WO2003094966A1 - Agent d'arterialisation et agent d'antiarterialisation - Google Patents

Agent d'arterialisation et agent d'antiarterialisation Download PDF

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WO2003094966A1
WO2003094966A1 PCT/JP2003/005901 JP0305901W WO03094966A1 WO 2003094966 A1 WO2003094966 A1 WO 2003094966A1 JP 0305901 W JP0305901 W JP 0305901W WO 03094966 A1 WO03094966 A1 WO 03094966A1
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activin
angiogenesis
vegf
angiogenic
agent
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PCT/JP2003/005901
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English (en)
Japanese (ja)
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Itaru Kojima
Kyoko Maeshima
Yuzuru Eto
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Ajinomoto Co.,Inc.
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Priority to AU2003231456A priority Critical patent/AU2003231456A1/en
Publication of WO2003094966A1 publication Critical patent/WO2003094966A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1796—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention provides an angiogenic agent and an anti-angiogenic agent effective for angiogenic therapy and anti-angiogenic therapy.
  • Follistatin a protein that binds to activin, was found to have an activity to promote follicle-stimulating hormone (FSH) production in cultured pituitary cells (Esch, FS et al., Mol. Endocrinol .; 1 (11): 849-55 (198 7)). Initially, it was thought that follistatin inhibited the secretion of FSH, and thus had an adverse effect on activin, which has a secretion promoting effect on FSH. Subsequent studies have shown that it is a specific binding protein of activin and binds to activin to inhibit activin activity (Nakamura, T. ei.al., Science, 247 (4944): 836 -8 (1990)).
  • FSH follicle-stimulating hormone
  • activin A activin A
  • activin AB activin AB
  • activin B activin B
  • activin As pharmacological effects of activin, in addition to the above-mentioned FSH secretion-promoting action, it has been reported that it promotes erythropoiesis, promotes bone formation, and promotes wound healing. It is known that an activin inhibitor has a therapeutic effect on kidney disease (Japanese Patent Application Laid-Open No. 2001-288111). However, it is not known that activin has an angiogenesis-promoting effect, that is, it is not only effective as an angiogenic agent for angiogenesis therapy, but rather, it has an activity of inhibiting angiogenesis. Having Have been reported.
  • follisutin As for the pharmacological effects of follisutin, it is known that, in addition to the aforementioned FSH secretion inhibitory action, local administration promotes liver cell proliferation (Kogure, K. et al., Hepatology; 24 ( 2): 361-6 (1996), Kojima, I., BIO Clonica; 883 (12), 43-46 (1997)). It has also been reported that follistatin antagonists promote healing with less scarring against wounds and fibrotic disorders (W097 / 15321), and that follastin inhibits erythropoiesis by systemic administration. ing. However, it is not known that foliostatin has an inhibitory effect on angiogenesis (Fanangiogenic effect), that is, it is not only effective for anti-angiogenic therapy, but also promotes angiogenesis. It is reported to have activity.
  • angiogenic agents applicable to angiogenesis therapy include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FG.F), EGF (epithelial cell growth factor), and platelet-derived growth factor ( Expectations are high for PDGF) and hepatocyte growth factor (HGF), but they have not achieved clinical efficacy.
  • VEGF vascular endothelial growth factor
  • b-FG.F basic fibroblast growth factor
  • EGF epihelial cell growth factor
  • HGF platelet-derived growth factor
  • thrombosbondin, angiostin, endostatin, interferon, i3, etc. are expected as anti-angiogenic agents applicable to anti-angiogenesis therapy, but they also have a clinical therapeutic effect. Has not been reached. Disclosure of the invention
  • the present invention examines in detail the presence or absence of the action of activin on angiogenesis, further examines the presence or absence of the action of follistatin on the activin action, and proposes a drug effective for angiogenesis therapy and anti-angiogenesis therapy.
  • the task is to provide.
  • activin has an effect of maintaining the survival of vascular endothelial cells and promoting proliferation. Furthermore, they found that activin promotes angiogenesis (lumen formation) in which vascular endothelial cells are cultured in collagen gel. By focusing on the fact that activin is involved in promoting angiogenesis, it was demonstrated using follistatin that the angiogenesis could be suppressed by suppressing its action, and the present invention was completed. Reached.
  • the present invention relates to an angiogenic agent containing an activin promoter as an active ingredient. is there.
  • the present invention provides the aforementioned angiogenic agent, wherein the activin promoting agent is activin.
  • the present invention also provides the angiogenic agent, which is administered together with a vascular endothelial growth factor.
  • the present invention provides an anti-angiogenic agent containing an activin inhibitor as an active ingredient.
  • the present invention also provides the anti-angiogenic agent used for treating an angiogenic disease involving an actipin effect.
  • the present invention provides the above anti-angiogenic agent, wherein the activin inhibitor is follistatin.
  • the present invention also provides an angiogenesis regulator comprising the angiogenic agent and the anti-angiogenic agent.
  • Angiogenesis is thought to be regulated by the well-balanced interaction of promoters and inhibitors.
  • vascular endothelial growth factor VEGF
  • basic fibroblast growth factor b-FGF
  • PDGF platelet-derived growth factor
  • HGF hepatocyte growth factor
  • EGF epithelial cell growth factor
  • angiopoietin suppressors such as thrombosbondin, angiostin, endostatin, interferon, and / 3 are known.
  • chemokines have a promoting effect and some have a suppressing effect.
  • Blood vessels are important structures for transporting blood to systemic tissues, and abnormalities in the vascular system are closely related to various lesions and cause their onset and progression.
  • angiogenesis is said to be involved in the development and exacerbation of the pathological condition in a living body after growth.
  • pathological conditions such as rheumatoid arthritis, cancer, and psoriasis vulgaris
  • suppression of angiogenesis leads to improvement of the above-mentioned angiogenic diseases Be expected.
  • angiogenesis is often a problem It is considered that diabetic retinopathy is one of the causes of excessive angiogenesis. On the contrary, it has been pointed out that angiogenesis is reduced in ischemic sites such as limbs of diabetic patients. As described above, abnormalities in angiogenesis are related to the onset and progress of many diseases, and thus inhibitors or promoters are promising as effective treatments.
  • Angiogenesis agents are used as angiogenesis therapies to promote angiogenesis, such as ischemic disease, atherosclerosis, atherosclerosis, obstructive arteriosclerosis, lower limb vascular obstruction, myocardial infarction, angina, cerebral infarction, intractable disease It can be used for the prevention and treatment of diseases in which the promotion of angiogenesis leads to improvement of the condition, such as skin ulcers, and for the prevention and treatment of diabetes through the regeneration of islands.
  • anti-angiogenic agents are used as anti-angiogenic therapies to prevent excessive angiogenesis. It can be used for the prevention and treatment of diseases whose progress is related to the onset and progression of disease states, so-called angiogenesis diseases.
  • FIG. 1 is a diagram showing the structure of a human forisintin cMA expression vector.
  • FIG. 2 is a diagram showing the purification of follistatin using HPLC.
  • the y-axis represents the ( ⁇ _D 28Q), showing a horizontal axis elution time (minute). Recombinant human foristin was eluted in the area indicated by the arrow.
  • FIG. 3 is a graph showing the activin inhibitory activity of purified follistatin.
  • the inhibitory activity against 5 n / ml of activin A was measured using EDF Atssay.
  • the vertical axis shows EDF activity, and the horizontal axis shows follistin concentration.
  • N 6 in each group.
  • FIG. 4 is a diagram (photograph) showing the promotion of angiogenesis by activin. ⁇ Aorta
  • the vascular endothelial cells were cultured on collagen gel and observed on the 7th day under a microscope. No addition (A), addition of 100 ng / ml VEGF (B), addition of 4 nM activin (C), addition of 100 ng / ml VEGF and 4 nM activin (D).
  • HE staining was performed on paraffin-embedded sections from experiments in which 4 nM activin was added (E).
  • FIG. 5 is a view quantitatively showing promotion of angiogenesis by actipin.
  • Aorta The vascular endothelial cells were cultured on collagen gel, and on the 5th day, 50 colonies were observed under a microscope and counted using image analysis software.
  • the vertical axis is the blood vessel length (tube length) (A), vascular structure (Bubble) (B). * Is significant at P ⁇ 0.05 compared to control.
  • FIG. 6 is a diagram (photograph) showing the inhibitory effect of follistatin on the promotion of angiogenesis by VEGF. ⁇
  • the aortic vascular endothelial cells were cultured on collagen gel, and microscopically observed on the seventh day. No addition (A), addition of 100 ng / ml VEGF (B), addition of 100 ng / ml VEGF and 10 nM follistatin (C).
  • FIG. 7 is a diagram (photograph) showing the inhibitory effect of TruncatedacactivinintyppeIIrececptor on the promotion of angiogenesis by VEGF.
  • the aortic vascular endothelial cells transfected with AdExLacZ (A, B) and AdextARII (C, D) were cultured on collagen gel and observed microscopically on day 7.
  • FIG. 8 is a diagram (photograph) showing the effect of activin on the expression of VEGF and VEGF receptor in vascular endothelial cells. ⁇ Culture the aortic vascular endothelial cells in the presence of 4 nM activin for 0-36 hours, and measure VEGF (A), F1t-1 (B), F1k-1 (B) by Western blotting did.
  • FIG. 9 is a diagram (photograph) showing the effect of VEGF on the expression of activin in vascular endothelial cells.
  • the aortic vascular endothelial cells were cultured in the presence of 100 ng / ml VEGF for 0 to 36 hours, and the amount of activin (j3A subunit) was measured by Western blotting.
  • BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
  • activin When activin was added to an experimental system for culturing vascular endothelial cells in a collagen gel, which is often used as a method for evaluating angiogenesis, a clear capillary network (lumen formation) was observed. In other words, it has been clarified that activin has an angiogenic effect, but this is the first discovery as far as the inventors know.
  • VEGF When VEGF was added to this experimental system, an angiogenic effect equivalent to that of actipin was observed, and the VEGF effect reported so far was confirmed.
  • Activin and VEGF Surprisingly, it was found that when both were added, they exhibited a more remarkable angiogenic action than when each was added alone.
  • follastin which is a specific binding protein of activin and is known to inhibit activin activity. investigated. In an experimental system for observing vascular endothelial cell lumen formation, it was found that the angiogenesis-promoting effect of VEGF was significantly attenuated by the simultaneous addition of follistatin. Not surprisingly, the angiogenic effect of activin was suppressed by foristin.
  • the angiogenic agent of the present invention contains an activin promoter as an active ingredient.
  • activin promoting agent enhances the physiological function of activin. It may be one that promotes the physiological function of activin or one that promotes the production of activin itself. Further, it may be one that promotes signal transduction caused by binding of activin to the activin receptor.
  • the anti-angiogenic agent of the present invention contains an activin inhibitor as an active ingredient.
  • activin inhibitor means a substance that reduces or eliminates the physiological function of activin, and may be one that directly binds to activin to inhibit the physiological function of activin. It may be one that inhibits its own production. Further, it may be one that inhibits signal transmission caused by binding of activin to an activin receptor.
  • the activin inhibitor include follistatin, an anti-activin antibody, an activin receptor inhibitor or an anti-activin receptor antibody, a signal transduction inhibitor related to the activin receptor, and an activin production inhibitor.
  • the inhibitor of the activin receptor include a protein or a compound having a structure similar to folistatin, which blocks the activin receptor and inhibits the binding between activin and follistatin.
  • the activin production inhibitor include antisense DNA, RNAi, and ribozyme against the activin gene. The effect of the activin enhancer can be determined by measuring the activity of the activin in the presence and absence of the activin.
  • the activity of activin is a differentiation-inducing effect on erythroid cells (EDF Atssei: Eto, Y. et. Al., Biochem. Biophys. Res. Commun .; 142 (3): 1095-103 (1987))
  • EDF Atssei Eto, Y. et. Al., Biochem. Biophys. Res. Commun .; 142 (3): 1095-103 (1987)
  • it can be measured by an in vitro test using the follicle stimulating hormone secretion promoting action on pituitary cells as an index.
  • the effect of an activin inhibitor can be determined by measuring the activity of the activin in the presence and absence of the activin inhibitor.
  • the activity of activin can be measured by an in vitro test using an index such as a differentiation-inducing effect on erythroid cells (EDF Atsusei) or a follicle-stimulating hormone secretion promoting effect on pituitary cells.
  • activin inhibitors are used regardless of the type or origin of follis-tin.
  • the effects of the present invention can be obtained as long as they have harmful effects.
  • actipin inhibitory effect not only human follistatin but also follistatin derived from animals such as pigs may be used. There may be.
  • Foristin is a glycoprotein with a molecular weight of the peptide portion of 30,000 to 40,000.It differs in the number of amino acid residues, such as 315, 303, 288, and the site of glycosylation. It is known that the numbers are different. In addition, the effects of the present invention can be obtained even in the case of having other structural changes as long as it has the ability to bind to activin and retains the same activity as an activin-binding protein.
  • the natural form of foristin is extracted from animal organs, such as the ovaries, and then purified.
  • Recombinant follistatin is prepared by incorporating the human or animal foristatin cDNA into an appropriate expression vector, introducing the gene into appropriate animal cells, and purifying the cells from a culture of the cells. .
  • Recombinant follistatin can be prepared using DNA encoding follistatin, according to conventional methods for producing a heterologous protein by recombinant techniques.
  • DNA encoding follistatin and a method for producing recombinant follistin using the DNA is disclosed in W089 / 01945.
  • those purified from a culture solution of CHO cells into which cDNA corresponding to human foristatin composed of 3 15 amino acids was incorporated were used.
  • the angiogenic agent of the present invention is provided as a medicament in the form of a pharmaceutical composition containing an activin promoter alone such as activin, or an activin promoter and a pharmaceutical additive.
  • the activin promoter may be used alone, or a plurality of activin promoters may be used in combination. Further, other angiogenic agents such as VEGF may be combined with the activin promoter.
  • the anti-angiogenic agent of the present invention is provided as a medicament in the form of a pharmaceutical composition containing an activin inhibitor alone, such as follistatin, or an activin inhibitor and a pharmaceutical additive.
  • the activin inhibitor may be used alone, or two or more activin inhibitors may be used in combination.
  • other anti-angiogenic agents such as anti-VEGF antibody may be combined with the actipin inhibitor.
  • An angiogenesis regulator is constituted by combining the above-mentioned angiogenesis agent and an anti-angiogenesis agent.
  • Examples of the dosage form of the angiogenic agent and the anti-angiogenic agent of the present invention include injections, sublinguals, transdermal patches, tablets, capsules, fine granules, syrups, suppositories, ointments, eye drops and the like. Can be Of these, injections, sublinguals, and transdermal patches are preferred.
  • pharmaceutically acceptable excipients for example, lactose, potato starch, calcium carbonate, sodium alginate and the like may be distributed according to the dosage form.
  • other ingredients usually used in pharmaceutical preparations for example, proteins such as serum albumin, salts for buffering action and osmotic pressure adjustment, carriers, excipients and the like may be added.
  • the dose of the angiogenic agent of the present invention varies depending on the age, symptoms, etc. of the patient when using activin.In general, the dose of activin is 0.1 lg / kg per adult per day for intravenous administration. ⁇ : L OmgZkg range, preferably 1 gZkg to 1 mgZkg.
  • the dosage of the angiogenic agent of the present invention varies depending on the age, symptoms, etc. of the patient when folistatin is used as an activin inhibitor.
  • the amount of follistatin is 0.
  • L OmgZkg preferably lg / kg-: Lmg / kg.
  • the angiogenic agent of the present invention is useful for the treatment and prevention of ischemic diseases.
  • diseases include lower limb vascular obstruction, but are not limited thereto.
  • the anti-angiogenic agent of the present invention is useful for the treatment and prevention of angiogenesis disease.
  • examples of such a disease include diabetic retinopathy and cancer, which are considered to be associated with the activin action. It is not limited to but is widely applied.
  • angiogenesis regulator of the present invention is useful for such diseases. That is, an angiogenic agent is administered when it is necessary to promote angiogenesis, and an anti-angiogenic agent is administered when it is necessary to inhibit angiogenesis. One is administered systemically and the other is administered Can also be administered topically.
  • the production of follistatin was measured using the activin neutralizing activity as an index.
  • the activin activity was determined using the EDF assay and prepared according to the method described in the literature (Eto, Y. et. Al., Biochem. Biophys. Res. Commun .; 142 (3): 1095-103 (1987)). 5 ng / m 1 of activin A
  • the inhibitory activity on (ED F) activity was determined.
  • the clone showing the highest production amount was selected and cultured using a roller bottle to obtain about 10 L of a culture solution.
  • concentration with an ultrafiltration membrane MW500 Ocut-off manufactured by Millipore
  • the mixture was charged into a heparin column (70 ml bed volume) equilibrated with 50% PBS.
  • the column was eluted with 50% PBS containing 1.5 M NaC1, and the protein elution portion was collected by a UV monitor.
  • One-fifth of this amount is HP LC (YMC Pack S -10 834 / 20mm) and eluted with an acetonitrile concentration gradient.
  • Figure 2 shows the elution pattern.
  • Angiogenesis was performed as follows.
  • the aortic vascular endothelial cells were suspended in a neutral collagen solution (manufactured by KOHKEN) at a cell density of 1 ⁇ 10 6 Zm 1 and seeded on a 24-well plate, and kept at 37C for incubation. After confirming that the collagen had turned into a gel, the above medium was added, and the test substance was added at the same time. Culture was performed while changing the medium every 3 to 4 days, and micrographs were taken.
  • RNA of aortic vascular endothelial cells was extracted using Trizol Reagent (manufactured by Gibeo ZB RL). From the total RNA, 1st-strand and DNA were prepared using Superscript Preamplification System (Gibco ZBRL), and the chromosomal DNA was removed with DNase. 5 of this total RNA was reacted with 1/1 of oligo dT at 70C for 10 minutes, 2 ⁇ 1 10 times PCR buffer, 1 / ⁇ 1 DTT (0.1 M), 2n1 d NTP mixed solution (1 OmM) and 21 MgCl 2 (25 mM) were added, reacted at 42 C for 5 minutes, and 11 reverse transcriptase was added.
  • This mixture was reacted at 42 C for 50 minutes, then at 70 C for 15 minutes, and 11 RNase H was added thereto and reacted at 37 C for 20 minutes.
  • PCR was performed using the primers described below according to the attached protocol (Perkin-Elmer).
  • the reaction solution was a 10-fold PCR buffer solution of 5 1, 21 MgC12 (5 OmM), 11 dNTP mixture, 1 1 3, 3 primer, 1 l 5 'primer, 0.51 TaQ polymerase, and 11 cDNA, and mix at 94C for 5 minutes using Perkin-Elmer DNA Thermal Cycler.
  • reaction was carried out at 94C for 30 seconds, at 53C or 54C or 56C or 63C for 30 seconds, at 72C for 90 seconds, and finally at 72C for 10 minutes. After confirming that the GAPDH gene expression level was constant, a reaction containing no cDNA was used as a negative control.
  • Ad eXt ARII (Ichika a, T., et.al., Hepatology 34: 918-925), a vector of adenovirus that incorporates Tr un catedtype II activin receptor (tAR II) cDNA. 2001)
  • Ad e XL ac Z (Ichikawa, T., et.al., Hepatology; Hepatology; 34)
  • an adenovirus vector incorporating E.co 1 i / S galactosidase (L ac Z) cDNA. : 918-925 (2001)).
  • vascular endothelial cells cultured in serum-deficient monolayers were infected with Adext ARII or AdExLac Z at the following ratio (MOI.) At 37 C for 2 hours and serum-free medium for 24 hours. Cultured. After washing with PBS, the cells were collected by trypsinization, and used for collagen angiogenesis. ) S-galactosidase activity was detected as follows. As described above, the vascular endothelial cells were infected with AdExLac Z and cultured for 3 days, washed with 0.25% 1 utaralde hyde for 5 minutes and PBS three times, and then stained with staining solution.
  • the reaction was performed at 37 C for 3 hours.
  • the lmgZm 1 5- promotion 4 black port one 3-indolyl one J3- D-galactoside (X- gal), 11! 11 ⁇ of 1 2, 3mM of K 3 F e (CN), the 3mM K 4 F e (CN), sodium phosphate 1 0 Omm containing KC 1 of 0. 1 T riton X- 100, 1 OmM with buffer solution.
  • ⁇ Galactosidase activity was determined by the formation of blue pigment.
  • the measurement of the capillary network formation was performed as follows. After culturing for 5 days, the capillary structure was observed using a phase contrast microscope, and the tube length of 50 colonies was measured using image analysis software (NIH Image) and quantitatively evaluated.
  • FIG. 4 is a photomicrograph on the 7th day of culture, with no addition (A).
  • A 100 ng Zm1 of VEGF alone was added
  • B 100 ng Zm1 of VEGF alone was added
  • C 4 nM activin alone
  • D simultaneous addition of both
  • E a section of collagen gel was prepared in an experiment in which 4 nM activin was added, and HE staining was performed. Success (E) was confirmed.
  • FIG. 5 when quantitative evaluation was performed using image analysis software, results supporting microscopic observation were obtained.
  • follistatin which has a neutralizing activity on activin.
  • the angiogenic activity observed with 100 ng / m 1 of VEGF (B) compared to the absence (A) was shown in Fig. 6. It was found that it disappeared with the simultaneous addition (C) of folium. From the above, it was found that activin alone promotes angiogenesis, has a synergistic or additive effect between activin and VEGF, and that the angiogenesis promoting effect of VEGF is suppressed by follistatin. That is, it was shown that activin is essential for the action of VEGF.
  • VEGF vascular endothelial cells
  • 100 ng Zm1 of VEGF was allowed to act on the aortic vascular endothelial cells, and changes in the expression level of activin were examined by Western blotting.
  • FIG. 9 it was found that the VEGF action increased the protein expression of activin (activin j3 A subunit) in vascular aortic endothelial cells.
  • activin gene inhibitor j3A gene
  • RT-PCR was performed for 30 cycles according to the method described in Example 2 using the following primers.
  • VEGF, VEGF receptor, and actipin had an effect on each other in vascular endothelial cells and promoted angiogenesis.
  • the anti-angiogenic anticancer test of follisutin was performed, for example, by the method reported in VEGF blocker (J. Holash et al .; Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 17, 11393). -11398, 2002).
  • 1 ⁇ 10 6 C 6 cells (glioma) or 2 ⁇ 10 6 A 673 cells (rhabdomyosarcoma) were suspended in serum-free medium, and 10-week-old male C. Inject into the flanks of B-17 SCID mice.
  • the angiogenesis inhibitory test of follistin for diabetic retinopathy was carried out, for example, by the method reported for a VEGF receptor tyrosine kinase inhibitor (H. Ozaki et al .; AM. J. Pathol. 156, 697, 2000). Perform according to.
  • the ischemic retinopathy model is prepared by rearing C57BLZ6J mice, 7 days old, with their mother mice in a 75% oxygen incubator for 5 days.
  • 1 group contains 10 animals, 0 (vehicle control), 20, 200, and 2000 ng Zg body weight of Forlis sutin 100 100% calcium phosphate buffered saline containing 5% homologous serum saline And subcutaneously administered once a day for one week. After necropsy, the eyes are enucleated and frozen sections are prepared. The cells are treated sequentially with serum, biotinylated lectin, and avidinated peroxidase according to the method described in the literature, and the number of lectin-positive cells on the retina is counted. Observe the dose-dependent angiogenesis inhibitory effect of follistin.
  • the test for inhibiting angiogenesis on the retinal vascular development process is performed as follows. Using newborn C57BL / 6J mice, 1 group of 10 mice, 0 (vehicle control), 20, 200, and 2000 ng / g body weight of Forlis evening tin 5% of 1001 Dissolve in calcium phosphate buffered saline containing serum and administer subcutaneously once a day for 1 week and 2 weeks. At necropsy, the retina is removed and the extent of angiogenesis on the retina is measured according to methods described in the literature. A dose-dependent angiogenesis inhibitory effect of follistin can be observed. Industrial applicability
  • the present invention provides novel angiogenic and anti-angiogenic agents.

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Abstract

L'invention concerne un agent d'artérialisation comprenant, en tant que principe actif, un accélérateur d'activine, notamment une activine ou un accélérateur de récepteur d'activine, un accélérateur permettant de signaler un système associé à un récepteur d'activine ou à un accélérateur de production d'activine; et un agent d'antiartérialisation comprenant, en tant que principe actif, un inhibiteur d'activine, notamment un inhibiteur pour follistatine, un anticorps antiactivine, ou un récepteur d'activine, un inhibiteur permettant de signaler un système associé à un anticorps de récepteur d'antiactivine ou à un récepteur d'activine, ou à un inhibiteur de production d'activine.
PCT/JP2003/005901 2002-05-10 2003-05-12 Agent d'arterialisation et agent d'antiarterialisation WO2003094966A1 (fr)

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Citations (1)

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WO2002022176A1 (fr) * 2000-09-15 2002-03-21 Genvec, Inc. Procede de modulation de neovascularisation

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2002022176A1 (fr) * 2000-09-15 2002-03-21 Genvec, Inc. Procede de modulation de neovascularisation

Non-Patent Citations (2)

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Title
DETLEF H. ET AL.: "The activin-binding protein follistatin regulates autocrine endothelial cell activity and induces angiogenesis", LABORATORY INVESTIGATION, vol. 76, no. 2, 1997, pages 267 - 276, XP002971893 *
RADETZKY S. ET AL.: "Activin a stimulates inflammatory corneal neovascularization", ARVO ANNUAL MEETING ABSTRACT SEARCH AND PROGRAM PLANNER, 2002, XP002971894 *

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