which an arbitrary functional group such as an alkyl group or a aryl group is bonded to the end of the side chain in addition to the acylation or acetylation
  • (5) is an example in which the side chain is added in addition to the acylation or acetylation
  • (6) is an example in which the double bond in the side chain is epoxidized in addition to acylation or acetylation
  • (7) is an example in which a double bond in a side chain is cleaved to bond an arbitrary functional group, for example, a propyloxyl group, and an aldehyde group may be bonded instead of a lipoxyl group.
  • (8) is obtained by substituting one methyl group at the terminal of the side chain with a hydrogen atom and substituting the other methyl group with an arbitrary functional group such as an alkyl group or a peryl group.
  • an arbitrary functional group such as an alkyl group or a peryl group.
  • a heavy bond is converted to a single bond
  • an arbitrary functional group for example, one or more hydroxyl groups is bonded to the same portion.
  • a dihydroxy cinnoside R in which two hydroxyl groups are bonded is shown. bt. In this case, only one hydroxyl group may be replaced with any hydrogen atom to create monohydroxyzine cenoside R t.
  • (10) is an example in which two molecules of the hydroxyl group described in (9) are dehydrated and epoxidized, that is, epoxyzine cenoside Rbi. Further, (11) protopanaxadiol. Any compound having protopanaxatriol, damarane or a reduced form thereof as a basic skeleton is included in the category of ginsenoside R t derivative. In this
  • Ginsenoside R Bi which has been subjected to D. ie 1 s — Alder reaction using a gen compound such as cyclopentene at the double bond of the side chain, or (13) Ginsenoside R b! The sugar chains of! are reduced to itols.
  • ginseng contains about 30 types of purified saponins, that is, natural ginsenosides or ginsenoside compounds in addition to ginsenoside Rbt (Junsho Shoji, Ginseng '95, PP251-26K edited by Akira Kumagai, Kyoritsu Publishing Co., Ltd.), and the chemical structure of these purified saponins, that is, ginsenosides, is similar to that of ginsenoside Rbi.
  • novel chemical derivatives that can be prepared by using ginsenoside Rbi as a lead compound are not limited to those described above.
  • purified saponins other than ginsenoside Rbi that is, natural ginsenosides (especially, protopanaxadiol and protopanaxatriol), reduce the side chain of the tamaran skeleton (steroid-like skeleton) or reduce the primary chain.
  • Chemical derivatives can be prepared in the same manner as in FIG.
  • the chemical derivatives of oranolic acid, including ginsenoside Ro have already been described.
  • Jinsenosai de such as Jinsenosai de R bt (including Jinsenosa Lee earth derivatives) are allowed to activate any transcription factor, cell death suppressing gene B c 1 - Expression of X L
  • the chemical structure of ginsenoside Rbi is described in PC TZ JP 00/04102 and the present specification.
  • PCR gene amplification reaction
  • ginsenoside Rbi enhances the expression of Be 1 — protein in neurons
  • a western blotting method was performed using an anti-Be 1 — protein antibody. After culturing rat cerebral cortical neurons for 48 hours in the presence or absence of ginsenoside Rbi, the cells were lysed with a sample buffer for electrophoresis and subjected to electrophoresis. Thereafter, the electrophoretic proteins were transferred to a two-nitrocellulose membrane and subjected to Western plotting. The results are shown in FIG. Figure 15 is a photograph replacing the drawing.
  • ginsenoside Rbi significantly increased neuronal Bc1-X expression by about 50% in the optimal concentration range of l to 100 fgm1 (Fig. 15, Fig. 15). 16 Figure).
  • Bel—X Promo Yuichi Z Luciferase Plasmid was created. That is, using DNA extracted from tail tissue of C57BL / 6 mouse as type I, the following primers LF1, Primer LR1 and Pyrobest DNA polymerase (pyrobest DNA polymerase) (Takara) were used. 30 cycles of the PCR reaction were performed.
  • the reaction solution was purified using QiaExII Gel Extraction Kit (Qiagen), digested with restriction enzymes XliOI and HindIII, and electrophoresed on a 2% agarose gel. A 0 bp DNA fragment was cut out. From the obtained gel slice, a DNA fragment was extracted using a QiaExII gel extraction kit (QiaExII Gel exraction kit) and inserted into pGL-2 Basic vector (Promega) (Bexx). promoter L) (Fig. 17). Similarly, using mouse MA as type II, 15 cycles of PCR reaction were performed using primer RF1 and primer RR1.
  • the solution A was used as type III, and a PCR reaction of 30 cycles was performed using each of the primer R F1 and the primer Mut-R, the primer Mut-F, and the primer R R1.
  • a PCR reaction of 30 cycles was performed using each of the primer R F1 and the primer Mut-R, the primer Mut-F, and the primer R R1.
  • DNA fragments of 312 bp and 309 bp were cut out, and a DNA fragment was extracted using a QiaExII gel extraction kit (QiaExII Gel exraction kit).
  • QiaExII Gel exraction kit QiaExII Gel exraction kit
  • the mixture is used as a ⁇ -type and subjected to a PCR reaction of 30 cycles with a primer RF2 and a primer RR2, and then eliminated with Sacl and BamHI. And inserted into the SacI / BgIII site of the pGL-2 Basic vector (Bcl-x promoter R (mutation) (Fig. 17)
  • Primer LF1 5 '-ATACTTCCCAGCCGCAAAACGC-3'
  • the primary culture of the astrocytes was performed as follows.
  • Astrocytes were isolated from a Wistar rat immediately after birth by a known method, cultured in a culture flask, and replanted two weeks later in a 12-well plate (12-well plate).
  • the astrocyte was published by the present inventors (Sakanaka, Tanaka) (Fujita, H. et al., Glia, 18, 269-281, 1996; Tanaka, J. et al.). , Gl ia, 20, 23-37, 1997; Tanaka, J. et al., Glia, 24, 198-215, 1998).
  • the gene transfer and luciferase atsey were performed as follows.
  • Transfection was performed for 5 hours in the presence of 10% FCS (fetal calf serum) using Lipofectamine (Invitrogen). Thereafter, the medium was replaced with a fresh medium, and the cells were cultured at 37 ° C. The next morning, 100 fg / ml of ginsenoside Rbi was added, and the cells were cultured at 37 ° C for 24 hours. After washing the cell surface twice with PBS, the cells were lysed with Luciierase Cell Culture Lysis eagent (Promega) 1001. The amount of luciferase in the solution was measured using a Luciferase Assaay System (Promega) and a luminescence sensor JNR (ATTO).
  • STAT5 signal transducer and activator of transcription
  • Interleukin 2 (IL-2), Interleukin 3 (IL-3), Inuichi Leukin 5 (1 L-5), Interleukin 7 (IL-7), Interleukin 9 (IL-9), interleukin 15 (IL-15), granulocyte 'macrophage' colony stimulating factor (GM-CSF)> growth hormone, tgrowth hormone, GH ), Prolactin, erythropoietin (EPO), etc., are activated by stimulation of site hormones and hormones, and are activated by Bel-X (including Bcl-xs, Bet-xj3), / 3-force zine, It is known to up-regulate the transcription of Oncos-Mintin M etc.
  • Bel-X including Bcl-xs, Bet-xj3
  • / 3-force zine It is known to up-regulate the transcription of Oncos-Mintin M etc.
  • the pharmaceutical composition exerts the same physiological actions, effects, and potencies as the above-mentioned cytokine hormone, Moreover, it can be said that the expression of B c 1 —x, —casein, oncostatin ⁇ , etc. is also induced.
  • IL_2 promotes ⁇ cell proliferation, induces natural killer ( ⁇ ) cells, cytotoxic ⁇ induces cells, and activates lymphokine (lymphokine activated killer) Induction of (LAK) cells, increase of B precursor cells It has effects such as inducing proliferation and differentiation, and is currently being marketed as an anticancer agent.
  • IL-13 promotes blood stem cell proliferative function and platelet progenitor cell proliferative function.
  • IL-5 has effects such as induction of B cell proliferation, promotion of differentiation of B cells into antibody-producing cells, induction of eosinophil proliferation and differentiation, and induction of expression of IL-2 receptor.
  • IL-17 has effects such as induction of proliferation and differentiation of precursor B cells, induction of proliferation and differentiation of precursor T cells, induction of LAK cells, and activation of monocytes.
  • IL-19 promotes the proliferation of platelet progenitor cells.
  • IL-15 promotes T cell proliferation and activates NK cells and LAK cells.
  • IL-115 also promotes B cell proliferation and, like IL-2, can be used for site-in therapy of cancer, sarcoma, and malignant neoplasms.
  • Erythropoietin (EPO) is used clinically as a therapeutic agent for renal anemia or secondary anemia because it differentiates and proliferates erythroid progenitor cells into erythrocytes.
  • GM-CSF promotes the proliferation and differentiation of neutrophils, eosinophils and macrophages, and induces megakaryocyte differentiation. Useful.
  • the above-mentioned site-inducing compounds have anti-cancer effect, anti-tumor effect, cancer metastasis suppressing effect, secondary anemia treatment effect, immunodeficiency disease (including AIDS) treatment effect, renal anemia treatment effect, aplasticity It can be said to show an anemia treatment effect, a thrombocytopenia treatment effect, or a neutrophil recovery promotion effect after cancer chemotherapy.
  • target organs for growth hormone include liver, kidney, adipocytes, muscle, lymphocyte, thymus, etc. In these organs or tissues, receptors for growth hormone are expressed.
  • Growth hormone has a growth promoting effect by stimulating the production and secretion of insulin-like growth factor (IGF-1, somatomedin C) in the liver, muscle and kidney. More specifically, growth hormone promotes the proliferation of chondrocytes, the synthesis of chondroitin sulfate, the hypertrophy of cells such as hepatocytes, and the assimilation of proteins. Therefore, growth hormone can also be used as a composition for regulating the growth of animals and plants, and as a pharmaceutical composition for inhibiting aging and degeneration of cartilage.
  • IGF-1 insulin-like growth factor
  • somatomedin C insulin-like growth factor
  • Target organs of prolactin include mammary gland, liver, kidney, adrenal gland, ovary, prostate, seminal vesicle, thymus, bladder, etc. In these organs or tissues, prolactin receptors are expressed.
  • Prolactin has a wide variety of physiological effects in a wide range of animals, including the promotion of mammary gland development, the promotion of casein synthesis, the stimulation of lactation, and the promotion of prostate and seminal vesicle gland development. Therefore, Prolactin is thought to be useful in the prevention, treatment, and treatment of diseases, conditions or disorders that exhibit insufficient lactation and poor mammary gland development. Prolactin may also be useful in preventing, treating, and treating sexual dysfunction.
  • Oncostatin M promotes blood cell production and proliferation of vascular endothelial-like cells. Therefore, Oncostin M is also considered useful for the prevention, treatment or treatment of anemia, immunodeficiency disease, thrombocytopenia, granulocytopenia, leukopenia, purpura, and DIC.
  • Power zein is the major protein in milk, and its production is induced by prolactin. Therefore, ⁇ -one force zein is considered to be useful for prevention, treatment or treatment of lactation deficiency.
  • the above-mentioned site force-in, hormone, oncostatin ⁇ or one-casein is a malignant neoplasm, cancer, sarcoma, anemia, immunodeficiency disease (including AIDS), renal anemia, secondary anemia, regeneration Aplastic anemia, hereditary spherocytosis, autoimmune hemolytic anemia, myelodysplastic syndrome, granulocytopenia, agranulocytosis, purpura, idiopathic thrombocytopenic purpura, disseminated intravascular coagulation (DIC), growth hormone deficient short stature, pituitary dwarfism, osteoarthritis of the knee, osteoarthritis of the hip, cervical spondylotic myelopathy, lumbar disc herniation, lactation deficiency or degenerative spine It is considered useful for prevention, treatment or treatment of the disease.
  • DIC intravascular coagulation
  • ginsenosides such as ginsenoside Rbi can be converted to site force-in (IL-2, IL-3, IL-5) through the activation of the transcription factor STAT5 in all cells, especially in the mouth opening site.
  • IL-2, IL-3, IL-5 site force-in
  • IL-7, IL-9, IL-15, GM-CSF, EP0 hormones (GH, prolactin), oncoscintin M or / 3-casein It can be said that it takes over the function of the substance.
  • ginsenosides such as ginsenoside Rbt
  • the pharmaceutical composition is useful for the prevention, treatment or treatment of blood
  • dihydrozincenoside R bi significantly increased the expression level of B e 1 -protein in neurons in the optimal concentration range of 1 to 100 fg Xm 1.
  • FIGS. 19 and 20 indicates P ⁇ 0.05, and the statistical analysis method is based on AN OVA + Fisher's PLSD.
  • dihydrodine senoside R b It can be said that ginsenoside derivatives, such as ginsenoside Rbi, also enhance neuronal Be1— expression.
  • dihydrozincenoside R bi and ginsenoside R bi have extremely similar pharmacological actions.
  • ginsenoside Rbi it is thought that the activation of transcription factor STAT5 also up-regulates the expression of Be1_ ⁇ ⁇ in nerve cells.
  • ginsenoside derivatives such as dihydroginsenoside Rb can be used to activate cytokines (IL-2, IL-3, IL-3) through the activation of the transcription factor STAT5 of cells such as the ostium mouth. 5, IL-7, IL-9, IL-15, GM-CSF, EP0), hormones (GH, prolactin), oncostatin M, or ⁇ -casein. It can be said that it takes over the function.
  • the pharmaceutical composition of the present invention is useful for prevention, treatment or treatment of blood-hema
  • the present inventors have already found that apoptosis or apoptosis-like cell death of a cell is strongly suppressed through an increase in the expression of B e1 — (WO0Z374781).
  • a group of prosavosine-related peptides which exhibit a potent anti-apoptotic action similar to ginsenoside Rbi, also exhibit Bc 1 -X expression-enhancing action, and have been tested in in vivo and in vitro experimental systems.
  • ginsenoside R bt it has been found to be useful for the prevention, treatment or treatment of cerebrovascular disorders (Japanese Patent Application No. 11-185, EP 0 03 05 05 04) No., Igase, K.
  • L-serine and / or glycine were enhanced through increased expression of a cell death suppressor gene product Bc1-1w, which also belongs to the Bc1-2 protein group. It has been shown to exert an anti-apoptotic effect on force (Yang, L. et al., Eurosci. Lett., 295, 97-100, 2000). By the way, 3-1 and 8 0 1 — ⁇ are expressed in cells of every tissue, and the physiological functions of both gene products are very similar, Again, it can be assumed that L-serine and / or glycine can also be used as a pharmaceutical composition for preventing, treating, or treating diseases or conditions that cause impaired blood flow.
  • the isocarbacycline group described in Japanese Patent Application No. 2000-4002639 also exhibits an anti-apoptotic effect by suppressing the expression of BaX which is one of the Be1-2 protein group.
  • the present inventors (Sakanaka) have shown that they are useful for the prevention, treatment or treatment of cerebrovascular disorders. Therefore, it can be said that the above-mentioned isocarpacycline group can also be used as a pharmaceutical composition for preventing, treating, or treating diseases or conditions that cause impaired blood flow. Therefore, a compound that inhibits apoptosis or apoptosis-like cell death by regulating the expression of the Be1-2 protein group is a pharmaceutical composition for preventing, treating, or treating a disease or condition that simultaneously impairs blood flow.
  • the present invention comprises administering a test substance to cultured cells and measuring the expression regulating action of the Be1-2 protein group, comprising preventing, treating or treating a disease or condition causing blood flow disorder. It also provides a method of searching for a pharmaceutical composition for treatment. Needless to say, the present invention relates to a disease which causes a blood flow disorder, comprising a compound having a Bc 1-2 protein group expression regulating action or a salt thereof when administered to cultured cells by the method described above. Alternatively, a pharmaceutical composition for preventing, treating or treating a disease state is provided.
  • the Bel-2 protein group has an inhibitory effect on apoptosis or apoptosis-like cell death (Bel-2, Bel-X, Bel-w, Mcl-l, Bfl-l ZB od, Nr—13, BRAG-1, Boo / Diva, Galectin—3) and those that promote apoptosis or apoptotic cell death (Bax, Bak, Bel) — Xs, Bad, Bik / Nbk, Bid, Bim / Bod, Hrk / DP5, BNI PZNix, Bok / Mtd, Blk, EGL-1) Organ apoptosis proofing method; Editing, Katsunori Otsuki, Takehiko Oji, Keiichi Watanabe, Nankodo, 2000, p16-224).
  • the compounds exhibiting the expression regulating action of the Bel-2 protein group those which promote the expression of the above-mentioned anti-apoptotic factors or the expression of the proapoptotic factors It is conceivable to suppress this.
  • B el - erythropoietin having x L expression enhancing action in evening Site forceins or growth factors such as 1-leukin 3 (Wen et al., J. Exp. Med. 188, 635-649, 1998; Japanese Patent Application No. 11-185185, EP No. 0 305 504) can also be used as a pharmaceutical composition for the prevention, treatment or treatment of diseases or conditions that cause impaired blood flow because they exhibit the activity of regulating the expression of the Bc1-2 protein group.
  • the compounds exhibiting the activity of regulating the expression of the Bel-2 protein group are not limited to those described above.
  • the present inventors have found that vitamin E and the like also enhance Bc1-XL expression in cells.
  • ginsenosides activate the transcription factor HIF-1 and induce VEGF expression. For this reason, the following experiment was performed using ginsenoside R bi as a representative ginsenoside of the present invention. Details of H I F-1 will be described later.
  • the present inventors conducted an experiment by selecting astrocyte as an example of cells in order to examine whether ginsenoside Rb increases the expression of VEGF mRNA in cells.
  • Astrocytes were isolated from the Wistar rat immediately after birth by a known method, cultured in a culture flask, and 12 days later, a 10 cm dish coated with poly-L-lysine was used.
  • RNA was extracted from the cultured astrocytes.
  • cDNA was prepared from 3 g of total RNA using oligo dT primer and reverse transcriptase.
  • the PCR reaction was carried out using Taq polymerase in accordance with the Tonenguchi article (Tonello, FEBS Letters, 442, 167-172, 1999).
  • the PCR-primer and PCR reaction conditions used are as follows. I3-actin is an internal standard.
  • Sense primer CCA TGA ACT TTC TGC TCT CTT G
  • FIG. Figure 21 is a photograph replacing the drawing.
  • ginsenoside As shown in Fig. 21, as compared with ginsenoside Rb-free astrocytes, ginsenoside; bi was added at a concentration of 100 f / m1 in the astrocytes and VEGF mRNA was not added. Expression was enhanced.
  • ginsenoside Rbi also induces VEGFFMRNA expression in human skin keratinocytes. For this reason, human skin keratinocytes are cultured in a monolayer, ginsenoside Rbi is added to the culture medium at a concentration of 0 to 10 pg / ml, and after 0, 1, 3, 6, and 12 hours. Total RNA was extracted from human skin keratinocytes. Thereafter, RT-PCR was carried out at a cycle number of 27 using the primers shown below. The results are shown in FIG. FIG. 22 is a photograph of R T—P C R instead of a drawing.
  • Sense primer TGG CAG AAG GAG GAG GGC AGA AT
  • ginsenoside Rb at a concentration of 1 fgZml to 10 pg / ml was added to cultured human skin keratinocytes, the expression of VEGF mRNA was induced within 12 hours.
  • ginsenoside Rb increased VEGF protein expression.
  • human skin keratinocytes are cultured in monolayer and 24 hours or 48 hours after addition of the cenoside Rb to the culture medium at a concentration of 0 to 10 pg / ml, the culture supernatant was collected. Thereafter, the VEGF concentration in the culture supernatant was measured using a human VEGFELISA kit (R & D system). The results are shown in FIG.
  • VEGF concentration in the culture supernatant increased, especially at a concentration of 100 fg / m 1 to 10 pg / m 1, as compared with the case without ginsenoside R bi.
  • ginsenoside R bi induces VEGF expression in cells such as the ostium-site keratinocytes. Therefore, the present inventors next examined whether ginsenoside R bi induces VEGF expression through activation of transcription factor HIF-1. For this reason, first, the B g1 II site on the multiple cloning site of the pGL-3 promoter, Oneichi (Promega), has a repeat sequence of “TACGTG” six times, ie, (TACGTG) 6. Was inserted. By the way,
  • TA CGTG is, HRE on DNA binding transcription factors HIF 1. - a common base Hai ⁇ of (hypox ia response element) (J (consensus sequence)
  • J consensus sequence
  • TAC GT G 6
  • the pGL-3 promoter vector into which is inserted is referred to as HRE leuciferase-plasmid .
  • the pGL-3 promoter-vector-1 was used as a control.
  • Astrocytes were isolated by a known method from a Wistar rat immediately after birth, cultured in a culture flask, and replanted two weeks later in a 12-well plate.
  • the astrocyte was published by the present inventors (Sakanaka and Tanaka) (Fujita, H. et al., Glia, 18, 269-281, 1996; Tanaka, J. et al., Glia, 20,
  • Nerve cells were isolated from the 17-day-old embryonic Wistar rat cerebral cortex by a known method and cultured on a 12-well plate coated with poly L-lysine. . Nerve on day 5 of culture Cells were used for gene transfer.
  • the amount of luciferase increased in the ginsenoside Rbi100 ig / ml-added example compared to the non-added example.
  • ginsenosides such as ginsenoside Rbi
  • HIF-1 and HRE hypo xi a-response element
  • ginsenoside Rbi activates the transcription factor HIF-1 even in human skin keratinocytes.
  • human skin keratinocytes are cultured in a monolayer on a 12-well type 1 collagen dish and transfection of plasmid DNA (ie, HRE-luciferase-plasmid or PGL-3 promoter vector) is performed. did.
  • plasmid DNA ie, HRE-luciferase-plasmid or PGL-3 promoter vector
  • the medium was changed and ginsenoside Rbi at a concentration of 0 to 1 ng / m1 was added.
  • the cells were lysed with Luciierase Cell Culture Lysis Reagent (Promega).
  • the amount of luciferase in the solution was measured using a Luciferase Assay System (Promega) and a luminescence sensor JNR (ATTO). The results are shown in FIG.
  • a control example PGL-3 promoter vector
  • the amount of luciferase was increased by the addition of ginsenoside Rb.
  • the increase in the amount of luciferase was remarkable when 10 fg / ml to lpg gZml of ginsenoside Rbi was added. Therefore, ginsenosides, such as ginsenoside Rb, activate the transcription factor HIF-11 in all cells, including the ostium mouth or human skin keratinocytes, and induce the binding of HIF-1 to HRE. It is thought to be.
  • Hypoxia Inducible Factor-1 is a heterodimer consisting of HIF-la and HIF-lj3. It is considered an activated factor.
  • HIF-1 activation by hypoxia to date (1) HIF-la mRNA is markedly induced by hypoxia-ischemia; (2) HIF-1a is ubiquitin-protein It is degraded in the lysosomal system, but its degradation is suppressed under hypoxia. (3) It is reported that HIF is regulated by three kinds of mechanisms; .
  • HRE hypoxia response element
  • EPO Erythropoietin
  • Transferrin is an iron transport protein, which transports iron to the bone marrow by binding to iron absorbed from the food into the body, where it can be used for hemoglobin synthesis.
  • VEGF Vascular endothelial growth factor
  • FLT-1 receptor FLT-1
  • Glycolytic enzymes such as aldolase A and C; phosphofrue tokinase L and C; phosphoglycerate kinase-1 (C) PGK-1)); Lactate dehydrogenase:)-ze-A (lactate dehydrogenase-A (LDH-A)); pyruvate kinase M (pyruvate kinase M) ; enora 1 A
  • Type 1 and type 3 glucose transporter overnight (GLUT 1; GLUT 3): Promotes the uptake of glucose into cells and enables the cells to undergo anoxic respiration.
  • Adenylate kinase 3 catalyzes the reaction of AMP + ATP ⁇ 2 ADP. This reaction is important as the first step of AMP-ATP, that is, the first step of ATP production.
  • Heme Oxygenase-1 Heme is decomposed into CO (—carbon oxide) and pyriberdine. The generated CO protects cardiomyocytes and nerve cells from low oxygen by vasodilator action.
  • Tyrosine dehydrogenase a catecholamine synthase.
  • HIF-1 can be used for (1) diseases or conditions that cause vascular injury or impaired blood flow through increased expression of VEGF and its receptor (eg, wounds, fractures, burns, hemorrhoids, radiation damage) Prevention, treatment or treatment of cerebrovascular disorders, angina pectoris, myocardial infarction, laser injuries, Reino's disease, collagen disease, atherosclerosis obliterans, skin ulcers, diabetic skin ulcers, heart failure, pressure sores, etc.) 2) Prevention, treatment or treatment of anemia, renal anemia and secondary anemia through increased expression of erythropoietin and transferrin, (3) glycolytic enzymes, glucose transporter, adenylate kinase 3 and Protects cells in a hypoxic environment through increased expression of HO-1 and thereby prevents, treats or treats cerebral infarction, angina pectoris, and myocardial infarction.
  • VEGF and its receptor eg, wounds, fractures, burns, hemorrhoids, radiation damage
  • compositions such as ginsenosides that activate the transcription factor HIF-1 also include wounds, bone fractures, burns, hemorrhoids, radiation damage, cerebrovascular disease, laser damage, Reino disease, Prevention, treatment or treatment of collagen disease, arteriosclerosis obliterans, skin ulcers, diabetic skin ulcers, pressure sores, anemia, renal anemia, secondary anemia, cerebral infarction, angina pectoris, cardiac infarction or myocardial infarction Will be useful.
  • mice lacking the hypoxia response element (IIRE) that binds to HIF-1 cause motor neuron cell death and exhibit a pathology similar to amyotrophic lateral sclerosis (ALS).
  • IIRE hypoxia response element
  • ALS amyotrophic lateral sclerosis
  • ginsenosides such as ginsenoside Rt or natural ginsenosides can be converted into erythropoietin, transferrin, transferrin receptor, VEGF, and VEGF through activation of transcription factor HIF-1.
  • FLT-1 Body (FLT-1), glycolytic enzymes (aldolase A, aldolase C, phospho rue tokinase L, phosp of r uctokinase C, phosphoglycerate kinase-1, lactate dehydrogenase A, pyruva te kinase M, enolase A) , Type 1 glucose transporter, type 3 glucose transporter, adenylate kinase 3, heoxygenase 1 (HO I) or tyrosine dehydrogenase .
  • glycolytic enzymes aldolase A, aldolase C, phospho rue tokinase L, phosp of r uctokinase C, phosphoglycerate kinase-1, lactate dehydrogenase A, pyruva te kinase M, enolase A
  • Type 1 glucose transporter type 3 glucose transporter
  • the pharmaceutical composition comprising the ginsenosides of the present invention comprises a wound, a bone fracture, a burn, a hemorrhoid through the action of the gene product or a physiologically active substance whose expression is induced by the transcription factor HIF-1.
  • Radiation injury, cerebrovascular disease, Reino's disease, collagen disease, arteriosclerosis obliterans, Baja disease, diabetic skin ulcer, skin ulcer, laser injury, pressure sore, anemia, secondary anemia, renal anemia It is considered to be useful for preventing, treating or treating diseases that cause blood flow disorders such as diseases involving nerve cell death, amyotrophic lateral sclerosis, cerebral infarction, angina, heart failure or myocardial infarction.
  • ginsenoside derivatives such as dihydrozincenoside Rbi induce the expression of VEGF mRNA in nerve cells. Then, the present inventors next examined whether the ginsenoside derivatives promoted the expression of VEGF mRNA in astrocytes in the same manner as ginsenoside Rb !. Therefore, as a representative example of the ginsenoside derivatives, an experiment was carried out by selecting a reduced derivative, that is, dihydrozincenoside R b represented by the above structural formula.
  • Astrocytes were isolated from the Wistar rat immediately after birth by a known method, cultured in a culture flask, and 12 days later, a 10 cm dish (10 cm) coated with poly-L-lysine was used. dish). Astrosites were cultured in DMEM medium containing 10% fetal calf serum (FCS). After 3 to 4 days, change to serum-free medium and The hydrozincenoside Rbi was added at a concentration of 0, lfg / ml, and 100 fg / m1, and cultured for 6 hours. The astrocyte was published by the present inventors (Sakanaka and Tanaka) (Fujita, H.
  • RNA was extracted from the cultured astrocytes.
  • cDNA was prepared from 3 ig of total RNA using oligo dT primer and reverse transcriptase.
  • the PCR reaction was performed using Taq polymerase according to Tonella's paper (Tonello, FEBS Letters, 442, 167-172, 1999).
  • the PCR-primer and PCR reaction conditions used are as follows.
  • ⁇ -actin is an internal standard. (1) ⁇ —
  • Ant isense primer TAC TTG CGC TCA GGA GGA GCA ATG
  • Sense primer CCA TGA ACT TTC TGC TCT CTT G
  • Ant isense primer GGT GAG AGG TCT AGT TCC CG
  • FIG. Figure 26 is a photograph replacing the drawing.
  • the dihydrozine senoside Rb was added at a concentration of 1 f / m1 or 100 fg / m1.
  • VEGF mRNA expression was enhanced in trocites.
  • the VEGF of the rat is divided into three subtypes, VEGF120, VEGF164, and VEGF188, corresponding to the number of constituent amino acids. At least two are allowed as shown in the figure.
  • the present inventors used Western blotting with an anti-VEGF monoclonal antibody (manufactured by Sunshine Cruz) to examine whether dihydrozincenoside R bi enhances not only VEGF mRNA but also VEGF protein expression in neuronal cells.
  • the law was implemented. Rat cerebral cortical neurons were cultured for 24 hours in the presence or absence of dihydrozincenoside Rbt, and the cells were lysed with a sample buffer for electrophoresis, and electrophoresis was performed. Thereafter, the electrophoretic protein was transferred to a Nitrocellulose membrane and subjected to Western blotting.
  • FIG. Figure 27 is a photograph of a Western plot that replaces the drawing.
  • dihydrozincenoside R bi significantly increased the expression level of VEGF protein in neurons in the optimal concentration range of 1 to 100 ig / ml.
  • * indicates P ⁇ 0.01
  • the statistical analysis method is based on ANOVA + Fisher's PLSD. From the above results, it can be said that ginsenoside derivatives such as dihydrozincenoside Rb also enhance VEGF expression in cells, like ginsenoside Rb.
  • ginsenoside derivatives such as dihydroginsenoside Rb can be used to activate erythropoietin, transferrin, and transferrin via activation of the transcription factor HIF-1 in cells such as neurons of the ostium.
  • VEGF vascular endothelial growth factor
  • FLT-1 VEGF receptor
  • glycolytic enzymes aldolase A, aldolase C, phosphofru ctokinase L, posphofuret okinas e C, phosphoglycerate kinase-1, lactate d ehydrogenase A, pyruvate kinase M, enolase A
  • type 1 glucose transporter type 3 glucose transporter
  • adenylate kinase 3 hemeoxygena It can be said to promote the expression of heme oxygenase 1 (HO I) or tyrosine dehydrogenase.
  • a pharmaceutical composition comprising the ginsenoside derivative of the present invention can be used for treating wounds, fractures, burns, hemorrhoids through the action of the gene product or a physiologically active substance whose expression is induced by the transcription factor HIF-1.
  • Radiation injury cerebrovascular disease, Reino disease, collagen disease, arteriosclerosis obliterans, Bajaja disease, diabetic skin ulcer, skin ulcer, laser injury, pressure sore, anemia, secondary anemia, renal anemia It is considered to be useful for preventing, treating or treating a disease associated with nerve cell death, a cerebral infarction, angina pectoris, a disease causing a blood flow disorder such as heart failure or myocardial infarction.
  • ginsenoside derivatives are also included in ginsenosides.
  • Example 1 Example for making dihydrozine cenoside Rbi
  • the present inventors first prepared dihydrozincenoside Rbi.
  • dihydrozincenoside R b The production examples and NMR data of dihydrozincenoside R b are shown below.
  • 10% Pd / C (palladium charcoal) 10.2 mg is weighed and placed in a two-necked flask with active oxygen.
  • the present inventors first examined the concentration at which the dihydrozine senoside Rbi obtained by the above-mentioned method exerts a favorable effect on cells in a usual culture experiment. For this reason, the present inventors examined the concentration at which apoptosis or apoptotic-like neuronal death of cultured neurons was suppressed by dihydrozine senoside Rbi.
  • the present inventors show that cultured neurons are exposed to the nitric oxide donor, sodium nitroprusside (SNP), for a short period of time. It has been reported that death is induced (Toku K. et al., J. Neurosci. Res., 53, 415-425, 1998). Using this culture experiment system, the present inventors have already found that ginsenoside R bi can induce apoptosis of neurons or apoptotic-like neuronal death in the optimal extracellular solution concentration range of 1 to 100 fg / 1. Have been found to be deterred (WO 00/374841). Therefore, the neuroprotective effect of dihydrozine senoside Rb was examined using a similar experimental system.
  • Nerve cells were isolated from the fetal cerebral cortex of a 17-day-old rat fetal cerebral cortex using trypsin EDTA, and plated on a polyerizin-coated 24-well plate. After culturing for 16 hours in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, the culture is cultured in neural cells containing insulin, transferrin, etc. The medium was replaced with a serum-free medium and cultured for 3 to 4 days. On the third or fourth day of the culture, sodium nitroprusside (SN ⁇ ) was added at a concentration of 300 ⁇ , and the mixture was incubated for 10 minutes.
  • DMEM Dulbecco's modified Eagle's medium
  • SN ⁇ sodium nitroprusside
  • FIG. 2 is a photograph in place of a drawing showing the result of the immobilization of microtuble-associated protein 2 (MAP 2).
  • the first lane from the left is a control cultured neuron, in which a clear MAP 2 band (ie, a band of nerve cells) was observed.
  • the SNP treatment caused many neurons to undergo apoptosis or apoptosis-like neuronal death, so that the MAP2 band was clearly weakened, as in the second lane from the left.
  • Fig. 3 shows the results of the above-described simulated noblot experiment of MAP2 repeated five times, and the results were analyzed by densitometry. As shown in Fig.
  • dihydrozincenoside R bi in the optimal extracellular fluid concentration range of 0.01 fg / ml to lng Zm 1 significantly decreased neuronal apoptosis or apoptotic-like neuronal cell death.
  • dihydrozincenoside Rbi has a favorable anti-cellular activity, particularly against nerve cells, in an optimal extracellular solution concentration range that is considerably wider than that of disenozide Rbi described in WO 00/37481. It is thought to exert an apoptotic effect.
  • ginsenoside derivatives such as dihydrozincenoside Rbi also promote regeneration and / or remodeling of nerve tissue by extending neurites.
  • ginsenoside derivatives such as dihydrozincenoside Rb have an extracellular fluid concentration of 100 g / m1 (about 90 M) or less, preferably 100 ng / m1 (about 90 nM) or less, more preferably 1 ng / m1 (about 0.9 nM) or less, and still more preferably 0.0000 0.001 fg / ml (about 0.0000 It is considered that by inhibiting apoptosis or apoptosis-like cell death of cells at fM) to 100000 fg / ml (approximately 900 fM), an excellent cytoprotective effect is exerted.
  • Ginsenoside derivatives such as dihydrozincenoside Rb cause cells of living tissues exposed to impaired blood flow to have anti-apoptotic effects. It is thought to protect through use.
  • Ginsenoside derivatives such as dihydroginsenoside R b, etc. are used for transplantation tissues and organ cells (stem cells, ES cells, skin keratinocytes, etc.), cells derived from all tissues, frozen cells for transplantation, blood cell components for blood transfusion, It may also be useful for the protection or preservation of platelets and germ cells (frozen eggs or sperm).
  • * indicates P ⁇ 0.0001
  • ** indicates P ⁇ 0.0001.
  • Statistical analysis is based on the post hoc test of AN OVA + Schedule.
  • Example 3 (Effect of dihydrozincenoside Rbi on cerebrovascular disorders)
  • ginsenoside derivatives such as dihydrozincenoside Rbi can be used for cells, particularly for neurons, in an extracellular solution concentration range that is considerably wider than the ginsenoside Rbi described in WO 00/37481. Inhibition of apoptosis or apoptosis-like neuronal cell death has been shown in an in vitro experimental system, but dihydrozine senocysis has also been demonstrated in an in vivo animal experimental system that actually causes blood flow disorders. The inventors of the present invention next examined whether or not ginsenoside derivatives such as de Rbi exhibited excellent effects similarly to the ginsenoside Rbt described in WO 00/37481.
  • cerebrovascular disorders are selected as the diseases that cause blood flow disorders as follows, and intravenous administration of dihydrozincenoside Rbi, one of the derivatives of ginsenosides. Treatment of cerebrovascular disorders, especially cerebral infarction I checked whether it is effective.
  • the results are shown in FIG.
  • the cerebral infarction area of the cerebral infarction group of the cerebral infarction group treated with dihydridine senoside R bi (2H-Rbi) was compared with that of the cerebral infarction group treated with the vehicle (saline).
  • the statistical analysis method in Fig. 6 is based on the Mann-Whitney test, and the ** mark indicates 0.01.
  • the therapeutic effect of dihydrozincenoside Rbi on cerebrovascular disorders is excellent enough to be comparable to the effect of ginsenoside Rbi and the like disclosed in WO0000374881.
  • ginsenoside derivatives such as dihydrozine cenoside Rbi are effective and efficacious in diseases or conditions that cause impaired blood flow such as cerebrovascular disorder and cerebral infarction.
  • ginsenoside derivatives such as dihydroginsenoside R bi promote regeneration and / or remodeling of cerebral blood vessels, similarly to ginsenoside Rb described in WO 00/48608. Was done.
  • the present inventor further increased the intravenous dose of dihydrozine senoside Rbi by a factor of 2 (single administration of 12 ig followed by continuous administration at a dose of 12 ig / day). After examining whether or not it could be obtained, no excellent effect was found as expected.
  • ginseng in W ⁇ 00/3 7 4 Noside Rb showed an excellent cerebral infarction treatment effect even at a dose of 6 / day on SH-SP rats weighing about 300 g, but ginsenoside derivatives such as dihydrozincenoside R bi It was considered that such a high dose did not necessarily exert a cerebral infarction therapeutic effect and / or a cerebral blood vessel regeneration / remodeling promoting effect.
  • the optimal dose of dihydrozincenoside; R for a cerebral infarction rat with a body weight of about 300 g is lower than the optimal dose of ginsenoside Rbi, and in particular, is preferably 60 igZ days or less. It was considered to be less than 12 zg / day.
  • dihydrozincenoside R bi inhibits apoptosis or apoptotic-like neuronal cell death of neurons in a broader concentration range than ginsenoside R bi, but in vivo.
  • Dihydrozincenoside Rb has excellent cerebral infarction treatment effect and cerebral vascular regeneration / regeneration in the same dosage amount as ginsenoside Rbi, or as low as 1/10 to 1/100 of that amount. It can be said that it has a construction promoting effect.
  • other ginsenoside derivatives such as dihydroxidine senoside R bi or epoxy ginsenoside R bi may be used at a dosage and concentration that is equivalent to or approximately 100 times higher than ginsenoside R bi. It is thought to show the same effect as bi.
  • Example 4 Example 4 (Experiment for judging the effect of dihydrozine cenoside Rbi on the treatment of spinal cord injury)
  • the present inventors have further investigated whether low doses of dihydrozine cenoside Rb also have a favorable effect on nerve trauma causing blood flow disorders.
  • neurological trauma such as spinal cord injury and head trauma also causes blood flow disorders due to rupture of blood vessels and edema of brain and spinal cord tissues, leading to irreversible higher-order neuropathy. Therefore, the present inventors took spinal cord injury as one of the diseases causing blood flow disorder, and dihydrozincenoside Rb! The effect of was decided to be investigated. For this reason, the following is an example of an experiment in which dihydridine senoside Rb! Was infused intravenously into a spinal cord injury rat (body weight: about 300 g) for 7 days at a dose of 1.2 g Z days.
  • Figs. 7 and 8 show the saline administration rats on the second day after spinal cord injury, and the right photographs of Figs. 7 and 8 show dihydrozincenoside Rb 1.2 gZ at the same time. Days) administration rats are indicated.
  • the saline administration rat with 20 g of pressure applied to the lower thoracic spinal cord for 20 minutes was used not only on the day of spinal cord injury but also after spinal cord injury. He also had paraplegia on both legs on day.
  • dihydrozine senoside Rbi was administered intravenously to spinal cord injury rats at a dose of 8 ii gZ days after a single dose of 8 / ig or 60 ⁇ g / day after a single dose of 60 Hg.
  • no excellent effect was observed.
  • ginsenoside derivatives such as dihydrozincenoside R bi are superior to the spinal cord in comparison with the ginsenoside R bi described in WO 00/46808.
  • Injury ⁇ It was found to be effective in treating nerve trauma.
  • the optimal dose of dihydrozincenoside Rb for spinal cord injury rats weighing 300 g was considered to be 6 days or less, preferably around 1.2 ig / day or less. That is, when dihydrozincenoside Rb is used as a pharmaceutical composition for treating nerve trauma, head trauma-spinal cord injury, the optimal dose is W ⁇ 00 / 468680 or PCT / JP0.
  • ginsenoside Rbi described in 0/04201 (about 60 ig _ / day for a rat weighing 300 g) is about 50% or less. It turned out to be.
  • dihydrozincenoside R can be prepared from high-purity ginsenoside Rb at a yield of 97%, so dihydrozincenoside Rb is more efficient than ginsenoside Rbi. It can be used for the prevention, treatment, and treatment of neurological trauma, brain that causes blood flow disorders such as stroke, and neurological diseases.
  • ginsenoside derivatives such as dihydrozincenoside Rbi indicate that blood flow disorders are caused by diseases such as cerebrovascular disorders and spinal cord injuries. It has been invented to be a pharmaceutical composition for prevention, treatment or therapy.
  • ginsenoside derivatives such as dihydrozincenoside Rbt inhibit diseases of blood flow by inhibiting cell apoptosis or apoptosis-like cell death and promoting the regeneration and remodeling of blood vessels, resulting in impaired blood flow.
  • Example 5 Open wound treatment with external preparation for skin containing dihydrozincenoside R b
  • each open wounds 0 respectively mercy Dorojinsenosai de R b. 0 0 0 1 wt% (1 0 4% by weight), 0.0 0 0 0 1 wt% (1 0 5 wt%), 0 . 0 0 0 0 1 wt% (1 0 6 wt%), 0.0 0 0 0 0 0 1 wt% (1 0-7 wt%), of at a concentration Puropeto (ophthalmic white (Cerine) was applied once daily for 0.1 day 9 days. Only the same amount of the plot was externally applied to the control. Immediately after the animal was euthanized by anesthesia, the wound skin was collected and photographed.
  • Puropeto ophthalmic white (Cerine)
  • FIG. 9 shows the results.
  • Figure 9 is a photograph replacing the drawing.
  • FIG. 9 shows four examples, in which the first example, the second example, the third example, and the fourth example are shown from above.
  • Each two left side there are traces of open wounds in total 5 places three increments to the right, from the top of the left 1 0 - For 4 wt%, in the case of 1 0 5 wt%, on the right from 1 0 _ 6 wt%, 1 0 - for 7 wt%, shows the case of 0% (control).
  • 9 0.0 0 0 0 1 wt% as shown in FIG.
  • the concentration of the composition in the external preparation is not more than 0.001% by weight or less, preferably not more than 0.001% by weight, more preferably not more than 0. . 0 0 0 0 0 1 wt% (1 0-7 wt%) to set the front and rear or less Therefore, for the prevention, treatment or treatment of diseases that cause impaired blood flow (for example, wounds, burns, pressure sores, laser injuries, skin ulcers, hemorrhoids, etc.), dihydrozincenoside R bi etc.
  • diseases that cause impaired blood flow for example, wounds, burns, pressure sores, laser injuries, skin ulcers, hemorrhoids, etc.
  • the concentration of the composition in a topical or topical skin preparation is 0.001% by weight or less, preferably less than 0.01% by weight.
  • 1 wt% (1 0 5 wt%) or less, more rather preferably has 0.0 0 0 0 0 0 1 wt% (1 0 7% by weight) is preferably set in the following.
  • the upper limit of the concentration of the ginsenoside derivative in the external preparation for skin or topical administration for treatment or therapy is 1% by weight or less, preferably 0.1% by weight or less.
  • the liquid concentration is 100 gZm1 or less, preferably 100 ng / ml or less, more preferably 1 ng1 or less, and even more preferably 0.0000 001/100 ng / ml.
  • vascular endothelial cells, vascular smooth muscle cells, epithelial cells, epidermal keratinocytes, stem cells, fibroblasts, etc. are actively dividing, as described in PCTZ JPO 0555554.
  • topical application of low-concentration ginsenoside derivatives not only promotes regeneration and reconstruction of cut blood vessels, but also enhances skin epidermal tissue and dermis. It is thought to promote regeneration and remodeling of connective tissue, dermal papillae, sebaceous glands, nerves, sweat glands, dermal papilla, pilo erectoris, hair follicles, etc., and accelerate wound treatment.
  • dihydrozincenoside Rbi at a low concentration is far superior to that of peptidic factors (PDGF, EGF, bFGF).
  • the ointment or topical preparation containing dihydrozincenoside Rb at a low concentration used in this experiment may be used not only on the skin but also on any organ, tissue (cornea, tissue, etc.) that has caused wounds, injuries, impaired blood flow, or histopathological changes.
  • External application to oral mucosa, outer ear, gastrointestinal mucosa, nasal mucosa, tympanic membrane, vagina, bladder, uterus, urethra, airway mucosa, rectum, .anus, etc.) to regenerate and regenerate diseased tissues including blood vessels Construction can be accelerated.
  • suppositories containing ginsenosides are used for the prevention, treatment or treatment of hemorrhoids.
  • Topical administration to the anus, z, or rectum provides excellent results.
  • the amount of ginsenoside derivatives, especially dihydrozinosine Rbi, per 10 g of the product mixed was 0.1 mg or less, and preferably 0.1 mg or less. did.
  • the optimal skin external dose of ginsenoside derivatives, particularly dihydrozinsenoside Rbi, to humans or vertebrates having skin diseases is considerably small.
  • a ginsenoside derivative such as dihydrozincenoside Rb
  • the upper limit thereof is 0.1 lg or less, preferably 0.1 OO lg. It is as follows.
  • Example 6 Promotion of Bcl-X LmRNA expression, suppression of VEGF mRNA expression or suppression of caspase 3 mRNA expression in neuronal cells by dihydrozincenoside Rb
  • the pharmaceutical composition of the present invention has anti-apoptosis It is thought to prevent, treat or treat diseases or conditions that result in impaired blood flow through its action as well as its revascularization-promoting remodeling action.
  • ginsenoside derivatives such as dihydrozincenoside Rbi expressed the main factors (molecular groups) involved in cell apoptosis or blood vessel regeneration and remodeling. I checked whether to adjust. Therefore, as a typical molecules involved in ⁇ Pot one cis or apoptosis-like cell death, B c 1 - a X L and caspase 3, taken up VEGF Exemplary molecules involved in revascularization and Roh or rebuilding, of We examined whether the expression of the molecular group was changed by dihydroginsenoside Rbi.
  • cDNA was prepared from 3 ⁇ g of total RNA using oligo dT primer and reverse transcriptase.
  • PCR reaction uses Taq polymerase I went.
  • the PCR-primers used and the PCR reaction conditions are as follows. Incidentally, 3-actin is an internal standard.
  • Ant isense prime TAC TTG CGC TCA GGA GGA GCA ATG
  • Sense primer AAG CGT AGA CAA GGA GAT GCA
  • Ant isense primer GGA GCT GAT CTG AGG AAA AAC C
  • Sense primer CCA TGA ACT TTC TGC TCT CTT G.
  • Ant isense primer GGT GAG AGG TCT AGT TCC CG
  • Sense primer GCT AAC CTC AGA GAG ACA TTC ATG
  • Ant isense primer TTA GTG ATA AAA GTA CAG TTC TTT
  • FIG. 11 and FIG. Figures 11 and 12 are photographs replacing the drawings.
  • VEGF As shown in Fig. 11, compared to the neurons without dihydrozincenoside Rb, Bc1 was found in the neurons supplemented with dihydrozine senoside Rb at a concentration of 1,100 fg / m1. - the expression of X L and VEGF m RNA is enhanced. In particular, the strongest expression of VEG FmRNA was observed in the 1 fg / m1 addition group.
  • the rat VEGF is configured According to the number of amino acids to be used, VEGF120, VEGF164, and VEGF188 can be divided into three subtypes, so that the VEGFmRNA band is at least 2 as shown in Fig. 11. This is recognized. Furthermore, as shown in Fig.
  • the caspases were higher in the neurons to which dihydrozinsenoside Rb was added at a concentration of 1,100 fg / m1. 3 mRNA expression was suppressed. In particular, the strongest suppression of caspase 3 mRNA expression was observed in the 1 fg / m1 addition group.
  • ginsenoside derivatives are composed of books such as B e 1 — or caspase 3 (Experimental Medicine, Vol. 17, No. 13, Planning, Shigeichi Nagata, 1999, Yodo Increasing, decreasing or regulating the expression of the apoptosis-related molecule group described in A. et al. Suppresses apoptotic or apoptotic-like cell death.
  • Ginsenoside derivatives, especially dihydroginsenoside R are described in the publications such as VEGF (Experimental Medicine, Vol. 17, No. 6, Planning, Masashi Shibuya, 1999, Yodosha). It is thought that increasing, decreasing or regulating the expression of the assembly-related molecules promotes the regeneration and remodeling of blood vessels.
  • ginsenoside derivatives such as dihydroxydine senoside Rb may also enhance, attenuate or regulate the action of Bc1-1 and / or caspase-3 transcription factors (eg, NFKB, STATS, etc.). .
  • the pharmaceutical composition of the present invention has an action of promoting the regeneration and remodeling of blood vessels in addition to the above-described anti-apoptosis action, and thus, through these two actions, a disease or a disease that causes impaired blood flow.
  • Example 7 (Ginsenoside derivatives, in particular, dihydroxyxinenoside Rbi or Prevention, treatment, and treatment of suture failure due to epoxy ginsenoside Rb) Diabetics, the elderly, immunodeficiency patients, malnutrition patients, cancer patients, etc. often develop suture failure after surgery. Preventing this from happening is considered to be of paramount importance. Therefore, ginsenoside derivatives, particularly dihydroxyzine senoside R bi or epoxy ginsenoside R bi, are usually added at a dose of 0.001 mg or more per day, preferably before or after surgery.
  • ginsenoside derivatives especially dihydroxyzinenoside Rb or epoxydinosenoside Rb, a water-soluble base, an ointment base, an ophthalmic base, a fat-soluble base
  • An external preparation for the skin cream, gel, poultice, spray, ointment, etc.
  • a liquid or eye ointment may be prepared and applied or instilled at the surgical wound site and around the wound until the wound is healed.
  • ginsenosides in particular, dihydroxyzine senoside R b or epoxy ginsenoside R bi may be locally administered during the operation.
  • the concentration of the extracellular solution of the ginsenoside derivative in the local part is 100 g / m 1 (about 90 M) or less, preferably 100 ng / 1 (about 90 nM) or less, more preferably Is less than or equal to Ing Zml (about 0.9 nM), more preferably less than or equal to 100 fg Zml (about 90 fM).
  • dihydrozincenoside Rbi may be used as one of the ginsenoside derivatives.
  • Example 8 Treatment and treatment of radiation damage or burns caused by ginsenoside derivatives, especially dihydroxydine cenoside Rb or epoxy ginsenoside Rb. Extensive degeneration of the tissue and the failure to achieve satisfactory results with skin culture sheet transplantation can jeopardize the patient's prognosis. For such patients, skin tissue regeneration from transplanted skin sheets Ginsenoside derivatives, especially dihydroxy ginsenoside R bi or epoxy ginsenoside to promote the regeneration and reconstruction of lesions due to cell division and proliferation, migration to lesions, differentiation, and adhesion of healthy skin tissue constituent cells.
  • Inject R bi at a dose of 0.001 mg or more, preferably 0.1 mg or more, more preferably 10 mg or more per day, or a single or continuous infusion into the vein every day until symptoms are improved.
  • ginsenoside derivatives in particular, dihydroxyzine senoside Rb or epoxydinsenoside Rbi were injected intravenously, and ginsenoside derivatives, especially dihydroxyzine, were added to a water-soluble base or a fat-soluble base.
  • E port Kishijinsenosai de R b 1 contaminating skin external agent cream, gel, lotion down, cataplasms, sprays or ointments, etc.
  • skin external agent cream, gel, lotion down, cataplasms, sprays or ointments, etc.
  • the extracellular fluid concentration of ginsenoside derivatives, especially dihydroxyxenosenoside Rb or epoxyginsenoside Rb, at the lesion site was 100 ⁇ g / m1 (about 90 ⁇ M).
  • the dinzenoside derivatives especially dihydroxyxenosenoside Rbi or epoxyzine senoside Rt ⁇ , mixed into the base is adjusted so as to obtain 0 fM).
  • Ginsenoside derivatives in external preparations for skin In particular, the concentration of dihydroxyxenosenoside R bi or epoxy ginsenoside R bi should be 0.1% by weight or less, preferably 0.01% by weight or less. Is preferred.
  • dihydrozincenoside Rbi may be used as one of the ginsenoside derivatives.
  • Example 9 Prevention, treatment, and treatment of pressure sores with ginsenoside derivatives, particularly dihydroxyxenineside R bi or epoxy ginsenoside R bi)
  • Ginsenoside derivatives especially dihydroxyxenosenoside R bi or epoxy ginsenoside R bi, mixed with a water-soluble base or a fat-soluble base containing or not containing glucose , A poultice or a soft patch) and apply it constantly to the area of the pressure sore and its surroundings until the healing, reduction or deterioration of the sore.
  • concentration of ginsenoside derivatives, especially dihydroxyzinenoside Rb or epoxyzinsenoside Rbi, in the external preparation for skin should be 0.1% by weight or less, preferably 0.01% by weight or less. Is preferred.
  • the extracellular concentration of the ginsenoside derivatives in the local area is 100 g / m 1 (about 90 M) or less, preferably 100 ng Zm1 (about 90 nM) or less, more preferably 1 ng / m1 (about 0.9 nM) or less, still more preferably 100 fgZml (about 90 fM) or less
  • the amount of the ginsenoside derivatives, especially dihydroxy ginsenoside R bi or epoxy ginsenoside R b mixed into the base is adjusted so that In addition, if necessary, intravenous administration of ginsenoside derivatives, particularly dihydroxyzine senoside R bi or epoxy ginsenoside R bi as described in Example 7 and Example 8 I do.
  • Ginsenoside derivatives such as dihydroxyzincenoside Rbt or epoxy ginsenoside Rbi, as described herein, inhibit the spread of pressure wound lesions through potent cytoprotection, It is thought that a superior therapeutic effect will be exerted on the depressed wound lesion in which skin tissue is deficient by promoting the regeneration and reconstruction of blood vessels or skin tissue.
  • dihydrozincenoside Rb ⁇ may be used as one of the ginsenoside derivatives.
  • Example 10 Treatment of Peptic Ulcer with Ginsenoside Derivatives, Especially Dihydroxyzine Senoside R b! Or Epoxy Ginsenoside R b ⁇
  • ulcer and duodenal ulcer H 2 receptor inhibitors, pro Tonpo pump inhibitor, although gastrointestinal mucosa protective agent is mainly used, temporarily by an agent Even if the ulcer lesion heals, the ulcer lesion often recurs when the drug is stopped. Ulcer lesions are also frequently seen in Crohn's disease and ulcerative colitis, which are designated as intractable diseases of the gastrointestinal tract, and worsen the prognosis of patients. After the onset of gastric ulcer, duodenal ulcer, ulcerative colitis, or Crohn's disease, apply ginsenoside derivatives as early as possible, especially with dihydroxyzine cenoside R bi or epoxy ginsenocyse, while applying usual treatment.
  • Diabetic skin ulcer is an intractable disease accompanied by impaired blood flow at the lesion and loss of skin tissue, etc., but ginsenoside derivatives, particularly dihydroxide, which have the effect of promoting the regeneration and reconstruction of blood vessels and skin tissue
  • the effect can be obtained by intravenous administration, local injection or topical application of cyginsenoside R bi or epoxy ginsenoside R bt. That is, for patients with diabetic skin ulcer, ginsenoside derivatives, especially dihydroxyzine senoside R bi or epoxy ginsenoside R bi are usually added to the patient daily in addition to the usual treatment.
  • a single or continuous infusion daily at a dose of OO lmg or more, preferably 0.1 mg or more, more preferably 10 mg or more.
  • an external preparation for skin containing dihydroxyxenosenoside R bi or epoxy ginsenoside R bi may be applied to the lesion and its periphery as described in Example 8.
  • a ginsenoside derivative in particular, a physiological saline solution or a glucose solution of dihydroxyzinenoside R bi or epoxy ginsenoside R bt may be injected into a lesion site.
  • the extracellular fluid concentration of the ginsenoside derivatives, particularly dihydroxyzine senoside Rbt or epoxyginsenoside Rb, in the lesion was 100 ⁇ g / m1 (about 90 M) or less.
  • the ginsenoside derivatives are preferably added to the base such that the concentration is 100 fg / m 1 (about 90 fM) or less. Adjust the amount of bi or the amount of local saline or glucose solution (dissolving agent) containing them.
  • dihydroginsenoside R bi may be used as one of the ginsenoside derivatives.
  • Example 1 2 (Induction of Bel—xi_ Expression in Neurons by Ginsenoside Rbt)
  • ginsenosides including ginsenoside derivatives
  • B e 1 - cell death suppressing gene B e 1 - was investigated whether to induce the expression of x L.
  • the experimental procedure was based on the paper of the present inventors (Sakanaka, Tanaka) (Wen T.-, et al., J.
  • FIG. Figure 14 is a photograph replacing the drawing.
  • ginsenoside R bi enhances the expression of Be 1 — protein in neurons
  • a western blotting method was performed using an anti-Be 1 — x L protein antibody. After culturing rat cerebral cortical neurons in the presence or absence of ginsenoside Rbi for 48 hours, the cells were lysed with a sample buffer for electrophoresis, and electrophoresis was performed. gave. Thereafter, the electrophoretic protein was transferred to a two-nitrocellulose membrane and subjected to Western blotting. The results are shown in FIG. Figure 15 is a photograph replacing the drawing.
  • ginsenoside Rb significantly increased the expression of Be1-X protein in neurons by about 50% in the optimal concentration range of 1 to 100 fg / ml (Fig. 15, (Fig. 16).
  • Example 13 Activation of transcription factor STAT5 by ginsenoside Rbi
  • the reaction solution was purified using QiaExII Gel Extraction Kit (Qiagen), digested with restriction enzymes XhoI and HindIII, and electrophoresed on a 2% agarose gel. A bp DNA fragment was cut out. From the obtained gel slice, a DNA fragment was extracted using a QiaExII gel extraction kit (QiaExII Gel exraction kit) and inserted into pGL-2 Basic vector (Promega) (Betrox promoter L). ) (Fig. 17).
  • PCR reaction of 15 cycles was performed using primers RF1 and RR1.
  • the reaction solution was diluted 1000-fold (Solution A), and the resulting mixture was subjected to PCR for 30 cycles with Primer RF2 and Primer RR2 as type III, digested with Sacl and BamHI, pGL- 2 Basic vector was inserted into the SacI / BgIII site of the vector (Belt x promoter R) (No. 17 Figure).
  • the solution A was used as a type III, and a PCR reaction of 3'0 cycle was performed using each of the primer R F1 and the primer Mut-R, and the primer Mut-F and the primer R R1. Electrophoresis was performed on a 2% agarose gel, DNA fragments of 312 bp and 309 bp were cut out, and DNA fragments were extracted using a Qia EXII gel extraction kit (QiaExII Gel exraction kit). . The extracted DNA fragments are mixed at a weight ratio of 1: 1.
  • Primer LF1 5 '-ATACTTCCCAGCCGCAAAACGC-3'
  • the primary culture of the astrocyte was performed as follows.
  • Astrocytes were isolated from a Wistar rat immediately after birth by a known method, cultured in a culture flask, and transplanted to a 12-well plate two weeks later.
  • the astrosite was published by the present inventors (Sakanaka, Tanaka) (Fujita, H. et al., Glia, 18, 269-281, 1996; Ta aka, J. et al., Glia). , 20, 23-37, 1997; Tanaka, J. et al., Glia, 24, 198-215, 1998).
  • the gene transfer and Lucifera Zeattsey were performed as follows.
  • Transfection was performed for 5 hours in the presence of 10% FCS (fetal calf serum) using Lipofectamine (Invitrogen). Then change the medium Then, the cells were cultured at 37 ° C., and 100 fg / m 1 of ginsenoside Rb was added the next morning, followed by culturing at 37 ° C. for 24 hours. After washing the cell surface twice with PBS, the cells were lysed with Luciierase Cell Culture Lysis Reagent (Promega) 1001. The amount of luciferase in the solution was measured using a Luciferase Assaay System (Promega) and a Noreluminescence Sensor-J NR (AT TO).
  • FCS fetal calf serum
  • Lipofectamine Invitrogen
  • the ginsenoside R t is activated through the activation of the transcription factor STAT5, which has a binding site upstream of exon 2 (Exon 2) of the Be 1 —X gene (Fig. 17). It can be said that it up-regulates the production of luciferase, one of the intergenes (Fig. 18).
  • B e 1 - to X L measured easy repo Isseki - implementing the promoter mediation Si in a form of replacing the Gene (Lucifera Ichize)
  • ginsenoside Rb promotes the expression of the Be1-X! _ Gene through activation of the transcription factor STAT5 in actual cells.
  • ginsenosides such as ginsenoside Rbi also increase the enzyme activity of JAK2, phosphorylating STAT5, and promoting homodimer formation and nuclear translocation of phosphorylated STAT5. It is thought to be. As a result, STAT5 is thought to function as a transcription factor.
  • Example 14 Simulation of neuronal Bc1-1 protein expression by dihydrozincenoside Rb
  • dihydrozincenoside Rbi significantly increased the expression level of Bc1-XL protein in neurons in the optimal concentration range of 1 to 100 fg / m1. Increased.
  • P ⁇ 0.05 indicates P ⁇ 0.05, and the statistical analysis method is based on AN OVA + Fisher's PLSD.
  • ginsenoside derivatives such as dihydrozincenoside Rbi also enhance the expression of Bc1-XL in neurons similarly to ginsenoside Rbi.
  • the pharmacological actions of dihydrozinsenoside R bi and ginsenoside R bi are extremely similar, so it is natural that dihydrozinsenoside R bt As well as ginsenoside Rb. It is thought that through activation of the transcription factor STAT5, Bcl-X expression in neurons is up-regulated.
  • Example 15 Promotion of VEGF mRNA expression of astrocytes by ginsenoside Rbt
  • the present inventors set the ginsenoside R b! In order to examine whether or not increases the expression of VEGF mMA in the cells, an experiment was performed by selecting an astrocyte site as an example of the cells.
  • Astrocytes were isolated from a Wistar rat immediately after birth by a known method, cultured in a culture flask, and transplanted 12 days later to a 10 cm dish coated with poly L-lysine.
  • the astrocytes were cultured in a DMEM medium containing 10% fetal calf serum (FCS). Three to four days later, the medium was replaced with a serum-free medium, ginsenoside Rbt was added at a concentration of 0.1000 fg / m1, and the cells were cultured for 6 hours.
  • FCS fetal calf serum
  • the astrosite was published by the present inventors (Sakanaka, Tanaka) (Fujita, H. et al., Glia, 18, 269-281, 1996; Tanaka, J.
  • cDNA was prepared from 3 g of the total RNA using oligo dT primer and reverse transcriptase.
  • the PCR reaction was carried out using T aq polymerase according to the paper by Tonello (Tonello, FEBS Letters, 442, 167-172, 1999).
  • Tonello Tonello, FEBS Letters, 442, 167-172, 1999.
  • the PCR-primer and PCR reaction conditions used are as follows.
  • -Actin is an internal standard. (1) ⁇ -actin
  • Antisense primer TAC TTG CGC TCA GGA GGA GCA ATG
  • Sense primer CCA TGA ACT TTC TGC TCT CTT G
  • Antisense primer GGT GAG AGG TCT AGT TCC CG 1) One cycle at 94 ° C for 5 minutes, 2) 35 cycles of 94 ° C for one minute, 62 ° C for 1.5 minutes, and 72 ° C for 1.5 minutes.
  • FIG. Figure 21 is a photograph replacing the drawing.
  • ginsenoside R b Compared to the unloaded astrocytes, the expression of VEGFmRNA was enhanced in the astrocytes to which ginsenoside Rb was added at a concentration of 100 fg / 1.
  • Example 16 Induction of VEGF Expression of Human Skin Keratinocytes by Ginsenoside Rbi
  • ginsenoside Rbi also induced VEGF mRNA expression in human skin keratinocytes.
  • human skin keratinocytes are cultured in a monolayer, and ginsenoside Rbt is added to the culture medium at a concentration of 0 to 10 pg / ml, and after 0, 1, 3, 6, and 12 hours, Total RNA was extracted from skin keratinocytes. Thereafter, RT-PCR was carried out at a cycle number of 27 using the primers shown below. The results are shown in FIG. FIG. 22 is a photograph of R T—P C R instead of a drawing.
  • Sense primer TGG CAG AAG GAG GAG GGC AGA AT
  • VEGF mRNA expression was induced within 12 hours. .
  • ginsenoside Rb increases VEGF protein expression.
  • human skin keratinocytes are cultured in a monolayer, and the ginsenoside Rbi is added to the culture medium at a concentration of 0 to 10 pg Zm1, and the culture supernatant is collected 24 or 48 hours later. did.
  • the VEGF ELISA kit human R & D system
  • the VEGF concentration in the culture supernatant was measured.
  • was measured The results are shown in FIG.
  • ginsenoside R bi causes VEGF expression induction through activation of transcription factor HIF-1. For this reason, the pGL-3 promoter—evening—vector ( pr0 mega) at the Bg1II site on the multiple cloning site
  • TAC GT G A six-time repeated sequence of “TAC GT G”, that is, (TAC GT G) 6 was introduced.
  • TAC GTG is a common U (consensus sequence) of HRE (hypoiar espons e element) on DNA that binds to transcription factor HIF-1. Therefore, in the present invention, the pGL-3 promoter overnight vector (TAC GTG) 6 is referred to as HRE-luciferase plasmid.
  • HRE-luciferase plasmid As a control, pGL-3 promoter vector-1 (plasmid) was used.
  • the primary culture of the astrocyte was performed as follows.
  • Astrosites were isolated from the Wistar rat immediately after birth by a known method, cultured in a culture flask, and relocated to a 12-well plate two weeks later.
  • the astrosite was published by the present inventors (Sakanaka, Tanaka) (Fujita, H. et al., Glia, 18, 269-281, 1996; Tanaka, J. et al., Glia). , 20, 23-37, 1997;
  • Nerve cells were isolated from the cerebral cortex of the 17th day of the embryo using Wistar rat cerebral cortex by a known method and cultured on a 12-well plate coated with poly L-lysine. . Inherited and derived neurons on day 5 of culture Used to enter.
  • the gene transfer and Lucifera Zeattsey were performed as follows.
  • plasmid DNA ie, HRE luciferase plasmid or pGL-3 promoter vector
  • Lipofectamine Invitrogen
  • the transfection was performed for 5 hours. Thereafter, the medium was replaced with a fresh medium and cultured overnight at 37 ° C. The next morning, 0 or 100 fg of ginsenoside Rbi was added, and cultured at 37 for 24 hours. After washing the cell surface twice with PBS (phosphate-buffered saline), the cells were lysed with Luciferase Cell Culture Lysis Reagent (Promega) 100 ⁇ 1. The amount of luciferase in the solution was measured using a Luciferase Assay System (Promega) and a luminescent sensor JNR (ATO). The results are shown in FIG.
  • the amount of luciferase was increased in the case where ginsenoside Rbi100fg / m1 was added as compared with the case where ginsenoside was not added.
  • ginsenosides such as ginsenoside Rb ⁇ ⁇ ⁇ ⁇
  • * In FIG. 24 indicates P ⁇ 0.01.
  • Statistical analysis is based on students' tests.
  • Example 18 (Activation of skin keratinocyte transcription factor HIF-1 by ginsenoside Rbi)
  • ginsenoside Rbi activates the transcription factor HIF-1 even in human skin keratinocytes.
  • human skin keratinocytes were cultured in a monolayer on a 12-well type 1 collagen dish, and plasmid DNA was added.
  • ginsenosides such as ginsenoside Rbt, activate the transcription factor HIF-1 in all cells, including fast mouth sites or human skin keratinocytes, and cause the binding of HIF-1 to HRE. It is considered something.
  • Example 19 Induction of astrocyte VEGF expression by dihydrozincenoside Rbi
  • ginsenoside derivatives promote VEGF raRNA expression in astrocytes, similarly to ginsenoside Rbi. Therefore, an experiment was carried out by selecting a reduced derivative, that is, dihydrozincenoside R b represented by the above structural formula, as a representative example of the ginsenoside derivatives.
  • Astrocytes were isolated by a known method from a Wistar rat immediately after birth, cultured in a culture flask, and 12 days later, a 10 cm dish (10 cm) coated with poly L-lysine was used. dish).
  • the astrocytes were cultured in a DMEM medium containing 10% fetal calf serum (FCS). After 3 to 4 days, the medium was replaced with a serum-free medium, and dihydrozincenoside Rb was added at a concentration of 0, 1 fg / ml and 100 fg / m1, and the cells were cultured for 6 hours.
  • FCS fetal calf serum
  • dihydrozincenoside Rb was added at a concentration of 0, 1 fg / ml and 100 fg / m1, and the cells were cultured for 6 hours.
  • the astrosite was published by the present inventors (Sakanaka, Tanaka) (Fujita, H.
  • cDNA was prepared from 3 g of the total RNA using oligo dT primer and reverse transcriptase.
  • the PCR reaction was carried out using Taq polymerase according to the paper by Tonello (Tonello, FEBS Letters, 442, 167-172, 1999).
  • Tonello Tonello, FEBS Letters, 442, 167-172, 1999.
  • PCR used primer And PCR reaction conditions are as follows. I3-actin is an internal standard. (1) — Actin
  • Antisense primer TAC TTG CGC TCA GGA GGA GCA ATG
  • Sense primer CCA TGA ACT TTC TGC TCT CTT G
  • Antisense primer GGT GAG AGG TCT AGT TCC CG
  • FIG. Figure 26 is a photograph replacing the drawing.
  • ginsenoside Rb was added at concentrations of 1 fg / m1 and 100 fg / m1 compared to ginsenoside Rbi-free astrocytes.
  • VEGF expression of VEGF mRNA was enhanced.
  • the VEGF of the rat is divided into three subtypes, VEGF120, VEGF164, and VEGF188, corresponding to the number of constituent amino acids. At least two are allowed as shown in the figure.
  • Example 20 Induction of VEGF protein expression in neurons by dihydrozincenoside Rb
  • the present inventors investigated whether the dihydrozincenoside R bi enhances not only VEGF mRNA but also VEGF protein expression in nerve cells by using an anti-VEGF monoclonal antibody (manufactured by Sanyo Cruz Co., Ltd.) by Western blotting.
  • Rat cerebral cortical neurons were cultured for 24 hours in the presence or absence of dihydrozincenoside Rbi, and the cells were lysed with a sample buffer for electrophoresis, and electrophoresis was performed. After that, the electrophoretic protein is transferred to a Nitrocellulose membrane and Western blotted. Was done. The details of the experimental procedure of the Western plot are described in detail by the present inventors.
  • FIG. Figure 27 is a photograph of a Western plot that replaces the drawing.
  • dihydrozincenoside Rbt significantly increased the expression level of VEGF protein in neurons in the optimal concentration range of 1 to 100 fg / m1. . * In FIG. 27 and FIG. 28 indicates P ⁇ 0.05, and the statistical analysis method is based on AN0VA + Fisher's PLSD.
  • ginsenoside derivatives such as dihydrozincenoside Rbt also enhance VEGF expression in cells, like ginsenoside Rbi.
  • the pharmacological actions of dihydrozincenoside R b and ginsenoside R bi are extremely similar, and, of course, dihydrozincenoseside R
  • ginsenoside derivatives such as b ′ are thought to upregulate VEGF expression in cells through activation of transcription factor HIF-1.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a ginsenoside derivative such as dihydrozincenoside Rb as an active ingredient for preventing, treating or treating a disease or condition causing blood flow disorder, or a medicament for promoting the regeneration and reconstruction of blood vessels.
  • a composition comprising a ginsenoside derivative such as dihydrozincenoside Rb as an active ingredient for preventing, treating or treating a disease or condition causing blood flow disorder, or a medicament for promoting the regeneration and reconstruction of blood vessels.
  • ginsenosides as a lead compound, particularly ginsenoside Rbh or natural products containing ginsenoside Rb or extracts thereof, are excellent in a low optimal extracellular solution concentration range.
  • a pharmaceutical composition having an anti-apoptotic action and a promoting action of regenerating and / or remodeling blood vessels has been invented.
  • Low-concentration and low-dose ginsenoside derivatives can be used in all diseases and conditions that cause impaired blood flow through vascular regeneration / remodeling promoting action and / or anti-apoptotic action. It is useful for prevention, treatment or treatment.
  • the pharmaceutical composition of the present invention promotes expression of B c 1 — ⁇ ⁇ Suppresses apoptosis or apoptosis-like cell death of all types of cells through suppression of expression of caspase-3 or caspase 3, and promotes vascular regeneration and remodeling through increased VEGF expression .
  • ginsenosides such as ginsenoside Rbt or dihydroginsenoside Rbi activate cell transcription factor STAT5 and / or transcription factor HIF-1 to express Bel-XL expression and And / or found to induce VEGF expression.
  • the Jinsenosai de such as Jinsenosai de R b t, natural products or their extracts containing Jinsenosai de R bi also it is thought to have similar effects, efficacy and usages and Jinsenosai earth derivative of the present invention.
  • the present invention relates to a medicament for preventing, treating or treating diseases causing blood flow disorders, which comprises administering a test substance to cultured cells and measuring the effect of regulating the expression of the Be1-2 protein group. It also provides a method of searching for a composition.

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Abstract

L'invention concerne des préparations intraveineuses efficaces, des préparations pour la peau à usage externe, ou des compositions médicinales contenant des dérivés de ginsenosides (en particulier le dihydroginsenoside Rb1), utilisées dans l'activation de la régénération et de la reconstruction vasculaire ou dans la prévention et le traitement de maladies ou d'états pathologiques entraînant une insuffisance circulatoire, ainsi que des ginsenosides (en particulier le dihydroginsenoside Rb1) qui sont également utilisés dans la prévention et le traitement de maladies ou d'états pathologiques entraînant une insuffisance circulatoire, via l'activation d'un facteur de transcription STAT5 et/ou d'un facteur de transcription HIF-1. L'invention concerne plus précisément des préparations intraveineuses, des préparations pour la peau à usage externe ou des compositions médicinales contenant des dérivés de ginsenosides (en particulier le dihydroginsenoside Rb1), particulièrement utilisées dans l'activation de la régénération et de la reconstruction vasculaire ou dans la prévention ou le traitement de maladies ou d'états pathologiques entraînant une insuffisance circulatoire. Ces compositions médicinales contiennent des ginsenosides tels que le dihydroginsenoside Rb1 et sont particulièrement utilisées dans la prévention et le traitement de maladies ou d'états pathologiques entraînant une insuffisance circulatoire, via l'activation d'un facteur de transcription STAT5 et/ou d'un facteur de transcription HIF-1.
PCT/JP2002/000368 2001-02-26 2002-01-21 Promoteurs de régénération vasculaire WO2002067950A1 (fr)

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JP2001-51151 2001-02-26
JP2001051151 2001-02-26
JP2001365282A JP2002322068A (ja) 2001-02-26 2001-11-29 血管再生促進剤
JP2001-365282 2001-11-29

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WO2002067950A1 true WO2002067950A1 (fr) 2002-09-06

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WO (1) WO2002067950A1 (fr)

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WO2002069980A2 (fr) * 2001-03-07 2002-09-12 Cambridge University Technical Services Limited Composes pharmaceutiquement efficaces et utilisation de ces composes
EP1575982A1 (fr) * 2002-12-26 2005-09-21 Amorepacific Corporation Promoteur destine a la production d'acide hyaluronique contenant un compose k ginsenoside
US8927033B2 (en) 2009-12-21 2015-01-06 Lion Corporation Hyperlipemia-ameliorating agent, anemia-ameliorating composition, uric-acid-level-reducing composition, and food or beverage
US11554161B2 (en) 2006-06-07 2023-01-17 Genzyme Corporation Gene therapy for amyotrophic lateral sclerosis and other spinal cord disorders

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US8124582B2 (en) * 2002-12-06 2012-02-28 Fibrogen, Inc. Treatment of diabetes
JP6386173B2 (ja) * 2015-07-16 2018-09-05 インテリジェント シンセティック バイオロジー センター 血管漏出症候群の予防または治療用組成物

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002069980A2 (fr) * 2001-03-07 2002-09-12 Cambridge University Technical Services Limited Composes pharmaceutiquement efficaces et utilisation de ces composes
WO2002069980A3 (fr) * 2001-03-07 2004-04-15 Univ Cambridge Tech Composes pharmaceutiquement efficaces et utilisation de ces composes
EP1575982A1 (fr) * 2002-12-26 2005-09-21 Amorepacific Corporation Promoteur destine a la production d'acide hyaluronique contenant un compose k ginsenoside
EP1575982A4 (fr) * 2002-12-26 2010-05-26 Amorepacific Corp Promoteur destine a la production d'acide hyaluronique contenant un compose k ginsenoside
US8173607B2 (en) 2002-12-26 2012-05-08 Amorepacific Corporation Promoter for the production of hyaluronic acid containing ginsenoside compound K
US11554161B2 (en) 2006-06-07 2023-01-17 Genzyme Corporation Gene therapy for amyotrophic lateral sclerosis and other spinal cord disorders
US8927033B2 (en) 2009-12-21 2015-01-06 Lion Corporation Hyperlipemia-ameliorating agent, anemia-ameliorating composition, uric-acid-level-reducing composition, and food or beverage

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