WO2010037892A1 - Composition comprising silibinin at determined concentrations and combined preparation comprising silibinin and a pi3k/akt pathway inhibitor for the treatment of cancer - Google Patents

Abstract

The present invention relates to the use of a composition comprising silibinin in the required proportion to attain concentrations in serum equal to or exceeding 250 μM, or alternatively silibilin and an Akt inhibitor, for the preparation of a medicament for the treatment of cancer, and a combined preparation of, at least, silibinin and an inhibitor of the PI3K/Akt pathway, for the separate, simultaneous or sequential use thereof in the treatment of cancer.

Description

 COMPOSITION THAT INCLUDES SILIBININ TO CERTAIN

CONCENTRATIONS AND COMBINED PREPARATION THAT

UNDERSTAND SILIBININ AND AN INHIBITOR OF THE PI3K / AKT ROAD FOR

THE TREATMENT OF CANCER.

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.

STATE OF THE PREVIOUS TECHNIQUE

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.

The main transcriptional regulator of the response to hypoxia is HIF-1 {hypoxia-inducible factor 1), which plays a central role in tumor growth and in angiogenesis. HIF-1 is a heterodimeric transcriptional factor formed by the HIF-1α and HIF-1β subunits. HIF-1β is constitutively expressed and its levels are not affected by changes in

The pÜ2. On the contrary, HIF-1α is rapidly and continuously degraded by the ubiquitin-proteasome system after its binding to von Hippel's protein.

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. Under hypoxic conditions, the hydroxylation of HIF-1α is inhibited, which leads to an increase in the stability of HIF-1α. Although the inhibition of prolyl hydroxylases in hypoxia is recognized as the primary mechanism of the accumulation of HIF-1α, it is evident that the expression of HIF-1α also depends on its de novo synthesis rate. Some growth factors, cytokines, and other signaling molecules can stimulate the synthesis of HIF-1α 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 (oligopyrímidine 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-1α regardless of the conditions of cellular oxygenation.

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. Several mechanisms of action of silibinin have been proposed, such as the inhibition of DNA synthesis, of cell growth by arrest in the G0 / G1 or G2 phase, or by a moderate increase in Ia expression of IGFBP-3 (insulin-like growth factorbinding protein-3), which may have an inhibitory effect on the mitogenic action of IGF-1. Recent studies seem to show that silibinin inhibits angiogenesis through the downward regulation of the Akt and NF-κB pathways (Mallikarjuna et al., 2004. Cancer Res. 64: 6349-6356). However, the molecular mechanism by which silibinin exerts its antitumor effects is not known exactly.

Recent data have shown that the 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).

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.

Notably, in addition to its role in promoting protein translation, it has been found that p70S6K represses the PI3K / Akt pathway by inhibiting the expression IRS1 (insulin receptor substrate-1) and IRS2 (Manning 2004. J CeII

Biol 167: 399-403). The mTORC2 complex contains mTOR, rictor

(rapamycin insensitive companion of mTOR), ml_ST8, and mSini (Guertin &

Sabatini 2007. CeI1 Cancer 12: 9-22), and recent studies have shown that mTORC2 induces Akt activity by direct phosphorylation of Ser ⁴⁷³ (Sarbassov et al., 2005. Science 307: 1098-10101).

Thus, the activation of the mTORCI pathway would suppress the signaling of

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).

Based on this mechanism, 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).

DESCRIPTION OF THE INVENTION

The doses used of silibinin, both to treat prostate cancer and other types of cancer, are between 150 mg and 450 mg per day (US 2002193424). Currently, 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 μM 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. Thus, for the treatment of a 70 kg patient, one vial (350 mg of silibinin) would be needed for each infusion. For this, 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. Administer the infusion for 2 hours. If we consider that an average individual weighing 70 kg has about 5 liters of blood, of which 3 liters would be serum, we have that the concentration of serum silibinin would be approximately 240 μM with a vial of 350 mg of silibilin.

In the examples of the present invention it is collected how, while the activation of Akt in the HeLa cell line increases with the dose of silibinin administered, in certain cell lines (AGS and Hep3B) silibinin concentrations greater than 250 μM can inhibit the activation of the Akt pathway, and in other cell lines (PC-3) the activation of Akt never occurs. In addition, it is seen as in all cell lines tested, the inhibition of VEGF production is greater at silibinin concentrations greater than 250 μM and even greater than 500 μM. And even more, when the inhibition of cell proliferation has been tested, it has been seen that in all cases the proliferation is inhibited from low concentrations, but in the case of cervical and gastric cancer cell lines, the proliferation inhibition is notably higher at concentrations greater than 250 μM.

Therefore, 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 μM, and even more preferably, of 500 μM, in serum.

Thus, 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 μM, 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 μM, for use in the treatment of cancer. In a preferred embodiment of this aspect of the invention, silibinin concentrations are kept substantially constant, the Cmin (minimum concentration) being in serum of at least 250 μM.

Silibinin (INN International Nonpropήetary Ñame), 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. By silibinin, herein, it is understood both the silibinin itself and any of its derivatives, salts, prodrugs, or the like, or any combination thereof.

Formula (I)

As used herein, the term "active substance", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" 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.

Pharmaceutically acceptable salts and derivatives of silibinin are known in the state of the art, and are described in numerous documents, such as, but not limited to, those included in US 6,699,900. As used herein, the term

"derivative" includes both pharmaceutically acceptable compounds, and pharmaceutically unacceptable derivatives, since these may be useful in

The preparation of pharmaceutically acceptable derivatives.

Also, within the scope of this invention are the prodrugs of silibinin. The term "prodrug" as used herein includes 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. Advantageously, 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.

The expression "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:

Fl = (Cmax - Cmin) / AUCτ / τ

in which 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), and τ is the length of the dose interval throughout the period τ. As inferred from the examples of the present invention, for the treatment of cancer, the Cmin would be 250 μM, and even more preferably, 500 μM. 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. , where silibinin is in the proportion necessary to reach serum concentrations (cellular) equal to or greater than 500 μM, or alternatively, to 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 μM, for use in the treatment of cancer. In a preferred embodiment of this aspect of the invention, silibinin concentrations are kept substantially constant, the serum Cmin being at least 500 µM.

In another preferred embodiment, silibinin is found as silibinin-C-2 ', 3-disodium dihydrogen succinate. In another preferred embodiment, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier. In another preferred embodiment of this aspect of the invention, the cancer is selected from the list comprising: cervical cancer, gastrointestinal cancer and liver cancer.

The proportion of 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. Thus, since silibilin is not really soluble in aqueous or lipophilic phases (Merck Index, Reference 8680), we can consider that the in vitro concentration would be equivalent to the plasma concentration, and if we take as a basis the current reference administration schedule, The necessary concentration of silibinin (of silibinin-C-2 ', 3-dihydrogen disodium succinate) to reach serum concentrations of 250 μM, is approximately 10.42 mg / ml (in 35 ml of solution for injection), so that The pharmaceutical composition must have silibilin values greater than 365 mg. Preferably the medicament comprises the necessary concentration of silibinin to reach serum concentrations equal to or greater than 500 μM, 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 μM, and even more preferably, concentrations greater than 500 μM 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.

Preferably, 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. In each infusion, therefore, more than 10 mg of silibinin per kg of body weight (more than 700 mg for a person weighing 70 kg) will be administered, thus reaching serum concentrations of approximately 500 μM. 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 μM, and even more preferably, substantially constant and equal to or greater than 500 μM.

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), In a preferred embodiment, 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).

On the other hand, 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.

The authors of the present invention have shown that silibinin inhibits the accumulation of hypoxia-induced HIF-1α in several cell lines.

Likewise, the authors describe that 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.

Thus, pharmaceutical 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.

Thus, 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 Ñame), also known as silibinin, is the main active constituent of silymarin, the mixture of flavolignans extracted from Silybum maríanum. 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. By silibinin, herein, it is understood both the silibinin itself and any of its derivatives, salts, prodrugs, or the like, or any combination thereof.

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. Between the PI3K 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).

Therefore, in a preferred embodiment of this aspect of the invention, 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 In a particular embodiment of this aspect of the invention, the PI3K / Akt pathway inhibitor is LY294002.

In the composition of the invention 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. As demonstrated in the examples, the combined use of silibinin with the PI3K / Akt inhibitor employed (LY294002) gives rise to a synergistic effect on certain tumor cell lines.

Thus, another aspect of the invention relates to the use of the composition of the invention for the preparation of a medicament for the treatment of cancer.

In a preferred embodiment of this aspect of the invention, the types of cancer are selected from the list comprising: cervical cancer, cancer gastrointestinal or liver cancer. In a particular embodiment of this aspect of the invention, 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.

The authors of the present invention have shown that 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.

Therefore, 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. It should be emphasized that 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. Thus, the expression "juxtaposed" implies that it is not necessarily a true combination, in view of the physical separation of the components.

A preferred embodiment of this aspect of the invention, 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. In a particular embodiment, the cancer is cervical cancer.

In this report "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. The term 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. 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.

It is generally said that 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.

It is said that 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. Here the malignant tumors fall within the definition of cancer within the scope of this definition as well. In general, 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, such as, for example, cancer of the tongue, lips, larynx, urinary bladder, uterine cervix, or penis, 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 ourselves, they can be:

I) cancer and tumor diseases of the bone marrow, and of cells derived from the bone marrow (leukemia),

II) endocrine and exocrine glands, such as thyroid, parathyroid, pituitary, adrenal glands, salivary glands, pancreas.

III) 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, ...

IV) The lung,

V) The stomach,

VI) The liver and spleen,

VII) The small intestine, VIII) The COlOn,

IX) 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,

X) the mouth, neck, larynx, and esophagus,

XI) The urinary bladder and the internal and external organs and structures of the male and female urogenital system, such as ovary, uterus, cervix, testicles and prosthetic gland, XII) The prostate,

XIII) The pancreas, such as ductal carcinoma of the pancreas,

XIV) Lymphatic tissue such as lymphomas and other tumors of lymphoid origin, XV) The skin,

XVI) Cancer and tumor diseases of all anatomical structures belonging to the respiratory system, including the thoracic muscles and the pleura,

XVII) primary and secondary cancer of the lymph nodes, XVIII) The tongue and bone structures of the palate and sinuses,

XIX) The mouth, cheekbones, neck and salivary glands,

XX) Blood vessels including the heart and its membranes,

XXI) The smooth or skeletal muscle, including its ligaments and membranes, XXII) The peripheral, autonomous and central nervous system, including the cerebellum,

XXIII) Adipose tissue.

Both the 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. Thus, 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. Alternatively, the compositions can be prepared for administration in solid form. The 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.

Such combined 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. In the sense used in this description, 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.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following Examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Silibinin inhibits the accumulation of hypoxia-induced HIF-1α 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 μmol / L). Protein levels of HIF-1α, HIF-1β 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). Proteins were detected by immunoblot. C, HeLa cells were transiently transfected with the reporter plasmid p9HIF1-Luc and then incubated for 8 h in a manner similar to that described in B. The transcriptional activity of HIF-1 was assayed by measuring the response to hypoxia dependent on the expression of the reporter gene as described in materials and methods. The experiments were performed in duplicate and the results shown are representative of three independent analyzes. The bars represent the mean ± SE. ^* P <0.05, ^** P <0.01, ^*** P <0.001, compared to their respective controls without silibinin. D, 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 μmol / L). The levels of the HIF-1α and actin proteins were detected by immunoblot as in A.

Fig. 2. Silibinin does not affect the degradation of the HIF-1α protein or the expression of the HIF-1α mRNA. A, HeLa cells were exposed to hypoxia (2% O2) for 2 h, and silibinin (500 μmol / 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-1α protein were measured by immunoblot. The lower panel shows the densitometric quantification of the levels of HIF-1α, with the values expressed as the percentage of the expression before reoxygenation (time 0). The values represent the mean ± SE of four independent experiments. B, CH13 KaO cells deficient in HIF-1α were transfected with the wild type HIF-1α, the mutant P402A / P564A-HIF-1α 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-1α 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 μmol / L) for the indicated times. Total RNA was isolated and analyzed for the expression of the HIF-1α mRNA by RT-PCR, using GAPDH as the control gene. 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 μmol / L), cyclohexamide (CHX, 100 μmol / L) or silibinin (500 μmol / 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 μmol / L) or silibinin (500 μmol / L), and HIF-1α 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 μmol / L), LY294002 (LY, 10 μmol / L), or rapamycin ( Rapa, 20 nmol / L) as indicated. C, 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 μmol / 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-1α are rapid and completely reversible. HeLa and Hep3B cells were exposed to hypoxia (2% O ₂ ) for 3 h, and silibinin (SiIi, 500 μmol / 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. B, VEGF was determined in HeLa cells treated for 12 h with the drugs and indicated oxygen conditions; Hypoxia (2% O2), Silibinin250 (250 μmol / L), SilibininδOO (500 μmol / L), LY294002 (LY, 10 μmol / L), rapamycin (Rapa, 20 nmol / L). The graph is representative of three experiments with similar results. The bars represent the mean ± SE. ^* , P <0.05, ^** , P <0.01, ^*** , P <0.001, significantly different compared to control cells in hypoxia without silibinin. #, P <0.01, compared to Silibinin25o- treatment

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,

250 or 500 μmol / L) for the indicated times. 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-1α 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 μM), and silibilin (SiIi, 500 μM) or a vehicle was added for the last 0 (untreated), 10, 20, 30 or 60 minutes of hypoxic incubation. HIF-1α 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. (a) The cells were cultured in normoxia (21% O2) for 3 hours in the presence of the indicated concentrations of silibinin (sili). (b) The cells were cultured in normoxia for 3 hours, and silibinin (SiIi, 500 μM) 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. (a) 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, (b) Transfected cells as described in (a), and treated for 3 hours in hypoxia (2% 02) with the indicated concentrations of silibilin or of the vehicle.

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 μM) 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.

EXAMPLES

Materials and methods.

Cell cultures and reagents. Human cervical adenocarcinoma (HeLa) and hepatocellular carcinoma (Hep3B) cells were obtained from the American Type Culture Collection (ATCC, Barcelona, Spain) and cultured in DMEM or MEM (Sigma, St. Louis, MO), respectively. CHO Ka13.5 cells deficient in HIF-1α 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. For exposure to hypoxia the cells were incubated at 37 ⁰ C in a humidified hypoxia chamber (COY Labs., Grasslake, Ml), insufflated with a mixture of 5% Cθ2 / 95% N2 (Air-Liquide, Madrid, Spain) and equipped with an O2 regulator to obtain the desired O2 concentration (21-0.1%). 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-1α and HIF-1β 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.

Cellular extracts and immunoblotting. 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 µl of lysis buffer composed of a CytoBuster ™ 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. The protein concentration was determined by the BCA protein assay (Pierce). Cell extracts (40-80 μg 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).

Plasmid construction. The human HIF-1α cDNA (GenBank accession U22431; SEQ ID NO: 1) was obtained from the ATCC (pCEP4 / HIF-1α). We subcloned the entire coding region of HIF-1α into the pcDNA4 / HisMax (Invitrogen) expression vector by PCR using Pfu DNA polymerase (Stratagene). The primers were designed to contain the restriction sites for Bam \ λ \ 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 \ λ \ and Not \, and ligated into pcDNA4 / HisMax previously digested with the same restriction enzymes. The resulting construct, pcDNA4 / HisMax-HIF-1α, was amplified and purified (Qiagen). The double mutant HIF-1α P402A / P564A was generated by sequentially directed mutagenesis (QuickChange II, Stratagene) using as a pattern pcDNA4 / HisMax-HIF-1α. 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-1α is not susceptible to hydroxylation by prolyl-4-hydroxylases and subsequent degradation. The uncertainty of the consírucciones was confirmed by DNA sequencing. The reporter plasmid p9HIF1-Luc, containing nine copies of the hypogene response (HRE) elemenium of the human promoter gene VEGF, was donated by Dr. Manuel O. Landázuri (Immunology Service, Hospital de la Princesa, Universidad Auíonoma de Madrid , Spain).

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 Lipofecíamine2000 (Invitrogen) and 1 μg of the DNA plasmid (wild type HIF-1α, P402A / P564A-HIF-1α, or the empty vector). For the luciferase reporter assay, the cells were cultured in 24-well plates and transfected with 0.3 μg per well of the reporter plasmid p9HIF1-Luc using the transfection agent FuGENE 6 (Roche) according to the manufacturer's instructions. After 24 hours, the medium was replaced and the cells were incubated for 6-8 hours with the drug and the indicated O2 concentrations. The transfection efficiency was monitored by cotransfection with 0.1 μg 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 μl). The results were normalized with respect to the amount of total protein per well.

RT-PCR Total RNA was isolated from tumor cells using the RNeasy Mini kit (Qiagen) and 0.5 μg were transcribed reverse with 50 U SuperScriptTM Il reverse transcriptase (Invitrogen), using a first oligo- (dT) according to the manufacturer's protocol. The cDNA was amplified by PCR using the following sets of primers: for human HIF-1α: SEQ ID NO: 8 and SEQ ID NO: 9 (GenBank NM_001530); Ia human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SEQ ID NO: 10 and SEQ ID NO: 11 (GenBank NM_002046). 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. Cell proliferation was 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. To measure apoptosis we use the Caspase-Glo® 3/7 Assay luminescent assay (Promega), which measures the activity of caspases-3 and -7. Both tests were carried out in 96-well plates according to the manufacturer's recommendations.

Statistic analysis. Where indicated, the experimental data was analyzed using Prism ™ GraphPad software (version 4.0). The significant differences were determined by the Student's t-test (with two tails) and the P values below 0.05 were considered significant.

Next, the invention will be illustrated by tests carried out by the inventors, which shows the specificity and effectiveness of the combined use of silibinin and inhibitors of the PI3K / Akt pathway for the treatment of cancer, especially those types of cancer in those that silibinin indirectly activates the PI3K / Akt pathway.

EXAMPLE 1. Silibinin inhibits the accumulation of hypoxia-induced HIF-1α in tumor cells HeLa and Hep3B. To examine the effect of silibinin on the expression of the HIF-1α and HIF-1β proteins, immunoblot experiments were first performed as a function of time. Hypoxia induced a time-dependent accumulation of HIF-1α protein in HeLa and Hep3B tumor cells that was noticeable after 1 h. Silibinin inhibited the accumulation of HIF-1α (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). In contrast, silibinin did not alter the levels of HIF-1β protein (Fig. 1A).

The dose-response experiments showed that the decrease in the expression of the HIF-1α protein produced by silibinin was dose-dependent (Fig. 1 B), demonstrating a comparable potency in HeLa and Hep3B cells (IC50 -150 μmol / L) . Similarly, silibinin fully advocated the accumulation of HIF-1α induced by the DMOG inhibitor (Fig. 1 B), a well-characterized mimetic hypoxic agent. Consistent with the inhibition of the accumulation of HIF-1α, 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).

To investigate whether the inhibitory effect of silibinin on the accumulation of HIF-1α was dependent on the severity of cellular hypoxia, we subjected the HeLa and Hep3B cells to increasing hypoxia in the presence or absence of silibinin. HIF-1α began to stabilize at O2 concentrations of 6%, with accumulation increasing markedly as the

O2 decreases until severe hypoxia is reached (0.1% 02). Silibinin prevented the accumulation of HIF-1α in all the tested O ₂ concentrations, demonstrating its ability to inhibit HIF-1α in any degree of cellular hypoxia (Fig. 1 D).

EXAMPLE 2. Silibinin does not affect the stability of HIF-1α or its degradation mediated by prolyl hydroxylases. HIF-1α is mainly degraded by the ubiquitin / proteasome system after Ia hydroxylation of prolines 402 and 564 by the specific prolyl hydroxylases of HIF-1α. To test the possibility that silibinin inhibits the accumulation of HIF-1α by promoting its degradation and / or reducing the half-life of the protein, 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. At the end of the hypoxic incubation (time 0) the cells were exposed to normoxia, and incubated for increasing periods until reaching 30 min. Under these conditions, the levels of HIF-1α would mainly reflect the degradation rate of HIF-1α. Although the accumulation of HIF-1α was slightly lower in silibinin-treated cells, the degradation rates of HIF-1α in untreated and treated cells were very similar, with protein half-life values of 13.5 ± 1.2 and 10.5 ± 1.0 min, respectively (Fig. 2A).

In a second experimental approach, the effect of silibinin on the stability of the double proline mutant P402A / P564A of HIF-1α (P402A / P564A-HIF-1α) was tested, which cannot be hydroxylated by prolyl hydroxylases and accumulates in cells independently of O2. To avoid the interference of endogenous HIF-1α, the P402A / P564A-HIF-1α or wild HIF-1α constructs were expressed in HIF-1α CHO Ka13.5 deficient cells. Silibinin was equally potent in the prevention of the accumulation of wild HIF-1α and the mutant P402A / P564A-HIF-1α (Fig. 2B), therefore eliminating the possibility that the prolyl hydroxylase of HIF-1 is involved in the effect of silibinin. Similar results were obtained in HeLa cells. Junios, these results demonstrate that silibinin does not affect the degradation of the HIF-1α protein. EXAMPLE 3. Silibinin does not affect the accumulation of the HIF-1α mRNA but decreases the translation of the HIF-1α protein. The lack of effect on the degradation of HIF-1α indicated the possibility that silibinin can reduce the production rate of HIF-1α. The RT-PCR analysis revealed that HIF-1α mRNA levels were still unaffected by treatment with silibinin in normoxia and hypoxia (2 or 4h) (Fig. 2C), thus indicating that silibinin does not affect Ia accumulation of the HIF-1α mRNA.

To examine whether the inhibition of the accumulation of HIF-1α mediated by silibinin was due to the reduction of the synthesis of the HIF-1α protein, the MG132 proteasome inhibitor was used to prevent ubiquitin-dependent degradation of HIF-1α. The treatment of cells with MG132 resulted in a pronounced accumulation of high molecular weight protein species of HIF-1α - ubiquitinated, in normoxia and in hypoxia (the upper panel of Fig. 2.a, lanes 2 and 6). On the contrary, the inhibition of protein synthesis with cyclohexamide totally prevented the accumulation of ubiquitinated HIF-1α in the presence of MG132 (lanes 3 and 7). Similarly, the addition of silibinin in the presence of MG132 resulted in a substantial reduction of ubiquitinated HIF-1α, in normoxia and hypoxia (lanes 4 and 8), suggesting interference with the protein synthesis machinery. In addition, silibinin mimicked the inhibitory effect of cyclohexamide on the accumulation of HIF-1α in cells that were incubated with these drugs under hypoxia for increasing periods (the lower panel of Fig. 2.a,). These results indicate that silibinin decreases the translation of HIF-1α.

EXAMPLE 4. The inhibition of the translation of the HIF-1α 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-1α protein, predominantly at the level of its translation. On the other hand, recent evidence indicates that hypoxia causes dephosphorylation and repression of mTOR, resulting in a decrease in the synthesis rate of HIF-1α. To see if silibinin inhibits the synthesis of HIF-1α protein in hypoxia through the regulation of the mTOR / p70S6K / 4E-BP1 pathway, we measure the phosphorylation status of mTOR and its effectors p70S6K, rpS6 and 4E-BP1 , as well as that of Akt. 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-1α in both types of tumor cell lines (Fig. 3A).

It was also found that concomitantly with the inhibition of the expression of HIF-1α and of the mTOR pathway in HeLa and Hep3B cells, the silibinin produced the activation of Akt, as indicated by the increase in the phosphorylation of Akt in Ser ⁴⁷³ (Fig. 3A). It was observed that the mTOR inhibitor rapamycin induced a pronounced increase in p-Akt similar to that observed with silibinin, under normoxic and hypoxic conditions (Fig. 3B). Notably, we observed a differentiated response between HeLa and Hep3B cells with respect to the effect of silibinin on Akt. While in HeLa cells the phosphorylation of Akt progressively increased with the increase in the concentration of silibinin, in Hep3B cells the induction of Akt was reversed at the highest dose (500 μmol / L) and the phosphorylation of Akt returned to baseline levels. (Fig. 3A).

This difference at high doses of silibinin had no implication for the inhibition of HIF-1α, which continued to be maximal in both cell varieties. The activation of p-Akt by silibinin required the activity of its upstream regulator PI3K, since the inhibitor LY294002 of PI3K blocked the phosphorylation of Akt induced by silibinin (Fig. 3B, the right panel, and Fig. 3C). On the other hand, it was found that LY294002 accentuated the inhibition of the expression of HIF-1α mediated by silibinin, as well as the repression of the mTOR pathway (reflected by the loss of p-p70S6K) (Fig. 3C). To better investigate the effect of silibinin on mTOR and the signaling of Akt in Io regarding the inhibition of HIF-1α expression, HeLa and Hep3B cells were exposed to hypoxia and subsequently treated with silibinin for the last 10 to 60 minutes of hypoxic incubation. Silibinin induced a rapid and potent inhibition of the accumulation of HIF-1α that reached 70-80% in Hep3B cells at 10 min after treatment and was complete after 60 min (Fig. 4). In HeLa cells, 70-80% inhibition was reached after 20 minutes of treatment and was also complete by 60 minutes. In both types of cells, the dephosphorylation of mTOR induced by silibinin and its effectors were detected within the term of 10-20 minutes of the treatment under hypoxic conditions, and resulted in the total loss of p-p70S6K after 20-30 minutes (Fig. 4). Similarly, there was a parallel reduction of p-rpS6 that was almost complete after 1 h of treatment. It is significant that the decrease in p-4E-BP1 was faster and more pronounced in Hep3B cells than in HeLa, probably due to the presence of higher basal levels of p-4E-BP1 in HeLa cells.

Consistent with the dose-response experiments, the level of p-Akt in HeLa cells was increased after exposure to silibinin for 20 minutes (500 μmol / L), while decreasing below baseline levels in Hep3B cells. Notably, the inhibition of the accumulation of HIF-1α 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). Likewise, after the elimination of silibinin, all the phospho-proteins analyzed returned to their basic phosphorylation levels. These results suggest that Ia Reduction of the mTOR / p70S6K / 4E-BP1 pathway is involved in the inhibition of the translation of the HIF-1α protein by silibinin.

EXAMPLE 5. 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. Silibinin dose-dependently inhibited the release of hypoxia-induced VEGF in both cell lines: 250 μmol / L of silibinin produced an inhibition of -25% in HeLa cells and an inhibition of -40% in Hep3B cells, while with 500 μmol / L Ia inhibition reached more than 90% in both cell types (Fig. 5A). No significant effects were observed at lower concentrations (50 and 100 μmol / L).

The effect of silibinin on the release of hypoxia-induced VEGF in comparison and in combination with PI3K / Akt or mTOR inhibitors was also examined. The PI3K / Akt LY294002 inhibitor (10 μmol / L) produced an inhibition of the release of VEGF (-30%) similar to that induced by a submaximal dose of silibinin (250 μmol / L), while the mTOR inhibitor Rapamycin (20 nmol / L) produced a weak inhibition of -15% (Fig. 5B). Interestingly, 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. In contrast, the combined treatment of silibinin and rapamycin did not produce any additive effect. At the maximum dose of silibinin (500 μmol / L), the inhibition of hypoxia-induced release of VEGF was greater than 90% regardless of the presence of additional inhibitors. EXAMPLE 6. Silibinin inhibits the proliferation of HeLa and Hep3B cells. The examination of the effect of silibinin on the proliferation of HeLa and Hep3B tumor cells revealed a dose-dependent inhibition in both cell lines that was evident after treatment for 2 h and increased with the incubation time (Fig. 6A). Compared to controls, treatment of HeLa cells with silibinin for 2 to 8 hours under hypoxic conditions resulted in reductions in the total number of cells that ranged from 35% (2 hours) to 45% (8 hours) with 250 μmol / L, and 60-75% during the same period with 500 μmol / L. There were no significant differences with 50 μmol / L, and only a weak inhibition of -10% with 100 μmol / L after 8 h. After a similar treatment of Hep3B cells growth was inhibited in 5-15% with 50 μmol / L, 15-25% with 100 μmol / L, 20-45% with 250 μmol / L, and 40-72% with 500 μmol / L. In normoxia, silibinin produced an identical inhibitory effect on cell growth (data not shown).

Claims

1. Use of a pharmaceutical composition comprising as active ingredient silibinin, a pharmaceutically acceptable salt, or a prodrug, derivative or analog thereof, for the preparation of a medicament for the treatment of cancer, where silibinin is in the necessary proportion to reach serum concentrations equal to or greater than 250 μM.

2. Use of a pharmaceutical composition according to the preceding claim, wherein silibinin is in the proportion necessary to reach serum concentrations equal to or greater than 500 μM.

3. Use of a pharmaceutical composition according to any of claims 1-2, wherein silibinin is found as silibinin-C-

2 ', 3-dihydrogen disodium succinate.

4. Use of a pharmaceutical composition according to any of claims 1-3, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

5. Use of a pharmaceutical composition according to any of claims 1-4, wherein the cancer is selected from the list comprising cervical cancer, gastrointestinal cancer and liver cancer.

6. A vial for infusion with lyophilized product comprising silibinin-C-2 ', 3-dihydrogen succinate disodium in values greater than about 550 mg.

7. An infusion vial according to the preceding claim, comprising silibinin-C-2 ', 3-dihydrogen succinate disodium in values greater than 1100 mg.

8. Composition comprising silibinin and an inhibitor of the PI3K / Akt pathway.

9. Composition according to the preceding claim for use as a medicine.

10. Composition according to any of claims 8-9, wherein the inhibitor of the PI3K / Akt pathway is selected from the list comprising: LY294002, Wortmannin, Triciribine (API-2, NSC-154020, TCN, Akt inhibitor V), A-443654, KP372-1, Akt lnhibitor Il (SH-5), Akt Inhibitor III (SH-6), Akt Inhibitor IV, Akt Inhibitor VIII (Isozyme-Selective, Akti-1/2), Akt Inhibitor X.

11. Composition according to any of claims 8-10. where the inhibitor of the PI3K / Akt pathway is LY294002.

12. Use of the composition according to any of claims 8-11, for the preparation of a medicament for the treatment of cancer.

13. Use of the composition according to the preceding claim wherein the cancer is selected from the list comprising: cervical cancer, gastrointestinal cancer or liver cancer.

14. Use of the composition according to claim 13 wherein the cancer is cervical cancer.

15. Combined preparation of at least silibinin and an inhibitor of the PI3K / Akt pathway for use separately, simultaneously or sequentially in the treatment of cancer.

16. Use of a combined preparation of at least silibinin and an inhibitor of the PI3K / Akt route separately, simultaneously or sequentially in the preparation of a medicament for the treatment of cancer.

17. Use of a preparation according to any of claims 15-16, wherein the cancer is selected from the list comprising: cervical cancer, gastrointestinal cancer or liver cancer.

18. Use of a preparation according to claim 17, wherein the cancer is cervical cancer.