WO2019200585A1 - Polythérapie contre le cancer avec de la naringénine et de l'acide asiatique - Google Patents

Polythérapie contre le cancer avec de la naringénine et de l'acide asiatique Download PDF

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WO2019200585A1
WO2019200585A1 PCT/CN2018/083741 CN2018083741W WO2019200585A1 WO 2019200585 A1 WO2019200585 A1 WO 2019200585A1 CN 2018083741 W CN2018083741 W CN 2018083741W WO 2019200585 A1 WO2019200585 A1 WO 2019200585A1
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cell
naringenin
cells
cancer
asiatic acid
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PCT/CN2018/083741
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English (en)
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Hui Yao Lan
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The Chinese University Of Hong Kong
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/191—Carboxylic acids, e.g. valproic acid having two or more hydroxy groups, e.g. gluconic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 

Definitions

  • Cancer is an umbrella term for a variety of potentially deadly diseases involving improper and uncontrolled cell proliferation with the potential of spreading or metastasis.
  • the latest year global cancer data are available, there were 14.1 million newly diagnosed cancer cases (among which there were 7.4 million cases in men and 6.7 million in women) and 8.2 million cancer deaths worldwide.
  • the global numbers are expected to reach 21.7 million new cancer cases and 13 million cancer deaths.
  • the cancer burden on the global population will probably be even larger in the future because of the adoption of western lifestyles, such as smoking, poor diet, physical inactivity, and fewer childbirths, in economically developing countries.
  • the total economic impact of premature death and disability from cancer worldwide was $895 billion in 2008, representing 1.5 percent of the world’s gross domestic product (GDP) .
  • GDP gross domestic product
  • naringenin and asiatic acid have been used to treat cancer and associated diseases (see, e.g., U.S. Patent Application No. 2004/0097463; U.S. Patent No. 5,145,839; WO 1999/01567; EP 0352147; WO 2001/051043) .
  • naringenin and Asiatic acid have been used for treating fibrosis, see, e.g., WO2014/063660.
  • the present inventor made the surprising discovery that, when naringenin and Asiatic acid are administered together, a synergistic effect is achieved in inhibiting cellular proliferation, especially cancer cell proliferation.
  • the present invention provides novel methods and compositions effective for the treatment and prevention of cancers at various anatomic sites.
  • the present invention is applicable to cancer patients who are not diagnosed with other conditions treatable with naringenin and Asiatic acid co-administration, e.g., fibrosis.
  • the invention provides a new method of inhibiting cell proliferation.
  • the method includes the step of contacting the cell with an effective amount of naringenin and Asiatic acid.
  • the cell may be a part of a tissue or an organ, such as liver, kidney, skin, or lung.
  • the contacting step comprises subcutaneous, intramuscular, intravenous, intraperitoneal, topical, or oral administration.
  • the effective amount of naringenin is about 1-10 mg/kg (e.g., about 2 or 5 mg/kg) body weight at the lower end and about 100 to 500 mg/kg (e.g., about 200 or 250 mg/kg) body weight at the higher end
  • the effective amount of asiatic acid is about 1-10 mg/kg (e.g., about 2 or 5 mg/kg) body weight at the lower end and about 15-50 mg/kg (e.g., about 20 or 25 mg/kg) body weight at the higher end.
  • Asiatic acid and naringenin are administered at a weight ratio of about 1: 1 or 2 at the higher end to about 1: 10 or 15 or 20 at a lower end, for example, the ratio may be about 1: 5.
  • naringenin and Asiatic acid are administered in a single composition.
  • naringenin and Asiatic acid are administered in two separate compositions.
  • Naringenin and Asiatic acid are administered in any appropriate form, including but not limited to, a solution, a powder, a gel, a cream/paste, a tablet, or a capsule.
  • the ratio of Asiatic acid to naringenin in weight varies from 1 to 0.001 or less; 0.001; 0.002; 0.005; 0.01; 0.025; 0.05; 0.1; 0.25; 0.50; 0.75; 1.0; 2.0; 2.5; 5.0; 7.5; 10; 20; 25; 50; 75; 100; 200; 250; 300; 400; 500; 600; 700; 800; 900; or 1,000, or even higher.
  • the invention provides a new composition that comprises (1) an effective amount of naringenin and Asiatic acid and (2) a pharmaceutically acceptable excipient.
  • Asiatic acid and naringenin are present in the weight ratio of about 1: 1 or 2 at one end to about 1: 10 or 15 or 20 at the other end, for example, the ratio may be about 1: 5.
  • the composition is formulated for subcutaneous, intramuscular, intravenous, intraperitoneal, topical, or oral administration.
  • the composition may be in the form of a solution, a powder, a gel, a cream, a paste, a tablet, or a capsule.
  • the ratio of Asiatic acid to naringenin in weight in the composition varies from 1 to 0.001 or less; 0.001; 0.002; 0.005; 0.01; 0.025; 0.05; 0.1; 0.25; 0.50; 0.75; 1.0; 2.0; 2.5; 5.0; 7.5; 10; 20; 25; 50; 75; 100; 200; 250; 300; 400; 500; 600; 700; 800; 900; or 1,000, or even higher.
  • the present invention provides the above-described compositions for use in the treatment and prevention of cancers at various anatomic sites.
  • the present invention is applicable to cancer patients who are not diagnosed with other conditions treatable with naringenin and Asiatic acid co-administration, e.g., fibrosis.
  • the compositions comprise an effective amount of naringenin and Asiatic acid, and the cancer to be treated may be liver, kidney, skin, or lung cancer, especially various types of melanoma and lung cancer.
  • the compositions are administered by subcutaneous, intramuscular, intravenous, intraperitoneal, topical, or oral meaning.
  • the effective amount of naringenin is about 1-10 mg/kg (e.g., about 2 or 5 mg/kg) patient body weight at the lower end and about 100 to 500 mg/kg (e.g., about 200 or 250 mg/kg) body weight at the higher end
  • the effective amount of asiatic acid is about 1-10 mg/kg (e.g., about 2 or 5 mg/kg) body weight at the lower end and about 15-50 mg/kg (e.g., about 20 or 25 mg/kg) body weight at the higher end.
  • Asiatic acid and naringenin are administered at a weight ratio of about 1: 1 or 2 at the higher end to about 1: 10 or 15 or 20 at a lower end, for example, the ratio may be about 1: 5.
  • naringenin and Asiatic acid are in a single composition to be administered.
  • naringenin and Asiatic acid are in two separate compositions to be administered.
  • Naringenin and Asiatic acid are formulated in any appropriate form, including but not limited to, a solution, a powder, a gel, a cream/paste, a tablet, or a capsule.
  • the ratio of Asiatic acid to naringenin in weight varies from 1 to 0.001 or less; 0.001; 0.002; 0.005; 0.01; 0.025; 0.05; 0.1; 0.25; 0.50; 0.75; 1.0; 2.0; 2.5; 5.0; 7.5; 10; 20; 25; 50; 75; 100; 200; 250; 300; 400; 500; 600; 700; 800; 900; or 1,000, or even higher.
  • the present invention provides a kit for inhibiting proliferation of cells, especially cancer cells.
  • the kit contains at least two containers: the first container contains a first composition, which comprises narigenin; and the second container contains a second composition, which comprises Asiatic acid.
  • the first composition is formulated for subcutaneous, intramuscular, intravenous, intraperitoneal, topical, or oral administration.
  • the second composition is formulated for subcutaneous, intramuscular, intravenous, intraperitoneal, topical, or oral administration.
  • the kit further includes an instruction manual for administration of the first and second compositions.
  • Asiatic acid to naringenin ratio in weight may vary from 1 to 0.001 or less; 0.001; 0.002; 0.005; 0.01; 0.025; 0.05; 0.1; 0.25; 0.50; 0.75; 1.0; 2.0; 2.5; 5.0; 7.5; 10; 20; 25; 50; 75; 100; 200; 250; 300; 400; 500; 600; 700; 800; 900; or 1,000, or even higher.
  • narigenin and Asiatic acid are achievable by substituting Asiatic acid with either of its two known analogs, madecassic acid and asiaticoside, in combined use with narigenin.
  • Fig. 1 Determination of a safe dosage of AA or NG in normal mice. Groups of 4 normal mice were treated with different dosages of asiatic acid (AA) or naringenin (NG) for two weeks and various parameters were measured to determine a safe dosage for the use as therapeutic dose in cancer-bearing mice. As indicated by a highlighted box, AA at 10mg/kg and NG at 50mg/kg was selected. Each bar represents the mean ⁇ SEM for groups of 4 mice. *p ⁇ 0.05, **p ⁇ 0.01 compared to untreated mice (BLK) .
  • AA asiatic acid
  • NG naringenin
  • Fig. 2 Combination of AA and NG produces a better inhibitory effect on tumor growth compared to monotherapy.
  • A-D Dose-dependent inhibitory effect of AA or NG on B16F10 melanoma volume and weight.
  • E, F Combination therapy with an optimal AA (10mg/kg/day) and NG (50mg/kg/day) produces a better inhibitory effect on B16F10 melanoma growth when compared to monotherapy.
  • G-J Combination therapy with an optimal AA (10mg/kg/day) and NG (50mg/kg/day) produces a better inhibitory effect on LLC lung carcinoma when compared to monotherapy as determined by tumor volume, tumor weight and bioluminescence imaging.
  • Each error bar represents the mean ⁇ SEM for groups of six to eight mice. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 compared to Ctrl; #p ⁇ 0.05, ##p ⁇ 0.01, ###p ⁇ 0.001 as indicated.
  • AA, NG, and combination therapy of AA and NG do not cause systemic toxicity including peripheral white blood cell counts, serum LDH, liver functions (AST and ALT) , and renal function (creatinine) on B16F10 melanoma-bearing mice.
  • Each bar represents the mean ⁇ SEM for groups of 6 mice.
  • Figs. 4A-4B Combination therapy of AA and NG produces a better therapeutic effect in rebalancing Smad signaling in both B16F10 melanoma and LLC lung cancer mouse models.
  • Western blot analysis shows that combination with AA and NG results in a further reduction of p-Smad3 but increasing Smad7 in both melanoma and lung cancer mouse models.
  • Each bar represents the mean ⁇ SEM for groups of six to eight mice. *p ⁇ 0.05, **p ⁇ 0.01 compared to untreated tumors (Ctrl) .
  • Fig. 5 Combination therapy of AA and NG produces a better inhibitory effect on LLC lung carcinoma proliferation by largely reducing Ki67+ cells, in vitro tumor colony formation, CD31+ angiogenesis, and vascular endothelial growth factor (VEGF) -producing cells in LLC invasive lung carcinoma.
  • Data represent for groups of 6 mice or at least three-independent experiments
  • Fig. 6 Determination of the effective ratio between AA or NG on tumor angiogenesis in vitro.
  • Tumor cells B16F10 were cultured with TGF-beta1 (2ng/ml) and VEGF mRNA expression by tumor cells was measured by real-time PCR. Results show that addition of AA at 1uM is able to significantly inhibit TGF-beta1-induced angiogenesis, which is enhanced by addition of NG from 0.5 to 250 uM.
  • Each bar represents the mean ⁇ SEM for 3 independent experiments. *p ⁇ 0.05, **p ⁇ 0.01 compared to TGF-beta 1-treated tumor.
  • Figs. 7A-7B Combination therapy of AA and NG produces a better inhibitory effect on B16F10 melanoma invasiveness in vitro.
  • Fig. 7A Wound healing assay and
  • Fig. 7B transwell assay for the inhibitory effect of AA, NG, and AA+NG (CB) on B16F10 melanoma invasion.
  • Each bar represents the mean ⁇ SEM for three-independent assays.
  • Fig. 8 Combination therapy of AA and NG produces a better inhibitory effect on the invasiveness of LLC lung carcinoma in vivo. Noted that the combined AA and NG therapy blocks the invasive activities of tumor such as MMP2, MMP9, and MMP13 of LLC lung tumor. Each bar represents the mean ⁇ SEM for groups of 6 mice. *p ⁇ 0.05, **p ⁇ 0.01 compared to untreated tumors (Ctrl) ; #p ⁇ 0.05 compared to NG treatment as indicated.
  • cytotoxic NK cells NK1.1+NKp46+
  • A Two-color immunofluorescence shows that numbers of cytotoxic NK cells (NK1.1+NKp46+) in LLC tumor microenvironment are largely increased in mice with the combination of AA and NG when compared to each single treatment.
  • NK1.1 green
  • NKp46 red
  • DAPI blue
  • B Two-color flow cytometry detects that numbers of cytotoxic NK cells (NK1.1+NKp46+) in peripheral blood of LLC tumor-bearing mice are largely increased in mice with the combination of AA and NG when compared to each single treatment.
  • Each bar represents the mean ⁇ SEM for groups of three mice. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 compared to Ctrl; ##p ⁇ 0.01, ###p ⁇ 0.001 as indicated.
  • Fig. 10 Combination with AA and NG enhances the production of IFN- ⁇ and granzyme B by NK cells both in vivo and in vitro.
  • A, B Two-color immunofluorescence detects NK1.1+IFN- ⁇ + and NK1.1+ granzyme B+ NK cells infiltrating the LLC tumor microenvironment. NK1.1+ cells (green) , IFN- ⁇ + or granzyme B+ cells (red) , DAPI (blue) .
  • Each error bar represents the mean ⁇ SEM for groups of three to four mice. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 compared to Ctrl; #p ⁇ 0.05, ##p ⁇ 0.01, ###p ⁇ 0.001 as indicated.
  • ELISA shows IFN- ⁇ and granzyme B levels in homogenized LLC tissue.
  • E, F Effect of AA (10 ⁇ M) , NG (100 ⁇ M) , and their combination on levels of IFN- ⁇ and granzyme B in supernatant of cultured splenic NK cells with TGF- ⁇ 1 (5ng/ml) stimulation for 24 hours detected by ELISA.
  • G NK cell cytotoxicity assay with AA/NG pre-treated bone marrow-derived NK cells as effector cells and LLC as target cells at E: T ratio of 5: 1, 10: 1 and 20: 1 in TGF- ⁇ 1 (5ng/ml) condition.
  • Each error bar represents the mean ⁇ SEM for groups of three independent experiments. *p ⁇ 0.05, ***p ⁇ 0.001 compared to TGF- ⁇ 1; ##p ⁇ 0.01, ###p ⁇ 0.001 as indicated.
  • Figs. 11A-11C Combination of AA and NG produces a better outcome in rebalancing Smad signaling in NK cells by inactivating Smad3 while upregulating Smad7.
  • Figs. 11A, 11B Two-color immunofluorescence detecting NK1.1+p-Smad3+ and NK1.1+Smad7+ NK cells in LLC tumor microenvironment. NK1.1+ cells (green) , p-Smad3+or Smad7+ cells (red) , DAPI (blue) . Each bar represents the mean ⁇ SEM for groups of three to four mice. **p ⁇ 0.1, ***p ⁇ 0.01 compared to Ctrl; #p ⁇ 0.5, ###p ⁇ 0.01 as indicated.
  • Figs. 12A-12B Combination of AA and NG attenuates TGF- ⁇ 1 induced inhibition on NK cell differentiation.
  • FIG. 12A Flow cytometry shows inhibitory effect of TGF- ⁇ 1 on NK cell differentiation as determined by NK1.1+ CD122+ populations in bone marrow-derived NK cells (9 days) . ***p ⁇ 0.01 compared to Ctrl; #p ⁇ 0.5 as indicated.
  • FIG. 12B Flow cytometry detects that the effect of AA, NG, and their combination on bone marrow-derived NK differentiation in response to TGF- ⁇ 1 (5ng/ml) . Each error bar represents the mean ⁇ SEM for groups of three independent experiments. ***p ⁇ 0.001 compared to TGF- ⁇ 1; ###p ⁇ 0.001 as indicated.
  • FIG. 13 Combination therapy with AA and NG reverses the suppressive effect of TGF- ⁇ 1 on Id2 and IRF2 expression.
  • A, B mRNA levels of Id2 and IRF2 in peripheral blood NK cells isolated from LLC bearing-mice detected by real-time PCR; **p ⁇ 0.01 compared to Ctrl; ##p ⁇ 0.01 as indicated.
  • C, D mRNA levels of Id2 and IRF2 in AA and NG treated bone marrow-derived NK cells detected by real-time PCR.
  • E Id2 and IRF2 expression in AA and NG treated bone marrow-derived NK cells measured by western blotting. Each bar represents the mean ⁇ SEM for groups of three to four mice or groups of three independent experiments. **p ⁇ 0.01, ***p ⁇ 0.001 compared to TGF- ⁇ 1; ##p ⁇ 0.01, ###p ⁇ 0.001 as indicated.
  • Fig. 14 Disrupted Id2 or IRF2 impairs AA and NG -induced expression of Id2 and IRF2 on NK cells in vitro by Western blotting. Bone marrow-derived NK cells were transfected with si-Id2 or si-IRF2 and then cultured with AA (10 ⁇ mol) and NG (100 ⁇ mol) under TGF- ⁇ 1 (5ng/ml) conditions for 6 days and collected for flow cytometry analysis. Each bar represents the mean ⁇ SEM for three independent experiments. **p ⁇ 0.01, ***p ⁇ 0.001 compared to non-treated group; ###p ⁇ 0.001 as indicated.
  • Figs. 15A Disrupted Id2 or IRF2 impairs the protective effect of AA and NG on NK cell differentiation and maturation in vitro as determined by by two –color flow cytometry with antibodies to NK1.1+NKp46+ cells or NK1.1+CD11b+ cells.
  • Fig. 15B Smad3 directly interacts with Id2 and IRF2 gene as a trancriptional repressor in NK cells as determined by predicted Smad binding site on Id2 and IRF2.
  • ChIP assay shows that addition of TGF- ⁇ 1 (5ng/ml) induces Smad3 directly binding to 3’UTR of both Id2 and IRF2 gene on bone marrow-derived NK cells.
  • inhibiting refers to any detectable negative or suppressing effect on a target biological or pathological process, such as the proliferation of cancer cells or progression of a cancer. Typically, an inhibition is reflected in a decrease of at least 10%, 20%, 30%, 40%, or 50%in a feature characteristic of the target process (e.g., the rate of cancer cell proliferation) when compared to a control.
  • a feature characteristic of the target process e.g., the rate of cancer cell proliferation
  • cancer encompasses any disease that involves improper and uncontrolled cellular proliferation resulting in the accumulation or growth of these improperly proliferating cells at an original anatomic site with a tendency of spreading to another anatomic site, a process called metastasis.
  • Cancers can arise from virtually any tissue type and can affect all organs and body parts, some high incidence cancers including lung cancer, colon and rectum cancer, breast cancer, prostate cancer, pancreatic cancer, brain cancer, stomach cancer, liver cancer, esophagus cancer, bladder cancer, kidney cancer, skin cancer (melanoma) , and various blood cancers such as leukemia, lymphoma, and myeloma. Humans of all ages can develop cancer.
  • the term “effective amount, ” as used herein, refers to an amount of a substance that produces therapeutic effects for which the substance is administered.
  • the effects include the prevention, correction, or inhibition of progression of the symptoms of a disease or condition and related complications to any detectable extent.
  • the “effective amount” may be expressed in more than one way.
  • the “effective amount” may be expressed in the total amount of all active ingredients, or expressed in a separate amount for each active ingredient, or expressed in a ratio (e.g., in weight or volume ratio) of one ingredient over another.
  • an “effective amount” will depend on the purpose of the treatment as well as the form and identify of the active substance, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992) ; Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999) ; and Pickar, Dosage Calculations (1999) ) .
  • Naringenin is a flavanone with the systematic (IUPAC) name 5, 7-dihydroxy-2- (4-hydroxyphenyl) chroman-4-one and synonym 4', 5, 7-trihydroxyflavanone (CAS Number 480-41-1) . Its chemical formula is C 15 H 12 O 5 and molecular weight is 272.257. In nature, naringenin is present most abundantly in grapefruits, oranges and tomato skin. High purity naringenin can be purchased from various commercial venders.
  • “Asiatic acid” is derived from an ancient, traditional herbal extract of the plant Centella asiatica, commonly called Gotu Kola. It is also known as dammarolic acid (CAS Number 464-92-6) . Its chemical formula (Hill Notation) is C 30 H 48 O 5 , and molecular weight is 488.70. Asiatic acid shares many similarities with its analogs, madecassic acid and asiaticoside. It is available through commercial suppliers such as Sigma-Aldrich.
  • administration encompasses any means of delivering a substance, e.g., an agent with therapeutic or prophylactic effects, to a subject, which may include but is not limited to, systemic, regional, and local applications.
  • a substance e.g., an agent with therapeutic or prophylactic effects
  • administration are injection (such as by subcutaneous, intramuscular, intravenous, or intraperitoneal means) , oral ingestion, intake through the nasal cavity or through the eyes or ears, inhalation, transdermal delivery, and anal or virginal deposit, etc.
  • pharmaceutically acceptable excipient and “physiologically acceptable excipient” may be used interchangeably to refer to an inert substance that is included in the formulation of a composition containing an active ingredient to achieve certain characteristics, such as more desirable pH, solubility, stability, bioavailability, texture, consistency, appearance, flavor/taste, viscosity, etc., but in itself does not negatively impact the intended therapeutic or prophylactic effects of the active ingredient.
  • tissue refers to an ensemble of cells that are similar in their biological attributes, such as morphology and biological activity, and are from the same origin, such that these cells together carry out a specific function.
  • An “organ” is a collection of different tissues joined in a structural unit to serve a common function.
  • a value of “about 10” can be any value within the range of 10 ⁇ 1, i.e., between 9 to 11.
  • Cancer is one of the leading cause for human deaths, yet the current treatment for cancer using cytotoxic drugs remains non-specific and ineffective with severe side-effects.
  • newly developed therapeutic compositions are described for effectively treating cancer by promoting the host immunity against tumor. It was discovered that a compound containing a component purified from the citric fruits called naringenin (NG) and an asiatic acid (AA) from a herb can produce more effective inhibition of tumor growth and invasiveness in two syngeneic mouse models of B16F10 melanoma and LLC invasive lung carcinoma, without causing toxicity to the normal body when compared to the individual use.
  • NG naringenin
  • AA asiatic acid
  • NK cell development and NK cell-dependent cancer-killing activities are the major therapeutic mechanism of this invention, although the combination therapy of AA and NG can also effectively inhibit tumor growth/proliferation and invasive/migration activities including angiogenesis (CD31 + ) and the expression of matrix metalloproteinases (MMPs) .
  • angiogenesis CD31 +
  • MMPs matrix metalloproteinases
  • This invention resides in the discovery that a mixed composition comprising naringenin and asiatic acid can produce more effective growth inhibition on cancer cells such as B6F10 melanoma and LLC invasive lung cancer cells while exhibiting little to no toxicity to the host body. It has also been revealed that the mixed composition can function as an immune regulator, which promotes NK cell development and NK cell-dependent cancer-killing activaties in the cancer microenvironment. Thus, this invention is advantageous over the current anti-cancer therapies, which use cytotoxic drugs and often produce many severe side-effects.
  • the present invention provides pharmaceutical compositions or physiological compositions comprising an effective amount of naringenin and Asiatic acid, which is effective for inhibiting undesirable proliferation of cells, especially cancer cells, in a tissue or organ in both prophylactic and therapeutic applications.
  • Such pharmaceutical or physiological compositions also include one or more pharmaceutically or physiologically acceptable excipients or carriers.
  • Pharmaceutical compositions of the invention are suitable for use in a variety of drug delivery systems. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985) . For a brief review of methods for drug delivery, see, Langer, Science 249: 1527-1533 (1990) .
  • the pharmaceutical compositions of the present invention can be administered by various routes, e.g., oral, topical, subcutaneous, transdermal, intramuscular, intravenous, intranasal, or intraperitoneal.
  • the preferred routes of administering the pharmaceutical compositions are local delivery to an organ or tissue suffering from or at risk of developing cancer (e.g., intraperitoneal injection to an organ) at daily doses of about 0.35 -17.5g, preferably 2.5-5.5g, of naringenin and about 0.1 -3.5g, preferably 0.5-1.0g, of Asiatic acid for a 70 kg adult human per day.
  • the appropriate dose may be administered in a single daily dose or as divided doses presented at appropriate intervals, for example as two, three, four, or more subdoses per day.
  • the pharmaceutical carrier can be either solid or liquid.
  • Solid form preparations include, for example, powders, gels/creams/pastes, tablets, dispersible granules, capsules, cachets, and suppositories.
  • a solid carrier can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material.
  • the carrier is generally a finely divided solid that is in a mixture with the finely divided active component, e.g., naringenin and/or Asiatic acid.
  • the active ingredient naringenin and/or Asiatic acid
  • the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • a low-melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into convenient-sized molds and allowed to cool and solidify.
  • Powders and tablets preferably contain between about 5%to about 70%by weight of the active ingredient of naringenin and/or Asiatic acid.
  • Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoa butter, and the like.
  • the pharmaceutical compositions can include the formulation of the active ingredient of naringenin and/or Asiatic acid with encapsulating material as a carrier providing a capsule in which the active ingredient (with or without other carriers) is surrounded by the carrier, such that the carrier is thus in association with the active ingredient.
  • encapsulating material as a carrier providing a capsule in which the active ingredient (with or without other carriers) is surrounded by the carrier, such that the carrier is thus in association with the active ingredient.
  • cachets can also be included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.
  • Liquid pharmaceutical compositions include, for example, solutions suitable for oral or parenteral administration, suspensions, and emulsions suitable for oral administration.
  • Sterile water solutions of the active component e.g., naringenin and/or Asiatic acid
  • sterile solutions of the active component in solvents comprising water, buffered water, saline, PBS, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like.
  • Sterile solutions can be prepared by dissolving the active component, such as naringenin and/or Asiatic acid, in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • the pH of the preparations typically will be between 3 and 11, more preferably from 5 to 9, and most preferably from 7 to 8.
  • compositions containing the active ingredient of naringenin and/or Asiatic acid can be administered topically to a patient in the case of treatment or prevention of skin cancer (melanoma) .
  • the compositions may be formulated as a gel, a cream, a paste, a powder, or a spray for the ease of use.
  • compositions containing the active ingredient of naringenin and/or Asiatic acid can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already suffering from a condition involving undesirable cellular proliferation such as cancer in an amount sufficient to prevent, cure, reverse, or at least partially slow or arrest the symptoms of the cancer and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.
  • Amounts effective for this use will depend on the severity of the disease or condition and the weight and general state of the patient, but generally range from about 0.35g to about 17.5g of naringenin and 0.1g to about 3.5g of Asiatic acid per day for a 70 kg patient, with dosages of from about 2.5g to about 5.5g of naringenin and 0.5g to about 1.0g of Asiatic acid per day for a 70 kg patient being more commonly used.
  • compositions containing naringenin and/or Asiatic acid are administered to a patient susceptible to or otherwise at risk of developing a cancerous disease or condition, in an amount sufficient to delay or prevent the onset of the cancer-related symptoms. Such an amount is defined to be a "prophylactically effective dose.
  • naringenin and Asiatic acid again depend on the patient's state of health and weight, but generally range from about 0.35g to about 5.5g of naringenin and 0.1g to about 3.5g of Asiatic acid for a 70 kg patient per day, more commonly from about 2.5g to about 5.5g of narigenin and 0.5g to about 1.0g of Asiatic acid for a 70 kg patient per day.
  • compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical formulations should provide a quantity of naringenin and/or Asiatic acid sufficient to effectively inhibit undesirable proliferation of cells (especially malignant cells) in the patient, either therapeutically or prophylactically.
  • kits for inhibiting cellular proliferation, especially undesirable proliferation such as cancer cell proliferation typically contain two containers: the first container contains a composition that comprises narigenin and the second container contains a composition that comprises Asiatic acid.
  • kits may include a container that contains a pharmaceutical composition having an effective amount of naringenin and Asiatic acid (such as a composition described herein in detail) , as well as informational material containing instructions on how to dispense the pharmaceutical composition, including description of the type of patients who may be treated (e.g., patients who are suffering from or are at risk of developing a hyperproliferative disease such as cancer) , the schedule (e.g., dose and frequency) and route of administration, and the like.
  • a pharmaceutical composition having an effective amount of naringenin and Asiatic acid such as a composition described herein in detail
  • informational material containing instructions on how to dispense the pharmaceutical composition, including description of the type of patients who may be treated (e.g., patients who are suffering from or are at risk of developing a hyperproliferative disease such as cancer) , the schedule (e.g., dose and frequency) and route of administration, and the like.
  • the current anti-cancer drugs are largely cytotoxic with severe side-effect on the body. It has been shown that naringenin can inhibit TGF- ⁇ signaling and has anti-cancer activities by inhibiting the tumor cell growth through the S-G2M-dependent mechanism (Patent No: EP2163247 B1; Abaza MS et al: Cancer Cell Int. 2015; 15: 46) . It is also reported that Asiatic acid has a wide arrange effect on inflammation, fibrosis, and cancer (Patent EP 1313462 B1; US 20040097463 A1; CN 102391351 B; CN 103467560 A) .
  • naringenin is a Smad3 inhibitor while Asiatic acid (AA) functions as a Smad7 agonist and the combination of both produces a better inhibitory effect on rebalancing TGF- ⁇ /Smad3 signaling and has better therapeutic effect on renal fibrosis (Meng et al: Oncotarget 2015; 6: 36984, Patent CN 104736151/WO2014063660A1) . It was tested whether a compound containing naringenin and asiatic acid can be used for a better treatment for cancer. Unexpectedly, it was discovered that the combined therapy with AA and NG at a ratio 1: 5 produces a better anticancer effect when compared to individual one.
  • this compound may function as a new immune regulator and suppresses cancer by promoting the host immunity against cancer, which is largely different from the current cytotoxic drugs.
  • a safe dosage of HLPC-purified naringenin (NG) and asiatic acid (AA) was first determined in normal mice by peritoneal injection with different dosages of AA or NG for toxicity assays and a dose of AA at 10 mg/kg body weight and NG at 50 mg//Kg body weight is selected for further examination of the therapeutic efficacy on cancer (Figure 1) .
  • TGF- ⁇ 1 plays a promoting role in tumor growth, invasion and metastasis via a mechanism associated with hyperactive Smad3 signaling while suppressing Smad7 in tumor microenvironment.
  • the present study revealed that rebalancing Smad3/Smad7 signaling with asiatic acid (AA, a Smad7 agonist) and naringenin (NG, a Smad3 inhibitor) can significantly inhibit tumor progression in two syngeneic mouse tumor models of invasive melanoma (B16F10) and lung carcinoma (LLC) . It was discovered that compared to the monotherapy, combination therapy with AA and NG produced a synergistic effect on inhibiting tumor growth on both B16F10 and LLC models.
  • TGF- ⁇ 1 signaling has been proved to be a tumor promoter in tumor growth, invasion, and metastasis (2-5) . Therefore, altering tumor microenvironment from supportive to inhibitory effect on cancer by targeting TGF- ⁇ 1 signaling may represent a prospective therapeutic target.
  • Smad3 As a vital transcriptional factor in TGF- ⁇ 1 downstream signaling, Smad3 is essential for tumor progression (6) . Previous studies have confirmed that enhanced expression of Smad3 is observed in human colorectal cancer (7) , while mice lacking Smad3 were resistant to chemical-induced skin carcinogenesis (8, 9) . A recent study also revealed that TGF- ⁇ 1/Smad3 signaling is essential for cancer growth and invasion via a mechanism associated with suppressing NK cell immunity against cancer in tumor microenvironment (10) . On the contrary, as an inhibitory Smad protein, Smad7 inhibits the phosphorylation of Smad2 and Smad3 via a negative feedback loop, thus preventing the over-activation of TGF- ⁇ 1/Smad3 signaling (11, 12) .
  • NK cells play a pivotal role in tumor immunosurveillance, which can rapidly respond to tumor formation and inhibit tumor progression independent from antigen presentation and T cell immunity (16-18) .
  • NK cell-mediated cytotoxicity on tumor cells is blunted by TGF- ⁇ 1 in tumor microenvironment (19) .
  • TGF- ⁇ 1 is responsible for NK cell immaturity during mouse infancy through inhibiting the expression of E4BP4, T-bet and GATA-3, which are crucial for NK cell differentiation and terminal maturation (10, 20) .
  • TGF- ⁇ 1 markedly impairs NK cell-mediated cytotoxicity via reducing cytokine productions of NK cells, such as granzymes and IFN- ⁇ (19, 21) .
  • Asiatic acid AA
  • Centella asiatica a triterpene from Centella asiatica
  • AA can function as a Smad7 agonist capable of inducing Smad7 to alleviate TGF- ⁇ 1-induced liver fibrosis (23)
  • NG naringenin
  • NG a natural predominant flavanone isolated of citrus, functions as an effective inhibitor for Smad3 in liver and pulmonary fibrosis (24, 25) .
  • the anti-tumor efficacy of combination therapy was further validated on LLC lung cancer mouse model, where a better anti-tumor effect on the tumor growth rate, bioluminescence imaging, and tumor weights over the monotherapy were achieved (Fig. 11G-J) .
  • Combination therapy enhances NK cell immune response in tumor microenvironment
  • TGF- ⁇ 1 markedly impairs NK cell-mediated cytotoxicity via inhibiting various cytokine productions including granzyme B (GB) and IFN- ⁇ (19) . It was also detected that treatment with AA or NG significantly increased granzyme B-secreting NK cells (GB + NK1.1 + cells) and IFN- ⁇ -producing NK cells (IFN- ⁇ + NK1.1 + cells) in the tumor microenvironment, which were further enhanced in LLC tumor-bearing mice receiving combination therapy (Fig. 13A, B) . Meanwhile, ELISA also detected a markedly increase in IFN- ⁇ and granzyme B in tumor tissue of mice treated with combination therapy when compared with those treated with control solvent and monotherapy with AA or NG (Fig. 13C, D) . This observation was further verified on cultured splenic NK cells treated with either AA or NG, or AA plus NG under high TGF- ⁇ 1conditions (Fig. 13E, F) .
  • Combination therapy with AA and NG attenuates the inhibitory effect of TGF- ⁇ 1 on NK cell development via rebalancing Smad3 and Smad7 signaling
  • NK cell development in peripheral blood and tumor microenvironment in AA and NG treated LLC mice was not associated with the proliferation or recruitment of NK cells (Fig. 20) .
  • the influence of combination therapy on bone marrow-derived NK cell development in vitro was examined.
  • addition of TGF- ⁇ 1 significantly suppressed the NK cell development in a dose-dependent manner.
  • TGF- ⁇ 1 at a dose of 5ng/ml largely reduced NK1.1 + CD122 + cell (immature NK cells) proportion from 80%to 11%.
  • Addition of AA or NG moderately attenuated this inhibitory effect, which was markedly enhanced by the combination treatment with AA and NG (Fig. 15B) .
  • NK cell differentiation and maturation are sterically regulated by various transcription factors including Id2 and IRF2 (28, 29) . It was then examined whether enhanced NK cell maturation by AA and NG is transcriptionally regulated by Id2 and IRF2.
  • Real-time PCR detected that expression of Id2 and IRF2 were significantly enhanced in NK cells isolated from peripheral blood of LLC lung carcinoma-bearing mice that received combination therapy with AA and NG when compared with control or monotherapy (Fig. 16A, B) .
  • TCM Traditional Chinese Medicine
  • the inventor provided the first evidence for rebalancing the TGF- ⁇ /Smad signaling in tumor microenvironment by a combination therapy with AA (a Smad7 agonist) and NG (a Smad3 specific inhibitor) to synergistically inhibit tumor progression in mouse models of melanoma and lung carcinoma.
  • AA a Smad7 agonist
  • NG a Smad3 specific inhibitor
  • NK1.1+CD122+ and NK1.1+NKp46+ are cell numbers (NK1.1+CD122+ and NK1.1+NKp46+) and anti-cancer activities such as production of IFN- ⁇ and granzyme B.
  • This finding is consistent with previous report that genetic disruption of Smad3 or pharmacological inhibition of Smad3 protects against cancer progression in mouse models of melanoma and lung carcinoma (10) .
  • NK cells exert rapid innate immune response to tumorigenesis and cancer progression by directly triggering tumor cell death (16, 18) .
  • NK cell immune response has been severely suppressed by hyperactive TGF- ⁇ 1/Smad signaling in tumor microenvironment characterized by markedly decreased NK cell accumulation and loss of NK cell cytotoxicity against cancer (20, 35) .
  • combination of AA and NG may serve as a potent therapeutic strategy to restore the NK cell immunity against cancer in TGF- ⁇ 1-abundant tumor microenvironment.
  • NK cell development is strictly programmed by a number of transcription factors and TGF- ⁇ 1 inhibits CD11b high CD43 high NK cells via suppressing both T-bet and GATA3 (20) .
  • TGF- ⁇ 1 via Smad3 promotes cancer progression by inhibiting E4BP4-mediated NK cell development (10) .
  • combination treatment with AA and NG promoted Id2/IRF2-dependent NK cell differentiation and maturation via rebalancing the Smad3/Smad7 signaling.
  • Id2 as the antagonist of E proteins, is indispensable for the development of NK cell precursor to immature NK cells, and also involved in NK maturation (30, 36, 37) ; meanwhile, IRF2 protects premature NK cells from apoptosis to ensure NK cells completing GATA3-induced terminal maturation as well as maintaining NK cell proliferation (31, 38) .
  • TGF- ⁇ 1 not only hampered NK cell terminal maturation by reducing CD11b + NK cell proportion via suppressing IRF2 signaling, but also impaired the production of immature NK via blocking Id2 dependent NK cell lineage commitment.
  • TGF- ⁇ 1 activates Smad2/3 together with ATF1 transcription factors to directly bind to the promoter region of both interferon- ⁇ and granzyme B to induce a significant reduction in cytokine productions (39, 40) .
  • suppressing hyperactive TGF- ⁇ 1/Smad signaling via rebalancing Smad3 and Smad7 signaling by combination therapy debilitated the inhibition of TGF- ⁇ 1 on IFN- ⁇ and granzyme B production in NK cells as noted in this study.
  • Cytotoxicity assay ultimately confirmed combination therapy with AA and NG not only promoted the production of cytokines, but effectively elevated NK cell cytotoxicity against tumor cells compared with either AA or NG monotherapy.
  • the present study provides a new, effective and safe TCM-based therapy for melanoma and lung carcinoma in mice with combination of AA and NG. It also identifies the underlying mechanism through which combined treatment with AA and NG synergistically promoted NK cell immunity against cancer by suppressing TGF- ⁇ /Smad3-mediated inhibition of Id2/IRF2-dependent NK cell differentiation and maturation. Thus, treatment with AA and NG can be a promising TCM-based therapeutic strategy for cancer clinically.
  • B16F10 melanoma syngeneic tumor model was generated in C57/Bl6 male mice (aged week 8 and 25 g body weight) via subcutaneous injection of 1 ⁇ 10 6 B16F10 cells.
  • Lewis Lung Cancer syngeneic tumor model was also generated in C57/Bl6 male mice via subcutaneous injection of 2 ⁇ 10 6 luciferase-labeled LLC cells.
  • mice were randomly divided into four groups respectively receiving drug administration as described below. Tumor progression was monitored by calculating tumor volume with vernier calipers as well as by bioluminescence imaging with IVIS Spectrum system (Xenogen, PerkinElmer, Inc., MA, USA) . All animal experiments were carried out by a protocol approved by Animal Ethics Experimental Committee at the Chinese University of Hong Kong.
  • TCM-based drugs Two TCM-based drugs were used in this study, including asiatic acid (95%HLPC-purified, Nanning, China) and naringenin (98%HLPC-purified, Xian, China) . Both drugs were dissolved in DMSO and Tween-80 at the ratio of 9: 1, which was further diluted 10 times with saline as working solution.
  • Tumor-bearing mice were randomly divided into groups for various treatments with AA at a dose of 10mg/kg body weight, NG at a dose of 50mg/kg body weight, or combination therapy with AA (10mg/kg) and NG (50mg/kg) every day via intraperitoneal injection. Studies were carried out by a protocol approved by Animal Ethics Experimental Committee at the Chinese University of Hong Kong.
  • Immunofluorescence staining was performed with 5 ⁇ m PLP fixed frozen sections of mouse tumor tissue to detect NK cell infiltration in tumor microenvironment and the protein levels of p-Smad3, Smad7, IFN- ⁇ and granzyme B in these tumor infiltrated NK cells.
  • Primary antibodies used for immunofluorescence staining include Alexa488 conjugated anti-mouse NK-1.1 (Biolegend, CA, USA) , PE conjugated anti-mouse NKp46 (eBioscience Inc., CA, USA) , anti-p-Smad2/3 (Santa Cruz Biotechnology, Santa Cruz, CA) , anti-Smad7, anti-interferon gamma and anti-granzyme B (Abcam, MA, USA) . All slides were mounted with DAPI containing mounting medium then analyzed with fluorescence microscope (Leica Microsystems, Wetzlar, Germany) .
  • bone marrow cells were isolated from C57BL/6 mice following the protocol as previously described (10) . Bone marrow cells were then seeded at the density of 1 ⁇ 10 6 cells/ml in MEM ⁇ medium (Gibco, Thermo Fisher Scientific Inc., MA, USA) containing 10%FBS supplemented with 1ng/ml IL-7, 10ng/ml Flt3L, 30ng/ml SCF, 50ng/ml IL-15 (Peprotech, NJ, USA) and 50mM ⁇ -Mercaptoethanol (Gibco, Thermo Fisher Scientific Inc., MA, USA) and cultured for 4 days. Then transferred cells to MEM ⁇ medium containing 10%FBS supplemented with 50ng/ml IL-15, 20ng/ml IL-2 and 50mM ⁇ -Mercaptoethanol to induce NK cells maturation for another 3 days.
  • MEM ⁇ medium Gibco, Thermo Fisher Scientific Inc., MA, USA
  • splenic NK cells were cultured in MEM ⁇ medium containing 10%FBS supplemented with 50ng/ml IL-15 and 20ng/ml IL-2.
  • Id2 and IRF2 were knocked down on freshly isolated mouse bone marrow cells via transfecting siRNA specific for IRF2 (5’-GCAAGCAGUACCUCAGCAATT-3’) , siRNA specific for Id2 (5’-GCACGTCATCGATTACATC-3’) and nonsense control (5’-UUCUCCGAACGUGUCACGUTT-3’) on day 0 and day 4 with Lipofectamine RNAiMAX transfection system (Life technologies, NY, USA) and Opti-MEM medium (Gibco, Thermo Fisher Scientific Inc., MA, USA) and cells were collected on day 6 for flow cytometry analysis..
  • B16F10 melanoma cells were harvested as target cells and bone marrow-derived NK cells treated with AA or NG for 24 hours were harvested as effector cells.
  • Cells were thoroughly washed and seeded in 96-well plates at the effector: target (E: T) ratios of 5: 1, 10: 1 and 20: 1.
  • E target
  • Peripheral blood NK cells and cultured NK cells were analyzed using various markers as previously described (10) , including rat anti-mouse CD16/CD32 antibody (BD Pharmingen, BD Biosciences, CA, USA) , APC-conjugated anti-mouse NK1.1, PE-conjugated anti-mouse NKp46, PE-conjugated anti-mouse CD122 (eBioscience Inc., CA, USA) and Alexa 488-conjugated anti-mouse/human CD11b Antibody (Biolegend, CA, USA) . After immunostaining, cells were resuspended with 200 ⁇ l staining buffer and analyzed with BD AccuriTM C6 Cytometer (BD Biosciences, CA, USA) .
  • BD AccuriTM C6 Cytometer BD Biosciences, CA, USA
  • Tumor homogenates and supernatants from cultured NK cells were measured for IFN- ⁇ and granzyme B levels by using enzyme-linked immunosorbent assay (ELISA) with Mouse IFN-gamma ELISA Kit (R&D Systems, Inc. MN, USA) and Mouse Granzyme B ELISA Kit (eBioscience Inc., CA, USA) .
  • ELISA enzyme-linked immunosorbent assay
  • Bone marrow-derived NK cells were stimulated with 5ng/ml TGF- ⁇ 1 for 1 hour and collected by centrifuge at 2000rpm for 5 minutes. After fixed by cross-linking with 37%formaldehyde, total chromatin was isolate with Enzymatic Chromatin IP Kit (Cell signaling, MA, USA) as instructed by manufacturer. Cross-linked Smad3-DNA complexes were precipitated with Smad3 (C67H9) antibody (Cell signaling, MA, USA) and normal anti-rabbit IgG (Cell signaling, MA, USA) . Targeted genomic regions were subsequently detected by PCR with specific primers for predicted conserved Smad binding site listed in Table 2 and analyzed with gel electrophoresis.
  • Primers used for Real-Time PCR were listed in Table 1.
  • NK cells were lysed with ice-cold RIPA buffer, and then protein was isolated as previously described (26) .
  • Membranes were blocked with 5%BSA in PBS for 1 hour at room temperature before incubated with primary antibodies against Smad7, Id2, ⁇ -actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) , p-Smad3 and IRF2 (Cell signaling, MA, USA) overnight at 4°C, subsequently incubated with IRDye 800-conjugated secondary antibodies (Rockland immunochemicals, PA, USA) .
  • Target protein expression was then detected by LiCor/Odyssey infrared image system (LI-COR Biosciences, NE, USA) and analyzed with Image J software (NIH, Bethesda, MD, USA) .
  • TGF-beta is responsible for NK cell immaturity during ontogeny and increased susceptibility to infection during mouse infancy. Nat Immunol 13, 843-850 (2012) .
  • TGF- ⁇ directly targets cytotoxic T cell functions during tumor evasion of immune surveillance. Cancer cell 8, 369-380 (2005) .

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Abstract

L'invention concerne une utilisation combinée de naringénine et d'acide asiatique pour inhiber la prolifération d'une cellule, et une composition ou un kit comprenant de la naringénine et de l'acide asiatique. La polythérapie avec l'acide asiatique et la naringénine inhibe de manière synergique la croissance du mélanome et du carcinome pulmonaire.
PCT/CN2018/083741 2018-04-19 2018-04-19 Polythérapie contre le cancer avec de la naringénine et de l'acide asiatique WO2019200585A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002017904A1 (fr) * 2000-08-29 2002-03-07 Government Of Malaysia Utilisation d'acide asiatique ou d'asiaticoside pour le traitement du cancer
EP2163247A1 (fr) * 2007-06-13 2010-03-17 The Institute of Biophysics Chinese Academy of Sciences UTILISATION DE NARINGÉNINE ET DE NARINGINE EN TANT QU'INHIBITEURS POUR LA TRANSFORMATION D'UNE VOIE DE SIGNALISATION DU FACTEUR DE CROISSANCE béta1
WO2014063660A1 (fr) * 2012-10-26 2014-05-01 The Chinese University Of Hong Kong Polythérapie de naringénine et d'acide asiatique pour le traitement d'une fibrose

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002017904A1 (fr) * 2000-08-29 2002-03-07 Government Of Malaysia Utilisation d'acide asiatique ou d'asiaticoside pour le traitement du cancer
EP2163247A1 (fr) * 2007-06-13 2010-03-17 The Institute of Biophysics Chinese Academy of Sciences UTILISATION DE NARINGÉNINE ET DE NARINGINE EN TANT QU'INHIBITEURS POUR LA TRANSFORMATION D'UNE VOIE DE SIGNALISATION DU FACTEUR DE CROISSANCE béta1
WO2014063660A1 (fr) * 2012-10-26 2014-05-01 The Chinese University Of Hong Kong Polythérapie de naringénine et d'acide asiatique pour le traitement d'une fibrose

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIN N. ET AL.: "Primary studies on the antitumor effects of asiatic acid combined with chemo therapeutics", JOURNAL OF JIANGSU UNIVERSITY (MEDICINE EDITION), vol. 18, no. 6, 30 November 2008 (2008-11-30), pages 494 - 497, ISSN: 1671-7783 *

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