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Researchers Submit Patent Application, "Pharmaceutical Composition Containing Oleanolic Acid Acetate as an Active Ingredient for Preventing Or...

July 14, 2014



Researchers Submit Patent Application, "Pharmaceutical Composition Containing Oleanolic Acid Acetate as an Active Ingredient for Preventing Or Treating Tlr- Or Il-6-Mediated Diseases", for Approval

By a News Reporter-Staff News Editor at Cancer Gene Therapy Week -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors Rho, Mun Chual (Daejeon, KR); Lee, Woo Song (Daejeon, KR); Oh, Hyun Mee (Daejeon, KR); Kim, Young Min (Daejeon, KR); Ryu, Young Bae (Daejeon, KR); Park, Su Jin (Daejeon, KR); Lee, Seung Woong (Daejeon, KR); Cho, Kyoung Oh (Gwangju, KR), filed on April 30, 2012, was made available online on July 3, 2014 (see also Korea Research Institute of Bioscience and Biotechnology).

The patent's assignee is Korea Research Institute of Bioscience and Biotechnology.

News editors obtained the following quote from the background information supplied by the inventors: "The present invention relates to a pharmaceutical composition including oleanolic acid acetate as an active ingredient for preventing or treating TLR- and IL-6-mediated diseases. More particularly, the present invention relates to a pharmaceutical composition including oleanolic acid acetate or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating TLR- and IL-6-mediated diseases, a pharmaceutical composition including an adzuki bean extract containing the compound or the salt thereof or a fraction thereof as an active ingredient for prevent ing or treating or TLR- and IL-6-mediated diseases, and a method for treating TLR- and IL-6-mediated diseases including the step of administering the composition to a subject suspected of having TLR- and IL-6-mediated diseases. Further, the present invention relates to a skin composition for external use, a heath functional food, and a personal hygiene product which include the active ingredient for preventing or improving TLR- and IL-6-mediated diseases.

"Immune responses are a series of reactions that are caused by activated immune cells against exogenous and endogenous substances (antigens). When microorganisms including bacteria, virus, etc. and foreign bodies enter the living body, host cells secrete factors such as cytokines associated with inflammation and induce inflammatory responses in order to overcome infections. When these microorganisms and foreign bodies are recognized by immune cells, immune cells are activated and many factors that are the causes of inflammation are secreted by the activated immune cells to cause inflammatory responses (Chen G. Y., Nat. Rev. Immunol., 10(12): 826-837, 2010).

"With the development of molecular biology and genetic engineering, inflammation-associated intracellular molecular targets have been revealed. In particular, as genes recognizing the specific region of a pathogen in the non-specific innate immune response in the early stage of infection have been identified, the new molecular mechanisms for initial inflammation events have been proposed. Recently, Toll-like receptor (TLR), which is a gene recognizing pathogens in the early stage of inflammation, has become known to recognize the plasma membrane components (lipopolysaccharide (LPS:endotoxin), peptidoglycan) and nucleic acid components (ds RNA, single strand RNA, GpG DNA etc.) of the pathogen, and thus many studies have been actively conducted on TLR (Brown J., et al, J. Dent. Res., 2010, Epub ahead of print: Palusinska-Szysz M., et al., Folia Microbiol. (Praha), 2010, 55(5), 508-14).

"TLR is a type I transmembrane signaling molecule and is mainly expressed in cells of the innate immune system. TLR in these cells recognizes PAMPs (pathogen-associated molecular patterns) or MAMPs (microorganism-associated molecular patterns) through an extracellular domain, and consequently, initiates inflammatory cell activation in the innate immune cells. Since the identification of TLRs, many efforts have been made to find PAMPs binding to TLRs. Until now, one or more ligands thereof have been identified, excluding those of TLR10, TLR12, and TLR13 (Kawai T. et al., Ann. N.Y. Acad. Sci., 2008, 1143, 1-20). Although TLRs are different from each other, they share common signaling pathways (Liew F. Y., et al., Nat. Rev. Immunol., 2005, 5(6), 446-458).

"The common signaling pathway of TLRs is NF-.kappa.B activation via TIR (Toll/interleukin-1 receptor-like) domain. In the signal transduction by TLR, it interacts with MyD88 having a TIR domain to form a complex. The TIR domain located at the C-terminus of MyD88 forms a complex with TLR, and the Death domain located N-terminus thereof forms a complex with the Ser/Thr kinase, IRAK (IL-1R associated kinase)-1 or IRAK-2. Subsequently, IRAK-1 activates NIK (NF-.kappa.B-inducing kinase) via interaction with TRAF6 (tumor necrosis factor receptor-associated factor 6), and NIK activation phosphorylates IKK (I.kappa.B kinase). In turn, the phosphorylated IKK phosphorylates I.kappa.B (inhibitory .kappa.B), leading to the activation of NF-.kappa.B. Activation of NF-.kappa.B results in its translocation to the nucleus, where it acts as a transcription factor to induce expression of cytokine and cell adhesion molecule-related genes (Brown J., et al, J. Dent. Res., 2010, Epub ahead of print: Palusinska-Szysz M., et al., Folia Microbiol. (Praha), 2010, 55(5), 508-14).

"In particular, TLR-7 is known to recognize ssRNA (single-stranded RNA) and its analogue polyI (polyinosinic acid). That is, TLR-7 recognizes ssRNA and then reacts with the adaptor protein MyD88 to activate the transcription factors NFkB and AP-1, resulting in production of inflammatory cytokines. In another TRIF-dependent mechanism, TLR transduces signals through TRIF, TBK1 (ANK-binding kinase 1), TRAF3 and the transcription factor IRF3, leading to the production of type I interferons. TLR-3 is known to recognize dsRNA (double-stranded RNA) and its analogue polyI:C (polyinosinic-polycytidylic acid) generated during RNA virus proliferation. That is, the leucine-rich repeat ectodomain of TLR-3 recognizes dsRNA, and then reacts with the adaptor protein TRIF to activate the transcription factors IRF3 and NF.kappa.B, leading to the production of inflammatory cytokines such as type I IFN, IL-6, IL-12 or the like (Alexopoulou L., et al, Nature, 2001, 413, 732-738). Excessive production of inflammatory mediators causes excessive immune responses, resulting in a worsening of various human diseases such as colitis, pancreatitis, rheumarthritis, asthma, or the like. Therefore, development of inhibitors of the inflammatory mediators such as TNF-.alpha., IL-1.beta., IL-6, and IL-10 will contribute to the treatment of various immune diseases and human diseases.

"STAT3 (signal transducers and activators of transcription) is known to act as another important transcription factor in autoimmune diseases including rheumarthritis, in addition to NF-.kappa.B. Particularly, STAT3 is known to be activated by an inflammation-mediating cytokine, interleukin-6 (IL-6), and also by epithelial growth factor (EGF) (Darnell Jr. J. E., et al., Science, 1994, 264, 1415-1421).

"IL-6 is a cytokine known as B-cell stimulating factor 2 or interferon .beta.2, and was found to be a differentiation factor involved in the activation of B-lymphocyte cells (Hirano T., et al., Nature, 1986, 324, 73-76), and later, it was also revealed that IL-6 is a multifunctional cytokine affecting T cell differentiation and proliferation, nerve cell differentiation, osteoclast formation, and protein synthesis in the liver (Akira S., et al., Adv. In Immunology, 1993, 54, 1-78).

"Two functionally different membrane receptor molecules are required for the induction of IL-6 activation. One of them is interleukin-6 receptor (IL-6R) having a molecular weight of approximately 80 KD which specifically binds to IL-6, and the other is gp130 having a molecular weight of approximately 130 KD which is involved in signal transduction. IL-6 and IL-6R form an IL-6/IL-6R complex, which binds to the other membrane protein gp130, leading to the induction of intracellular IL-6 signal transduction (Taga T., et al., J. Exp. Med., 1987, 196, 967). It was revealed that during the induction of intracellular IL-6 signal transduction, phosphorylation of gp130 and JAK (Janus kinase) is essential for STAT3 transcriptional activation (Heinrich P., et al., Biochem. J., 2003, 374, 1-20). Binding of IL-6 to receptors results in activation of intracellular JAK2 (Janus Kinases 2) by phosphorylation, and several tyrosine residues in its cytoplasmic domain are phosphorylated by the phosphorylated JAK2, providing the docking site for cytoplasmic proteins such as STAT3 having SH2 or other phosphotyrosine-binding motif. STAT3 binding to the cytoplasmic domain of the receptor is phosphorylated by JAK2, and then released from the receptor. The activated STAT3 bind to each other in the cytoplasm, and then form homo- or heterodimers that translocate to the nucleus where they bind to the transcriptional activation region of a target molecule to promote transcription (Levy, D. E., et al., Nat. Rev. Mol. Cell Biol., 2002, 3, 651-62: Darnell, J. E., Science, 1997, 277, 1630-1635). It has been revealed that excessive production of IL-6 is involved in various diseases such as rheumarthritis, castleman syndrome, Crohn's disease, osteoporosis, cancer cachexia, arteriosclerosis, diabetes or the like (Akira S., et al., Adv. Immunology, 1993, 54, 1-78: Kallen K., et al., Exp. Opin. Invest. Drugs, 1997, 6, 237-266). Furthermore, constitutive activation and overexpression of STAT3 appear to be involved in several forms of cancer, including myeloma, breast carcinomas, prostate cancer, brain tumors, head and neck carcinomas, melanoma, leukemias and lymphomas, particularly chronic myelogenous leukemia and multiple myeloma (Niu, et al., Cancer Res., 1999, 59, 5059-5063).

"Previously, there have been reports that a compound found to have an antioxidant activity, such as inhibition of iNOS activity, could show therapeutic effects on inflammatory diseases. However, it has since been reported that antioxidants have no therapeutic effect on the diseases classified as inflammatory diseases, such as rheumarthritis and lupus (Karen H. Costenbader, et al., American Journal of Epidemiology, 172(2):205-216, 2010). Thus, there is a problem that antioxidant effects cannot be directly applied to the treatment of inflammatory diseases.

"Meanwhile, atopic dermatitis is a representative autoimmune skin disease. Atopic dermatitis is a chronic recurrent skin disease, accompanied by wheal, eczema, itching, and inflammation. Its pathological and genetic mechanisms have not been clarified yet. Atopy is a multifactorial immune response arising from susceptible genetic background, environment, damage of skin barrier, and immunological factors. Atopic dermatitis is defined as a typical Th2-type immune disease (Mamessier, E., et al, European Journal of Dermatology, 2006, 16(2), 103), because Th2 immune response plays an important role in the onset of atopic dermatitis. When allergens functioning as an antigen enter the body through the skin, antigen presenting cells in the skin recognize and react with it. At this time, allergens specifically induce Th2-type immune response, and Th2-phenotype APCs directs differentiation of T cells to Th2 cells. Th2 cells express large amounts of Th2-type cytokines, and Th2-type cytokines activate mast cells to promote secretion of soluble mediators such as histamine and stimulate B cells to promote IgE production. These mediators recruit immune cells into the site of inflammation to induce more enhanced Th2-type immune response, consequently leading to atopic dermatitis. Candidate target genes for the treatment of atopic dermatitis include FceR1b with high IgE affinity, Th2-type cytokines such as IL-4, IL-5, and IL-13, and chemokines such as eotaxin and RANTES, which play a critical role in atopic dermatitis (Hoffjan, S., et al., Journal of Molecular Medicine, 2005, 83(9), 682-692: Cookson, W., Nat. Rev. Immunol, 2004, 4(12), 978-988). Therefore, development of compounds capable of regulating gene expressions of Th2-type cytokines and chemokines greatly will greatly contribute to the treatment of various immune diseases including atopic dermatitis and human diseases.

"The present inventors have made many efforts to find a therapeutic substance for TLR- and IL-6-mediated diseases. As a result, they demonstrated that an adzuki bean (Phaseoli angularis Wight or Phaseolus calcaratus Roxburgh) extract from the natural resources, a fraction thereof, and a compound purified therefrom have the effects of inhibiting TLR-3 and TLR-7 activation induced by the synthetic analogues of dsRNA and ssRNA, Poly(I:C) and Poly(I) and blocking the NF-.kappa.B signaling pathway, the effect of inhibiting transcriptional activation and phosphorylation of an inflammation-related transcription factor STAT3 which is activated by IL-6, and the effect of inhibiting DNCB-induced atopic dermatitis in NC/Nga mouse and collagen-induced arthritis, thereby completing the present invention for developing a pharmaceutical composition for the prevention and treatment of TLR- and IL-6-mediated diseases."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "An object of the present invention is to provide a pharmaceutical composition including oleanolic acid acetate or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating TLR- and IL-6-mediated diseases.

"Another object of the present invention is to provide a pharmaceutical composition including an adzuki bean extract containing the compound or the pharmaceutically acceptable salt thereof, or a fraction thereof as an active ingredient for preventing or treating TLR- and IL-6-mediated diseases.

"Still another object of the present invention is to provide a method for treating TLR- and IL-6-mediated diseases, including the step of administering the composition to a subject suspected of having TLR- and IL-6-mediated diseases.

"Still another object of the present invention is to provide a skin composition for external use, a health functional food, and a personal hygiene product including, as an active ingredient, oleanolic acid acetate or the pharmaceutically acceptable salt thereof; or the adzuki bean extract containing the compound or the pharmaceutically acceptable salt thereof, or the fraction thereof, for preventing or improving TLR- and IL-6-mediated diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

"FIG. 1A is a chromatogram showing the results of HPLC of the methanol extract, the hexane fraction, the ethyl acetate fraction and the water fraction of Phaseoli angularis Wight;

"FIG. 1B is a chromatogram showing the results of HPLC of the methanol extract, the hexane fraction, the ethyl acetate fraction and the water fraction of Phaseolus calcaratus Roxburgh;

"FIGS. 2A and 2B are a graph and a table showing the inhibitory effects of the methanol extract, the hexane fraction, the ethyl acetate fraction and the water fraction of adzuki bean on SEAP activity increased by poly(I) treatment in THP-1-blue cells;

"FIG. 3 is a graph showing the inhibitory effect of the methanol extract of adzuki bean on poly(I:C)-induced SEAP activity in THP-1-blue cells;

"FIGS. 4A and 4B are Western blot images showing the inhibitory effect of the adzuki bean methanol extract on NF.kappa.B P50 that is activated by poly(I) treatment and translocates to the nucleus (FIG. 4A) and the inhibitory effect on C-Jun activity of AP-1 (FIG. 4B) in THP-1 cells;

"FIGS. 4C, 4D and 4E are Western blot images showing the inhibitory effect of the adzuki bean methanol extract on poly(I)-induced IKK .alpha./.beta. (FIG. 4C), I.kappa.B (FIG. 4D), p38 and JNK (FIG. 4E) phosphorylations in THP-1 cells;

"FIGS. 5A and 5B are Western blot images showing the inhibitory effect of the adzuki bean methanol extract on NF.kappa.B P65/p50 that is activated by poly(I:C) treatment and translocates to the nucleus in THP-1 cells (FIG. 5A) and the inhibitory effect on C-Jun activity of AP-1 (FIG. 5B);

"FIGS. 5C, 5D and 5E are Western blot images showing the inhibitory effect of the adzuki bean methanol extract on poly(I:C)-induced IKK .alpha./.beta. (FIG. 5C), I.kappa.B (FIG. 5D), p38 and JNK (FIG. 5E) phosphorylations in THP-1 cells;

"FIG. 5F is a Western blot image showing the inhibitory effect of the adzuki bean methanol extract on poly(I:C)-induced IRF3 phosphorylation in THP-1 cells;

"FIGS. 6A, 6B, 6C, 6D and 6E are graphs showing the inhibitory effects of the adzuki bean methanol extract on mRNA expressions of a poly(I)-induced inflammatory cytokine TNF.alpha. (FIG. 6A), an inflammatory cytokine IL-6 (FIG. 6B), a chemokine RANTES (FIG. 6C), IFN.beta. (FIG. 6D), and a chemokine MCP-1 (FIG. 6E) in THP-1 cells;

"FIGS. 7A, 7B, 7C and 7D are a graph showing the inhibitory effect of the adzuki bean methanol extract on mRNA expressions of a poly(I:C)-induced inflammatory factor TNF.alpha. (FIG. 7A), an inflammatory factor IL-6 (FIG. 7B), a cell adhesion factor ICAM-1 (FIG. 7C), and a chemokine MCP-1 (FIG. 7D);

"FIG. 8A is a photograph showing the inhibitory effect of the adzuki bean methanol extract on IL-6-induced STAT3 phosphorylation in Hep3B cells;

"FIG. 8B is a photograph showing the inhibitory effect of the compound of Chemical Formula 1 isolated and purified from adzuki bean on IL-6-induced STAT3 phosphorylation in Hep3B cells;

"FIG. 9 is a photograph showing the back skin of NC/Nga mouse, in which the adzuki bean methanol extract showed the effect of improving the symptoms of DNCB-induced atopic dermatitis in NC/Nga mouse;

"FIG. 10 is a graph showing skin severity, in which the adzuki bean methanol extract showed the effect of improving the symptoms of DNCB-induced atopic dermatitis in NC/Nga mouse;

"FIG. 11 is a graph showing the effect of the adzuki bean methanol extract on the blood eosinophil count according to DNCB-induced atopic dermatitis of NC/Nga;

"FIG. 12 is a photograph showing the forelegs and hind legs of normal DBA/1 OlaHsd and CIA-induced mouse model;

"FIG. 13 is a CIA index showing the improvement effect of the adzuki bean methanol extract on the symptoms of CIA-induced arthritis of DBA/1 OlaHsd mice;

"FIG. 14 is a graph showing the inhibitory effect of each concentration of oleanolic acid or oleanolic acid acetate on IL-6-induced STAT3 luciferase activity in Hep3B cells;

"FIG. 15 is a photograph showing the inhibitory effects of the adzuki bean methanol extract on differentiation of osteoclasts from the bone marrow macrophages isolated from mouse bone marrow cells; and

"FIG. 16 is a photograph showing the inhibitory effects of oleanolic acid acetate on differentiation of osteoclasts from the bone marrow macrophages isolated from mouse bone marrow cells."

For additional information on this patent application, see: Rho, Mun Chual; Lee, Woo Song; Oh, Hyun Mee; Kim, Young Min; Ryu, Young Bae; Park, Su Jin; Lee, Seung Woong; Cho, Kyoung Oh. Pharmaceutical Composition Containing Oleanolic Acid Acetate as an Active Ingredient for Preventing Or Treating Tlr- Or Il-6-Mediated Diseases. Filed April 30, 2012 and posted July 3, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2258&p=46&f=G&l=50&d=PG01&S1=20140626.PD.&OS=PD/20140626&RS=PD/20140626

Keywords for this news article include: Kinase, Hygiene, Alcohols, Methanol, Oncology, Chemicals, Chemistry, Cytoplasm, Viral RNA, rev Genes, Immunology, Bone Marrow, Dermatology, Macrophages, Viral Genes, Interleukins, Therapeutics, Bone Research, Immune System, Skin Diseases, Atopic Dermatitis, Genetic Phenomena, Genome Components, Membrane Proteins.

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Source: Cancer Gene Therapy Week


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