WO2010037892A1 - Composition comprenant de la silibinine à des concentrations déterminées et préparation combinée comprenant de la silibinine et un inhibiteur de la voie pi3k/akt pour le traitement du cancer - Google Patents

Composition comprenant de la silibinine à des concentrations déterminées et préparation combinée comprenant de la silibinine et un inhibiteur de la voie pi3k/akt pour le traitement du cancer Download PDF

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WO2010037892A1
WO2010037892A1 PCT/ES2009/070415 ES2009070415W WO2010037892A1 WO 2010037892 A1 WO2010037892 A1 WO 2010037892A1 ES 2009070415 W ES2009070415 W ES 2009070415W WO 2010037892 A1 WO2010037892 A1 WO 2010037892A1
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silibinin
cancer
akt
hif
cells
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PCT/ES2009/070415
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English (en)
Spanish (es)
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Jesús MATEO DE CASTRO
Patricia GARCÍA MACEIRA
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Fundación Centro Nacional De Investigaciones Cardiovasculares Carlos Iii (Cnic)
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    • 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
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics

Definitions

  • the present invention is within the field of medicine, and refers to a composition comprising silibinin at certain concentrations, and to said composition that also comprises an Akt inhibitor, for the treatment of cancer.
  • hypoxia is a common characteristic of most solid tumors. As the tumor cells proliferate, the demand for nutrients and oxygen grows to the point where the diffusion of oxygen from the blood vessels becomes limiting, resulting in hypoxia. Cancer cells adapt to this hypoxic environment through the activation of a number of cell pathways that stimulate glycolysis, proliferation, over-regulation of numerous survival factors, and neovascularization (angiogenesis). These processes provide the tumor with sufficient energy and a blood supplement to allow growth under hypoxic conditions.
  • HIF-1 hypoxia-inducible factor 1
  • HIF-1 hypoxia-inducible factor 1
  • HIF-1 ⁇ hypoxia-inducible factor 1
  • HIF-1 ⁇ hypoxia-inducible factor 1
  • HIF-1 ⁇ is rapidly and continuously degraded by the ubiquitin-proteasome system after its binding to von Hippel's protein.
  • HIF-1 ⁇ Lindau (pVHL), in a process that depends on the hydroxylation of proline residues 402 and 564 by a family of enzymes known as prolyl-4-hydroxylases that are dependent on O2, iron and oxoglutarate.
  • prolyl-4-hydroxylases that are dependent on O2, iron and oxoglutarate.
  • the hydroxylation of HIF-1 ⁇ is inhibited, which leads to an increase in the stability of HIF-1 ⁇ .
  • prolyl hydroxylases in hypoxia is recognized as the primary mechanism of the accumulation of HIF-1 ⁇ , it is evident that the expression of HIF-1 ⁇ also depends on its de novo synthesis rate.
  • Some growth factors, cytokines, and other signaling molecules can stimulate the synthesis of HIF-1 ⁇ protein through the activation of the phosphatidylinositol-3-kinase (PI3K) / Akt / mTOR (mammalian target of rapamycin) pathway.
  • mTOR regulates the translation of proteins containing sequences 5 '-terminal TOP through the increase of the phosphorylation of p70S6K effectors (ribosomal protein S6 kinase) and rpS6 (ribosomal protein S6), whichever results in the increase of the translation of mRNAs (oligopyr ⁇ midine tract) in its 5 ' -UTR.
  • mTOR also phosphorylates the 4E-BP1 factor (eukaryotic translation initiation factor 4E-binding protein-1), which results in the activation of elF4E (eukaryotic initiation factor 4E) and induces cap-dependent translation. Recent data suggest that the activity of mTOR is necessary for the expression of HIF-1 ⁇ regardless of the conditions of cellular oxygenation.
  • 4E-BP1 factor eukaryotic translation initiation factor 4E-binding protein-1
  • Silibinin is an antioxidant flavonoid isolated from Silybum marianum L. Gaertn., which is used clinically for its hepatoprotective properties and as an antihepatotoxic agent for the treatment of various liver diseases, also being sold as a dietary supplement. Recently, its preventive, antiproliferative and pro-apoptotic effects have been proven in several cancer cells, mainly in skin, breast, lung, colon, pancreas and prostate cancer.
  • silibinin inhibits angiogenesis through the downward regulation of the Akt and NF- ⁇ B pathways (Mallikarjuna et al., 2004. Cancer Res. 64: 6349-6356).
  • the molecular mechanism by which silibinin exerts its antitumor effects is not known exactly.
  • mTOR protein kinase can form two multiproteic complexes that regulate different aspects of mTOR signaling: the mTOR 1 Complex (mTORCI) and the mTOR 2 Complex (mTORC2).
  • mTORCI mTOR 1 Complex
  • mTORC2 mTOR 2 Complex
  • the mTORCI complex is composed of mTOR, raptor (regulatory-associated protein of mTOR), and ml_ST8, and regulates cell growth and proliferation by modulating processes such as ribosomal biogenesis and protein translation through its effectors p70S6K, rpS6 and 4E-BP1.
  • the mTORC2 complex contains mTOR, rictor
  • Akt PI3K / Akt, while mTORCI inhibition would activate Akt through the negative feed-back produced by p70S6K. It is accepted that rapamycin and its analogs are universal inhibitors of mTORCI (and p70S6K), while they are inhibitors of mTORC2 depending on the cell type, and so on Akt (Sabatini 2006. Nat Rev Cancer 6: 729-734).
  • the tumor cells in which the inhibition of mTOR results in the deactivation of Akt would be those in which a rapamycin-sensitive mTORC2 complex is expressed, while the cells in which Akt is activated or unaffected by mTOR inhibitors they could be those that express a rapamycin insensitive mTORC2 complex, as demonstrated in HeLa cells (Sarbassov et al., 2006. Mol CeII 22: 159-68).
  • silibinin both to treat prostate cancer and other types of cancer
  • silibilin is marketed in the form of 150 mg capsules, or as a lyophilized injectable solution containing the equivalent of 350 mg of silibinin.
  • Studies in mice have shown that administering 2 g of silibinin per kg of body weight orally reaches up to 160 ⁇ M of silibinin in plasma (Agarwal et al., 2003. Oncogene 22, 8271-8282).
  • the recommended dose for injectable administration is 20 mg of silibinin per kg of body weight and day, divided into 4 IV (intravenous) infusions of 2 hours each, and with 4 hours of interval between them, controlling the balance of liquids In each infusion, therefore, 5 mg of silibinin per kg of body weight will be administered.
  • one vial 350 mg of silibinin
  • the contents of the vial are dissolved in 35 ml of the infusion solution to be administered (0.9% sodium chloride solution or 5% glucose) and the amount of reconstituted solution (1 ml «is added 10 mg silibinin), necessary, depending on the weight of the patient, the rest of the saline or glucose.
  • treatment with silibinin in most types of cancer will be more effective if high doses of the same are administered to the patient, which allow reaching concentrations in cells greater than 250 ⁇ M, and even more preferably, of 500 ⁇ M, in serum.
  • a first aspect of the invention refers to the use of a pharmaceutical composition, hereinafter the first composition of the invention, comprising as active ingredient silibinin, a pharmaceutically acceptable salt, or a prodrug, derivative or analog thereof, for Ia elaboration of a medicine for the treatment of cancer, where silibinin is in the proportion necessary to reach serum concentrations (cellular) equal to or greater than 250 ⁇ M, or alternatively, to a pharmaceutical composition comprising as active ingredient silibinin, a pharmaceutically acceptable salt, or a prodrug, derivative or analogue thereof in the proportion necessary to reach serum concentrations (cellular) equal to or greater than 250 ⁇ M, for use in the treatment of cancer.
  • silibinin concentrations are kept substantially constant, the Cmin (minimum concentration) being in serum of at least 250 ⁇ M.
  • Silibinin (INN International Nonprop ⁇ etary ⁇ ame), also known as silibin or silibinin, is the main active constituent of silymarin, the mixture of flavolignans extracted from Silybum marianum. They have as CAS number 22888-70-6, and as chemical formula IUPAC (International Union of Puré and Applied Chemistry) 3,5,7-trihydroxy-2- (3- (3-hydroxy-4- methoxyphenyl) -2- ( hydroxymethyl) -2,3 dihydrobenzo [b] [1,4] dioxin-6-yl) chroman-4- one.
  • silibinin herein, it is understood both the silibinin itself and any of its derivatives, salts, prodrugs, or the like, or any combination thereof.
  • active substance means any component that potentially provides a pharmacological activity or other different effect on the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.
  • the term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form provided that provides the specific activity or effect.
  • derivative includes both pharmaceutically acceptable compounds, and pharmaceutically unacceptable derivatives, since these may be useful in
  • prodrugs of silibinin include any compound derived from silibinin, for example, esters, including carboxylic acid esters, amino acid esters, phosphate esters, metal salt sulphonate esters, etc., carbamates, amides , etc., which, when administered to an individual, is capable of providing, directly or indirectly, said compound of formula (I) in said individual.
  • said derivative is a compound that increases the bioavailability of the compound of formula (I) when administered to an individual or that enhances the release of the compound of formula (I) in a biological compartment.
  • the nature of said derivative is not critical, as long as it can be administered to an individual and provides the compound of formula (I) in a biological compartment of an individual.
  • the preparation of said prodrug can be carried out by conventional methods known to those skilled in the art.
  • substantially constant with respect to the serum concentration of the active ingredient (silibilin, pharmaceutically acceptable salts, derivatives or analogs, or any combination thereof) means that the serum profile after the administration of the formulation does not essentially have values substantial peak. This can also be expressed mathematically in relation to the "fluctuation index" (Fl) for the serum concentration of the active substance (not bound) (or the sum of the active ingredients when applicable), where the Fl fluctuation index is calculated how:
  • Cmax and Cmin are the maximum and minimum concentrations, respectively, of active ingredient
  • AUC_ is the area under the serum concentration profile (concentration versus time curve)
  • ⁇ is the length of the dose interval throughout the period ⁇ .
  • the Cmin would be 250 ⁇ M, and even more preferably, 500 ⁇ M.
  • Ways to achieve substantially constant concentrations of the active ingredient in serum are known in the state of the art, such as, but not limited to, by the use of controlled release pharmaceutical forms.
  • the formulation of the present invention is not restricted to any particular type of formulation. For this reason, the embodiment of the present invention can be used various types of controlled or sustained release formulations, such as, for example, osmotic tablets, gelatinous matrix tablets, coated pellets, etc.
  • a preferred embodiment of this aspect of the invention refers to the use of a pharmaceutical composition comprising as active ingredient silibinin, a pharmaceutically acceptable salt, or a prodrug, derivative or analogous thereof, for the preparation of a medicament for the treatment of cancer.
  • silibinin is in the proportion necessary to reach serum concentrations (cellular) equal to or greater than 500 ⁇ M
  • a pharmaceutical composition comprising as active ingredient silibinin, a pharmaceutically acceptable salt, or a prodrug, derivative or analog thereof in the proportion necessary to reach serum concentrations (cellular) equal to or greater than 500 ⁇ M, for use in the treatment of cancer.
  • silibinin concentrations are kept substantially constant, the serum Cmin being at least 500 ⁇ M.
  • silibinin is found as silibinin-C-2 ', 3-disodium dihydrogen succinate.
  • the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.
  • the cancer is selected from the list comprising: cervical cancer, gastrointestinal cancer and liver cancer.
  • silibinin necessary to reach said cellular concentrations will depend on the formulation of the drug, and on the pharmaceutically accepted vehicles that allow an adequate release of silibinin. Suitable formulation methods are known in the state of the art. Methods of extrapolation of drug concentrations In vitro serum values in vivo are also known in the state of the art, such as, but not limited to, those described in Gülden & Sebet 2003. Toxicology 189: 211-222.
  • the medicament comprises the necessary concentration of silibinin to reach serum concentrations equal to or greater than 500 ⁇ M, which, following the above reasoning, would be equivalent to 20.82 mg / ml in 35 ml, so that the pharmaceutical composition must have values of Silibilin greater than 730 mg.
  • Other methods of calculating the necessary concentration of silibinin to achieve serum concentrations greater than 250 ⁇ M, and even more preferably, concentrations greater than 500 ⁇ M are known and well documented in the state of the art, and as said, will depend on The formulation used, the route of administration, and other factors, such as the weight and age of the patient.
  • the recommended dose for injectable administration is greater than 40 mg of silibinin per kg of body weight and day, divided into 4 IV (intravenous) infusions of 2 hours each, and with an interval of 4 hours between them, controlling the balance of liquids.
  • IV intravenous
  • more than 10 mg of silibinin per kg of body weight will be administered, thus reaching serum concentrations of approximately 500 ⁇ M.
  • Another alternative would be the elaboration of controlled release forms that maintain the concentrations of the active substance. (silibilin, pharmaceutically acceptable salts, derivatives or analogs, or any combination thereof) in serum substantially constant and equal to or greater than 250 ⁇ M, and even more preferably, substantially constant and equal to or greater than 500 ⁇ M.
  • the route of administration of the injectable solution can be, without limitation, intravenous, but preferably, it is administered by direct injection into the tumor, since it allows the therapeutic effect to be concentrated at the level of the affected tissues, or by any form of known controlled release. in the state of the art that allows to maintain in serum the concentrations of the active principle described herein.
  • Said controlled release pharmaceutical form can also be located or implanted in the area of interest (in the tumor).
  • Another aspect of the invention relates to a vial for infusion with lyophilized product comprising silibinin-C-2 ', 3-dihydrogen disodium succinate in values of approximately 550 mg (551, 15 mg of silibinin-C-2', 3- disodium dihydrogen succinate, equivalent to 365 mg of silibinin (INN),
  • the lyophilized product infusion vial comprising silibinin-C-2 ', 3-dihydrogen disodium succinate in values of approximately 1100 mg (1102, 3 mg of silibinin-C-2 ', 3-dihydrogen disodium succinate, equivalent to 730 mg of silibinin (INN).
  • the present invention provides a useful treatment for cancer, especially for those types of cancer in which silibinin indirectly activates the PI3K / Akt pathway, by administering silibinin and an inhibitor of the PI3K / Akt pathway.
  • silibinin inhibits the accumulation of hypoxia-induced HIF-1 ⁇ in several cell lines.
  • silibinin represses the activity of mTOR and its effectors p70S6K, rpS6 and 4E-BP1, increasing the phosphorylation of Akt, and that the combined use of silibinin with inhibitors of the PI3K / Akt pathway has a synergistic effect in the treatment of certain types of cancer, by preventing Ia activation of PI3K / Akt that produces silibinin in certain cell types.
  • compositions comprising silibinin and an inhibitor of the PI3K / Akt pathway are more useful in those types of cancer in which silibinin produces an activation of the PI3K / Akt pathway.
  • another aspect of the invention relates to a composition, hereinafter second composition of the invention, comprising silibinin and an inhibitor of the PI3K / Akt pathway.
  • Another aspect of the invention refers to the use of the composition of the invention as a medicine, or alternatively, to the second composition of the invention for use as a medicine.
  • Silibinin (INN International Nonproprietary ⁇ ame), also known as silibinin, is the main active constituent of silymarin, the mixture of flavolignans extracted from Silybum mar ⁇ anum. They have the chemical formula IUPAC (International Union of Puré and Applied Chemistry) 3,5,7-trihydroxy-2- (3- (3-hydroxy-4-methoxyphenyl) -2- (hydroxymethyl) -2,3 dihydrobenzo [b] [1, 4] dioxin-6-yl) chroman-4-one.
  • IUPAC International Union of Puré and Applied Chemistry
  • the PI3K / Akt pathway has been widely studied and has been recognized as a promising target for anticancer therapies since its activation is a key cellular event during tumorigenesis. Once the PI3K and Akt kinases have been activated under apoptotic stress, signals are translated into a series of downstream regulators.
  • Akt inhibitors include LY294002 (CAS 154447-36-6) and Wortmannin (CAS 19545-26-7), and among Akt inhibitors, Triciribine (API-2, NSC-154020, TCN, Akt inhibitor V ; CAS 35943-35-2), A-443654 (imidazole-pyridine based), KP372-1, Akt Inhibitor Il (SH-5), Akt Inhibitor III (SH-6), Akt Inhibitor IV (CAS 681281-88- 9), Akt Inhibitor VIII, Isozyme-Selective, Akti-1/2 (hydrated salt of 1,3-Dihydro-1- trifluoroacetate (1 - ((4- (6-phenyl-1 H-imidazo [4,5- g] quinoxalin-7-yl) phenyl) methyl) -4-piperidinyl) -2H-benzimidazol-2-one), Akt Inhibitor X (CAS 925681-41-0).
  • the Akt inhibitor is selected from the list comprising: LY294002, Wortmannin, Triciribine (API-2, NSC-154020, TCN, Akt inhibitor V), A-443654 (imidazole-pyridine based), KP372-1, Akt Inhibitor Il (SH-5), Akt Inhibitor III (SH-6), Akt Inhibitor IV, Akt Inhibitor VIII, Isozyme-Selective, Akti-1/2, Akt Inhibitor X
  • the PI3K / Akt pathway inhibitor is LY294002.
  • silibinin has an mTOR inhibitory effect, useful in the treatment of cancer, and at the same time inhibitors of the activation of the PI3K / Akt pathway are useful in those types of cancer in which silibinin, at Like other mTOR inhibitors, they activate this pathway.
  • the combined use of silibinin with the PI3K / Akt inhibitor employed (LY294002) gives rise to a synergistic effect on certain tumor cell lines.
  • composition of the invention for the preparation of a medicament for the treatment of cancer.
  • the types of cancer are selected from the list comprising: cervical cancer, cancer gastrointestinal or liver cancer.
  • the cancer is cervical cancer.
  • Another aspect of the invention relates to a combined preparation of at least silibinin and an inhibitor of the PI3K / Akt pathway, from now on combined preparation of the invention, for its separate, simultaneous or sequential use in the treatment of Cancer.
  • silibinin acts as an anticancer by inhibiting mTOR, and since the function of mTOR and its regulation by the PI3K / Akt pathway is a specific tumor, the treatment of silibinin cancer sometimes activates the pathway PI3K / Akt. The activation of
  • the PI3K / Akt pathway is necessary for cell differentiation, and probably has a great physiological relevance, since it couples vital processes such as cell differentiation and survival.
  • the combination of silibinin with a PI3K / Akt inhibitor synergistically increases the response, as shown in the examples of the invention, not representing a mere aggregate of known agents, but a new combination that provides a new effective treatment. against cancer
  • Silibinin and the PI3K / Akt inhibitor could be administered to a patient separately, simultaneously or sequentially, depending on the most appropriate administration schedule for each case.
  • Said combined preparation of silibinin and an inhibitor of the PI3K / Akt route separately, simultaneously or sequentially would be useful in the preparation of a medicament for the treatment of cancer.
  • the term "combined preparation” or also called “juxtaposition”, herein means that the components of the combined preparation need not be present as a union, for example in a composition, in order to be available for separate application or sequential.
  • the expression “juxtaposed” implies that it is not necessarily a true combination, in view of the physical separation of the components.
  • the combined preparation of the invention is used for the treatment of a series of cancers that are selected from the list comprising: cervical cancer, gastrointestinal cancer and liver cancer.
  • the cancer is cervical cancer.
  • cancer means a set of diseases in which the body produces an excess of malignant cells (also known as cancer or cancer), with typical behavioral and uncontrolled growth traits (growth and division beyond the limits normal, invasion of surrounding tissue and sometimes metastasis). It comprises any disease of an organ or tissue in a mammal, preferably man, characterized by a poorly controlled, or uncontrolled, multiplication of normal or abnormal cells in said tissue, and their effect on the entire body.
  • cancer within this definition, includes benign neoplasms, dysplasias, hyperplasias, as well as neoplasms that show metastases, or any other transformation such as, for example, leukoplasias that often precede the outbreak of cancer.
  • Cancer cells and tissues are cancerous when they grow and replicate more quickly than normal, moving or dispersing in the surrounding healthy tissue or any other body tissue, which is known as metastasis, assumes forms and abnormal sizes, shows changes in its nucleocytoplasmic ratio, nuclear polychromasia, and finally ceases. Cancer cells and tissues can affect the body as a whole causing paraneoplastic syndromes, or if cancer occurs in a vital organ or tissue, its normal function being interrupted or damaged, with possible fatal results. The final result of the evolution of a cancer that involves a vital organ, either primary or metastatic, is the death of the affected mammal. The cancer tends to spread, and its degree of extension is normally related to changes in the survival of the disease.
  • the cancer is in one of three growth stages: early or localized, when the tumor is still confined in the tissue of origin, or in its primary location; direct extension, when the cancer cells of the tumor have invaded adjacent tissue or have spread only to regional lymph nodes; or metastasis, when cancer cells have migrated to distant parts of the body from the primary location, through the circulatory or lymphatic system, and has been established in secondary locations.
  • a cancer is malignant because of its tendency to cause death if it is not treated.
  • Benign tumors usually do not cause death, although they can do so if they interfere with the normal function of the body due to its characteristics or location, size or paraneoplastic effects.
  • the malignant tumors fall within the definition of cancer within the scope of this definition as well.
  • cancer cells divide at a higher rate than normal cells, but the distinction between the growth of normal and cancerous tissues is not so much that cell division is much faster, such as partial or complete loss of stopping growth and differentiate into a useful and limited tissue, of the type that characterizes the functional balance of normal tissue growth.
  • the cancerous tissue can express certain receptor molecules and are probably influenced by susceptibility and immunity, and it is known that certain prostate and breast cancers, for example, depend on certain hormones.
  • the term "cancer” herein is not simply limited to benign neoplasms, but also includes other benign or malignant neoplasms such as: 1) Carcinoma, 2) Sarcoma, 3) Carcinosarcoma, 4) Blood-forming tissue cancers, 5) tumors of nerve tissues, including the brain, 6) cancer of skin cells.
  • Carcinosarcoma occurs in the epithelial tissues, which cover the external face of the body (the skin) and the mucous membranes and the internal cavity of the structure of the organs, such as the breasts, the lung, the digestive and gastrointestinal tract, the glands endocrine, and the genitourinary system.
  • Ductal or glandular elements may persist in epithelial tumors, as well as in adenocarcinomas, such as thyroid adenocarcinoma, gastric adenocarcinoma, uterine adenocarcinoma.
  • Cancers of the epithelium of paved cells of the skin and certain mucous membranes can be called squamous cell carcinoma of the respective tissues, and are also within the definition of cancer in this memory.
  • Sarcoma develops in connective tissues, including fibrous, adipose tissue, muscle, blood vessels, bone, and cartilage such as osteogenic sarcoma, liposarcoma, fibrosarcoma, and synovial sarcoma.
  • Carcionosarcomas develop in both epithelial and connective tissue.
  • the cancer can be primary or secondary. Primary indicates that The cancer has originated in the tissue that has been found, rather than having been established after metastasis from another region.
  • Cancer and tumor diseases can also be benign or malignant, and can affect the anatomical structures of a mammalian body. For example, but without limiting our, they can be:
  • endocrine and exocrine glands such as thyroid, parathyroid, pituitary, adrenal glands, salivary glands, pancreas.
  • breast such as benign and malignant tumors in the mammary glands of both men and women, mammary ducts, adenocarcinoma, medullary carcinoma, carcinoma carcinoma. Paget's disease of the nipple, inflammatory carcinoma of young women, ...
  • Bone, and its connective and supportive tissues such as malignant or benign bone tumor, for example, osteogenic malignant sarcoma, benign osteoma, cartilage tumors; such as malignant chondrosarcoma or benign chondroma; Bone marrow tumors, such as malignant myeloma, or benign eosinophilic granuloma, as well as metastatic tumors of bone tissues in other locations of the body,
  • pancreas such as ductal carcinoma of the pancreas
  • Lymphatic tissue such as lymphomas and other tumors of lymphoid origin
  • XV Lymphatic tissue
  • XVII primary and secondary cancer of the lymph nodes
  • XVIII The tongue and bone structures of the palate and sinuses
  • XXI The smooth or skeletal muscle, including its ligaments and membranes
  • XXII The peripheral, autonomous and central nervous system, including the cerebellum
  • compositions of the present invention and the combined preparation can be formulated for administration to an animal, and more preferably to a mammal, including man, in a variety of ways known in the state of the art.
  • they can be, without limitation, in sterile aqueous solution or in biological fluids, such as serum.
  • Aqueous solutions may be buffered or unbuffered and have additional active or inactive components. Additional components include salts to modulate the ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelants, and the like, and nutrients including glucose, dextrose, vitamins and minerals.
  • the compositions can be prepared for administration in solid form.
  • compositions can be combined with various vehicles or excipients inert, including but not limited to; binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or corn starch; lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint or methyl salicylate.
  • binders such as microcrystalline cellulose, gum tragacanth, or gelatin
  • excipients such as starch or lactose
  • dispersing agents such as alginic acid or corn starch
  • lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide
  • sweetening agents such as sucrose or saccharin
  • flavoring agents such as peppermint or methyl salicylate.
  • compositions or preparations and / or their formulations may be administered to an animal, including a mammal and, therefore, to man, in a variety of ways, including, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intracecal, intraventricular, oral, enteral, parenteral, intranasal or dermal.
  • the dosage to obtain a therapeutically effective amount depends on a variety of factors, such as, for example, the age, weight, sex, tolerance, ... of the mammal.
  • the expression "therapeutically effective amount” refers to the amount of silibinin, prodrugs, derivatives or analogs of silibinin that produce the desired effect and, in general, will be determined, among other causes, by the characteristics typical of said prodrugs, derivatives or analogs and the therapeutic effect to be achieved.
  • the pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the vehicles known to those skilled in the art.
  • Fig. 1 Silibinin inhibits the accumulation of hypoxia-induced HIF-1 ⁇ protein and transcriptional activation of HIF-1.
  • A. The HeLa and Hep3B tumor cells were exposed to hypoxic conditions (2% O2) for the times indicated in the absence or presence of silibinin (SiIi, 500 ⁇ mol / L). Protein levels of HIF-1 ⁇ , HIF-1 ⁇ and actin were detected by immunoblot from total cell extracts as described in materials and methods.
  • B Hep3B cells were treated for 4 h in normoxia (21% O2, lane 1), hypoxia (2% O2), or with the hypoxia-mimetic agent DMOG (1 mmol / L) in the presence of silibinin in the indicated concentrations, or with the vehicle (0).
  • HeLa cells were exposed to the indicated hypoxia levels ([O 2 ], 6, 3, 1 and 0.1%) for 4 h in the absence or presence of silibinin (SiIi, 500 ⁇ mol / L).
  • the levels of the HIF-1 ⁇ and actin proteins were detected by immunoblot as in A.
  • Fig. 2 Silibinin does not affect the degradation of the HIF-1 ⁇ protein or the expression of the HIF-1 ⁇ mRNA.
  • A HeLa cells were exposed to hypoxia (2% O2) for 2 h, and silibinin (500 ⁇ mol / L) or the vehicle (control) were added 15 min before the end of the hypoxic incubation. The cells were then exposed to normoxia (reoxygenation) for the indicated periods, and the levels of the HIF-1 ⁇ protein were measured by immunoblot.
  • the lower panel shows the densitometric quantification of the levels of HIF-1 ⁇ , with the values expressed as the percentage of the expression before reoxygenation (time 0). The values represent the mean ⁇ SE of four independent experiments.
  • HIF-1 ⁇ CH13 KaO cells deficient in HIF-1 ⁇ were transfected with the wild type HIF-1 ⁇ , the mutant P402A / P564A-HIF-1 ⁇ or with the empty vector as described in Materials and Methods. After 24 h, the cells were exposed to normoxia or hypoxia (2% O 2 ) for 4 h in the presence of the indicated concentrations of silibinin (SiIi), and the recombinant HIF-1 ⁇ was detected by immunoblot.
  • C HeLa cells were cultured under normoxic (21% O 2 ) or hypoxic (2% O2) conditions in the absence or presence of silibinin (500 ⁇ mol / L) for the indicated times.
  • D Upper panel, HeLa cells were incubated for 2 h under normoxic conditions (21% O 2 ) or hypoxic (2% O 2 ). The cells were then treated for an additional hour under the same atmospheres in the presence of MG132 (20 ⁇ mol / L), cyclohexamide (CHX, 100 ⁇ mol / L) or silibinin (500 ⁇ mol / L), followed by immunoblot analysis.
  • Lower panel HeLa cells were incubated for 3 h in hypoxia (2% O 2 ). Subsequently, the cells were treated in hypoxia for the additional times indicated with the vehicle (control), CHX (100 ⁇ mol / L) or silibinin (500 ⁇ mol / L), and HIF-1 ⁇ was detected by immunoblot.
  • Fig. 3 Silibinin inhibits mTOR signaling and increases the phosphorylation of Akt in HeLa and Hep3B cancer cells.
  • A The cells were incubated in hypoxia (2% O 2 ) for 3 h in the presence of the indicated concentrations of silibinin (SiIi). Lane 1 of each panel shows the baseline levels in normoxia of the proteins under study.
  • B HeLa cells were incubated for 4 h in normoxia (21% O2) or hypoxia (2% O 2 ) in the presence of silibinin (500 ⁇ mol / L), LY294002 (LY, 10 ⁇ mol / L), or rapamycin ( Rapa, 20 nmol / L) as indicated.
  • Hep3B cells were incubated as in B, and treated with the indicated concentrations of silibinin (SiIi) in the absence or presence of LY294002 (LY, 10 ⁇ mol / L).
  • the proteins in AC were detected by immunoblot using the specific antibodies described in Materials and Methods.
  • Fig. 4 The effects of silibinin on the mTOR pathway, the activation of Akt and the accumulation of HIF-1 ⁇ are rapid and completely reversible.
  • HeLa and Hep3B cells were exposed to hypoxia (2% O 2 ) for 3 h, and silibinin (SiIi, 500 ⁇ mol / L) was added during the last 0 (untreated), 10, 20, 30 or 60 minutes of hypoxic incubation.
  • Lanes 7 and 8 of each panel represent the cells treated with silibinin during the last 60 min of hypoxic incubation, followed by two washes of the extracellular medium to eliminate the silibinin, and the incubation for an additional period of 30 or 60 min under hypoxia. study the reversibility of the effects.
  • Lane 1 of each panel shows the baseline normoxia levels of the analyzed proteins, which were detected by immunoblot.
  • Fig. 5 Silibinin inhibits the release of hypoxia-induced VEGF in tumor cells HeLa and Hep3B.
  • A The cells were incubated for 12 h in normoxia or hypoxia (2% O2) in the presence of the indicated silibinin concentration, and the extracellular medium was recovered for the determination of VEGF by ELISA as described in Materials and Methods. The values represent the mean ⁇ SE from three independent experiments. ** , P ⁇ 0.01, *** , P ⁇ 0.001, significantly different compared to control cells in hypoxia without silibinin.
  • VEGF vascular endothelial growth factor
  • Fig. 6 Effect of silibinin on the proliferation and apoptosis of HeLa and Hep3B tumor cells.
  • A The cells were incubated in hypoxia (2% O2) with increasing concentrations of silibinin (0, 50, 100,
  • Viable cells were quantified fluorimetrically by converting resazurin to resorufin, as described in Materials and Methods.
  • B The cells were incubated for 8 h as in A, and apoptosis was quantified by measuring caspase-3 and -7 activities with a luminescent analysis, using staurosporin as a positive control (Staur, 500 nmol / L). The bars represent the mean ⁇ SE. * , P ⁇ 0.05, ** , P ⁇ 0.01, *** , P ⁇ 0.001, significantly different compared to the untreated control at each time.
  • Fig. 7 Effect of silibinin on the signaling of mTOR and the phosphorylation of Akt in the AGS cell line of gastrointestinal cancer.
  • AGS cells were cultured in hypoxia (2% O 2 ) for 3 h in the presence of the indicated concentrations of silibinin (SiIi). Lane 1 shows the baseline levels in normoxia of the proteins under study. The proteins indicated were detected by immunoblot using the specific antibodies described in Materials and Methods.
  • Fig. 8 Effect of silibinin on the signaling of mTOR and the phosphorylation of Akt in the PC-3 cell line of prostate cancer.
  • the PC-3 cells were cultured in hypoxia (1.5% O2) for 3 h in the presence of the indicated concentrations of silibinin (SiIi).
  • Lane 1 shows the baseline levels in normoxia of the proteins under study.
  • the proteins indicated were detected by immunoblot using the specific antibodies described in Materials and Methods.
  • Fig. 9 Silibinin rapidly decreases the accumulation of the HIF-1 ⁇ protein under hypoxia despite the inhibition of proteosomal degradation of the protein.
  • HeLa cells were exposed to hypoxia (2% O2) for 2 hours. Subsequently, the cells were treated for an additional hour in the same atmosphere, in the presence or absence of MG132 (20 ⁇ M), and silibilin (SiIi, 500 ⁇ M) or a vehicle was added for the last 0 (untreated), 10, 20, 30 or 60 minutes of hypoxic incubation. HIF-1 ⁇ and Actin were detected by western blot analysis.
  • Fig. 10 Silibilin affects mTOR signaling and Akt forphosphorylation in HeLa and Hep3B cancer cells under normoxic conditions.
  • the cells were cultured in normoxia (21% O2) for 3 hours in the presence of the indicated concentrations of silibinin (sili).
  • silibinin SiIi, 500 ⁇ M was added for the last 0 (untreated), 10, 20, 30 or 60 minutes of incubation.
  • Lanes 6 and 7 of each panel represent the cells treated with silibinin during the last 60 minutes of incubation followed by two washes of the extracellular medium to remove the silibinin, and the additional incubation for 30 or 60 minutes to study the reversibility of the effects. After the treatments, the indicated proteins were detected by western blot analysis in the complete cell extracts.
  • Fig. 11 The TSC1 / TSC2 complex is necessary for the Akt-induced phosphorylation (Ser 473 ) but not for the mTOR / p70S6K / 4E- suppression.
  • BP1 HeLa cells were transfected with TSC2 or control siRNA. 48 hours after transfection, the cells were treated for 3 hours in normoxia (21% O2) with the indicated concentrations of silibinin (SiIi), rapamycin (Rapa, 2OnM) or the vehicle. The cells were subsequently collected and a western blot analysis was performed to detect the indicated proteins,
  • Fig. 12 Effect of silibinin and rapamycin on the proliferation of non-tumor epithelial cells.
  • Primary fibroblasts of adult human skin and human embryonic renal cells (HEK293) were cultured in hypoxia (2% O 2 ) with increasing concentrations of silibinin (50, 100, 250, 500 ⁇ M) or rapamycin (1, 10, 100 nM) for 8 hours
  • the fluorometrically viable cells were quantified as described in the materials and methods.
  • the bars represent the mean ⁇ SE. * , P ⁇ 0.05, ** , P ⁇ 0.01, *** , P ⁇ 0.001, significantly different compared to untreated controls.
  • HeLa Human cervical adenocarcinoma
  • Hep3B hepatocellular carcinoma
  • ATCC American Type Culture Collection
  • DMEM fetal calf serum
  • CHO Ka13.5 cells deficient in HIF-1 ⁇ were grown in a Ham's F-12 nutritive mixture (Invitrogen, Bacelona, Spain). Culture media were supplemented with 10% inactivated FBS (Sigma) and penicillin (100 IU / ml) / streptomycin (100 .mu.g / ml), and the cells were grown at 37 0 C in humidified incubator containing 5% CO2.
  • a humidified hypoxia chamber COY Labs., Grasslake, Ml
  • O2 regulator Air-Liquide, Madrid, Spain
  • All cell treatments were carried out in a growth medium containing 3% FBS and without antibiotics.
  • Silibinin, cyclohexamide, staurosporine and Z-Leu-Leu-Leu-al (MG132) were purchased from Sigma.
  • Dimethyloxalyl glycine (DMOG) was purchased from Alexis Biochemicals (Lausen, Switzerland).
  • HIF-1 ⁇ and HIF-1 ⁇ were from BD Transduction Laboratories (BD Biosciences, Madrid, Spain).
  • LY294002 rapamycin and antibodies against phospho-Akt (Ser473), phospho-mTOR (Ser2448), phospho-p70S6 kinase (Thr389), phospho-ribosomal protein S6 (Ser235 / 236) and phospho-4E-BP1 (Ser65) CeII Signaling Technology (Beverly, MA).
  • Anti- actin was obtained from Sigma.
  • the cells were seeded in 60 mm culture plates and allowed to adhere for 24 hours. Immediately after the treatments, the cells were washed with cold PBS and recovered by scraping in 1-ml of cold PBS supplemented with a protease inhibitor cocktail (Roche Diagnostics, Barcelona, Spain). The cell pellets were homogenized in 50 ⁇ l of lysis buffer composed of a CytoBuster TM protein extraction reagent (Novagen) supplemented with protease inhibitor cocktails (Roche) and phosphatases (Sigma). After incubation on ice (15 min), the used ones were stirred and centrifuged (16,000 xg, 10 min, 4 ° C), and the supernatants collected as total cell extracts.
  • lysis buffer composed of a CytoBuster TM protein extraction reagent (Novagen) supplemented with protease inhibitor cocktails (Roche) and phosphatases (Sigma). After incubation on ice (15 min), the used ones were stirred and centrifuged (16,000 xg,
  • the protein concentration was determined by the BCA protein assay (Pierce). Cell extracts (40-80 ⁇ g of proteins) were separated by SDS-PAGE and transferred to nitrocellulose membranes, and the immunoblot was carried out as previously described. When required, the intensity of the bands was quantified with the Quantity One v4.6 software (Bio-Rad, Hercules, CA).
  • HIF-1 ⁇ cDNA GenBank accession U22431; SEQ ID NO: 1
  • ATCC accession U22431
  • SEQ ID NO: 1 The human HIF-1 ⁇ cDNA was obtained from the ATCC (pCEP4 / HIF-1 ⁇ ).
  • pcDNA4 / HisMax The primers were designed to contain the restriction sites for Bam ⁇ ⁇ ⁇ and Not ⁇ at terminations 5 ' and 3 ' , respectively.
  • Primers SEQ ID NO: 2 and SEQ ID NO: 3 were used.
  • the amplified PCR product was purified (QIAquick PCR Purification Kit, Qiagen, Valencia, CA), digested with Bam ⁇ ⁇ ⁇ and Not ⁇ , and ligated into pcDNA4 / HisMax previously digested with the same restriction enzymes.
  • the resulting construct, pcDNA4 / HisMax-HIF-1 ⁇ was amplified and purified (Qiagen).
  • the double mutant HIF-1 ⁇ P402A / P564A was generated by sequentially directed mutagenesis (QuickChange II, Stratagene) using as a pattern pcDNA4 / HisMax-HIF-1 ⁇ .
  • the mutagenic oligonucleotides were, for the P402A mutation SEQ ID NO: 4 and SEQ ID NO: 5; for the P564A mutation, SEQ ID NO: 6 and SEQ ID NO: 7).
  • This mutant of HIF-1 ⁇ is not susceptible to hydroxylation by prolyl-4-hydroxylases and subsequent degradation.
  • the uncertainty of the cons ⁇ rucations was confirmed by DNA sequencing.
  • Transient transfection and reporter trial of HRE-dependent luciferase Transffective transfection of CHO Ka13.5 cells was carried out in 60 mm culinary plates using Lipofec ⁇ amine2000 (Invitrogen) and 1 ⁇ g of the DNA plasmid (wild type HIF-1 ⁇ , P402A / P564A-HIF-1 ⁇ , or the empty vector).
  • the cells were cultured in 24-well plates and transfected with 0.3 ⁇ g per well of the reporter plasmid p9HIF1-Luc using the transfection agent FuGENE 6 (Roche) according to the manufacturer's instructions.
  • the transfection efficiency was monitored by cotransfection with 0.1 ⁇ g of the control plasmid pRL-TK (Promega) carrying the Renilla luciferase gene.
  • the activities of firefly and Renilla luciferase were tested using the Dual-Glo Luciferase Assay System (Promega), and the firefly luciferase activity was normalized to the activity of Renilla luciferase.
  • VEGF quantification The concentration of human VEGF in the conditioned cell medium was measured with an ELISA kit (Pierce). The cells were seeded in 6-well plates and cultured to reach 80-90% confluence. The medium was replaced and the cultures were treated as indicated. The secreted VEGF was quantified after 12 h in the extracellular medium (50 ⁇ l). The results were normalized with respect to the amount of total protein per well.
  • the PCR conditions were established in pilot experiments to ensure the linearity of reaction rates GAPDH was used as the internal standard.
  • the PCR products were separated on 1.5% agarose gels and visualized by staining with ethidium bromide. The gels were photographed using an image analyzer GeI DOC 2000 (Bio-Rad).
  • Cell proliferation and apoptosis were studied with the Cell-Blue® CeII Viability Assay fluorimetric assay (Promega), which exploits the ability of the resazurin indicator to measure the metabolic capacity, an indication of cell viability.
  • Cell-Blue® CeII Viability Assay fluorimetric assay Promega
  • Caspase-Glo® 3/7 Assay luminescent assay Promega
  • EXAMPLE 1 Silibinin inhibits the accumulation of hypoxia-induced HIF-1 ⁇ in tumor cells HeLa and Hep3B.
  • hypoxia induced a time-dependent accumulation of HIF-1 ⁇ protein in HeLa and Hep3B tumor cells that was noticeable after 1 h.
  • Silibinin inhibited the accumulation of HIF-1 ⁇ (Fig. 1A), and this inhibition was complete within the first hour of treatment with silibinin in both cell types, and persisted while the drug was present in the medium (at least up to 16 h , data not revealed).
  • silibinin did not alter the levels of HIF-1 ⁇ protein (Fig. 1A).
  • silibinin fully advocated the accumulation of HIF-1 ⁇ induced by the DMOG inhibitor (Fig. 1 B), a well-characterized mimetic hypoxic agent. Consistent with the inhibition of the accumulation of HIF-1 ⁇ , silibinin also produced the dose-dependent inhibition of the transcriptional activity of HIF-1 in cells exposed to hypoxia or treated with DMOG, as determined using a reporter construct of the response to hypoxia (Fig. 1C).
  • HIF-1 ⁇ did not stabilize at O2 concentrations of 6%, with accumulation increasing markedly as the
  • Silibinin does not affect the stability of HIF-1 ⁇ or its degradation mediated by prolyl hydroxylases.
  • HIF-1 ⁇ is mainly degraded by the ubiquitin / proteasome system after Ia hydroxylation of prolines 402 and 564 by the specific prolyl hydroxylases of HIF-1 ⁇ .
  • various experimental strategies were employed. First, reoxygenation experiments were performed. The cells were exposed to hypoxia, and the silibinin or vehicle was added during the last 15 minutes of hypoxic incubation.
  • the MG132 proteasome inhibitor was used to prevent ubiquitin-dependent degradation of HIF-1 ⁇ .
  • the treatment of cells with MG132 resulted in a pronounced accumulation of high molecular weight protein species of HIF-1 ⁇ - ubiquitinated, in normoxia and in hypoxia (the upper panel of Fig. 2.a, lanes 2 and 6).
  • the inhibition of protein synthesis with cyclohexamide totally prevented the accumulation of ubiquitinated HIF-1 ⁇ in the presence of MG132 (lanes 3 and 7).
  • EXAMPLE 4 The inhibition of the translation of the HIF-1 ⁇ protein by silibinin implies the repression of mTOR and its effectors p70S6K, rpS6 and 4E-BP1.
  • the PI3K / Akt / mTOR pathway has been implicated in the regulation of the expression of the HIF-1 ⁇ protein, predominantly at the level of its translation.
  • hypoxia causes dephosphorylation and repression of mTOR, resulting in a decrease in the synthesis rate of HIF-1 ⁇ .
  • silibinin inhibits the synthesis of HIF-1 ⁇ protein in hypoxia through the regulation of the mTOR / p70S6K / 4E-BP1 pathway
  • the treatment of HeLa and Hep3B cells with silibinin under hypoxic conditions produced a dose-dependent dephosphorylation of mTOR in Ser 2448 , which was correlated with the inhibition of phosphorylation of p70S6K, rpS6 and 4E-BP1, and with the reduction of the accumulation of HIF-1 ⁇ in both types of tumor cell lines (Fig. 3A).
  • the level of p-Akt in HeLa cells was increased after exposure to silibinin for 20 minutes (500 ⁇ mol / L), while decreasing below baseline levels in Hep3B cells.
  • the inhibition of the accumulation of HIF-1 ⁇ was reversed rapidly after two changes of the medium to eliminate silibinin from the cells. This reversal was evident after 30 minutes and was complete after 60 minutes under the same hypoxic atmosphere (Fig. 4, lanes 7 and 8).
  • all the phospho-proteins analyzed returned to their basic phosphorylation levels.
  • Silibinin inhibits hypoxia-induced secretion of VEGF in tumor cells. Silibinin has demonstrated anti-angiogenic characteristics in several experimental models. Since HIF-1 is the main regulator of VEGF in hypoxia, the effect of silibinin on the production of VEGF in vitro was analyzed. Compared to normoxia, exposure of HeLa or Hep3B cells to hypoxia for 12 h produced a 2-3-fold increase in VEGF secretion to the extracellular medium.
  • PI3K / Akt LY294002 inhibitor 10 ⁇ mol / L
  • the PI3K / Akt LY294002 inhibitor produced an inhibition of the release of VEGF (-30%) similar to that induced by a submaximal dose of silibinin (250 ⁇ mol / L)
  • the mTOR inhibitor Rapamycin (20 nmol / L) produced a weak inhibition of -15% (Fig. 5B).
  • the combined treatment with these doses of silibinin and LY294002 resulted in a synergistic effect, reducing the release of VEGF near the control levels in normoxia.

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Abstract

La présente invention concerne l'utilisation d'une composition qui comprend de la silibinine dans une proportion nécessaire pour atteindre des concentrations en sérum supérieures ou égales à 250 μM, ou dans une variante, de la silibinine et un inhibiteur de Akt, pour l'élaboration d'un médicament destiné au traitement du cancer, et une préparation combinée au moins de silibinine et d'un inhibiteur de la voie PI3K/Akt, pour son utilisation séparée, simultanée ou séquentielle dans le traitement du cancer.
PCT/ES2009/070415 2008-10-02 2009-10-02 Composition comprenant de la silibinine à des concentrations déterminées et préparation combinée comprenant de la silibinine et un inhibiteur de la voie pi3k/akt pour le traitement du cancer WO2010037892A1 (fr)

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GARCIA-MACEIRA P. ET AL.: "Silibinin inhibits hypoxia-inducible factor-1 alpha and mTOR/p70S6K/ 4E-BP signalling pathway in human cervical and hepatoma cancer cells: implications for anticancer therapy", ONCOGENE, vol. 28, no. 3, 22 January 2009 (2009-01-22), pages 313 - 324 *
MOMENY M. ET AL.: "Effects of silibinin on cell growth and invasive properties of a human hepatocellular carcinoma cell line, HepG-2, through inhibition of extracellular signal- regulated kinase 1/2 phosphorylation", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 591, no. 1-3, 4 September 2008 (2008-09-04), pages 13 - 20 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102942551A (zh) * 2012-12-04 2013-02-27 贵阳医学院 含有3-硝基丙酰基的黄烷醇和含其的药物组合物及其在制药中的应用

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