EP3429568A1

EP3429568A1 - Pharmaceutical composition for use in the therapeutic treatment of cancer and complications of cancer

Info

Publication number: EP3429568A1
Application number: EP17808579.1A
Authority: EP
Inventors: Guy Faustin MONKAM NITCHEU
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-10-31
Filing date: 2017-10-25
Publication date: 2019-01-23
Also published as: CN109562080A; CA3043456A1; IL266623A; KR20190077449A; FR3058059A1; MX2019003685A; FR3058059B1; FR3058058A1; CN109562080B; MA44413A; WO2018078539A1; ZA201902910B

Abstract

The invention relates to a pharmaceutical composition, characterised in that it comprises, as an active substance, a combination of beta-elemene, lupeol and a pharmaceutically active agent selected from cinnamaldehyde, 2-hydroxycinnamaldehyde, 2'- benzoyloxycinnamaldehyde, beta-sitosterol, curcumin and the mixtures thereof.

Description

Pharmaceutical composition used for the therapeutic treatment of cancer and its complications.

[Technical field of the invention]

The present invention relates to a pharmaceutical composition which can be used as a medicament, in particular for the therapeutic treatment of cancers and in particular hepatocellular carcinoma.

[Prior Art]

Effective palliative treatment for hepatocellular carcinoma (HCC) has long been considered difficult because of the usual resistance of this type of tumor to conventional cytotoxic chemotherapy. In addition, aggressive chemotherapy with several non-selective cytotoxic molecules can often not be offered to cirrhotic patients with compromised liver function due to the high risk of systemic side effects.

Improved knowledge of the molecular processes involved in oncogenesis has led to the identification of new targets for the treatment of cancers. Thus, new so-called "targeted" therapies are currently available in the treatment of HCC and other tumors. They intervene mainly in the transduction of the signals (signals which ask the cell to multiply). The so-called tyrosine kinase pathway is the best known to date. This pathway can be blocked by monoclonal antibodies (Mabs) or enzymatic inhibitors (inib). These drugs include VEGF inhibitors (vascular endothelial growth factor, VEGF is secreted by most tumor cells, partly due to lack of local oxygenation and promotes angiogenesis), inhibitors EGFR Receptor (Epidermal Growth Factor Receptor), Inhibitors of Several Tyrosine Kinase-Activating Receptors That Parallelize Cellular Angiogenesis and Proliferation, Kinase Inhibitors, IGF-IR Receptor Inhibitors, Inhibitors of mTOR and inhibitors of the MEK-ERK signaling pathway.

However, these targeted therapies for the most part give relatively modest results in terms of efficiency and survival in HCC and especially in advanced HCC. On the other hand, the effectiveness of targeted therapies in HCC is still limited by resistance phenomena. Some cancer cells possess or acquire the possibility of circumventing the action mechanisms of drugs, while others are sensitive at first but develop resistance capacities during treatment. It should also be noted that with these targeted therapies, significant side effects can cause discomfort, and thus do not offer patients a better quality of life: myelosuppression (blood tests are performed regularly to check red blood cells, white blood cells and platelets), alopecia (which can be difficult to live psychologically for patients because it is a concrete and visible sign of the disease), skin disorder and hand-foot syndrome, diarrhea, increased blood pressure, proteinuria, hyperglycemia, hypercholesterolemia, allergic reactions, cytokine release syndrome, tumor lysis syndrome that can self-sustain the tumor, etc. On the other hand, several observations point to the existence of a close link between chronic inflammation and the risk of developing cancer. One of the best examples of this correlation is undoubtedly the dramatic (20-fold) increase in the risk of colon cancer in people with inflammatory bowel disease, particularly ulcerative colitis. This is also the case of cancer of the stomach, which develops following an inflammation caused by the presence of a bacterium, Helicobacter pylori. Prostate cancer is caused by an inflammation of the prostate (prostatitis). In CHC, the hypothesis is that chronic inflammation increases DNA mutations and that increased proliferation of hepatocytes in regenerated nodules increases the likelihood of attachment of oncogenic mutations. In addition, pre- and post-infusion medication combining a corticosteroid and an antihistamine is strongly recommended to reduce the risk of allergic reactions related to certain targeted therapies.

While biomarkers have been validated for breast cancer (HER copy number), lung cancer (EGFR mutations) or colon cancer (Kras mutations), there are currently no biomarkers Validated prognostic for HCC under targeted therapy.

Numerous studies have shown that reptin and / or pontine are overexpressed during carcinogenesis; their nucleo-cytoplasmic localization was variable according to the type of cancer and was not necessarily a factor of poor prognosis. In contrast, overexpression of reptin and pontine is a poor prognostic factor in HCCs and a high level of pontine mRNA correlates poor prognosis [(Haurie et al., 2009). Hepatology. 2009 Dec; 50 (6): 1871-83. doi: 10.1002 / hep.23215]. In addition, it is known that reptin is overexpressed in prostate cancer, hepatocellular carcinoma (HCC), gastric cancer, kidney cancer and breast cancer.

It is also known that pontine is overexpressed in hepatocellular carcinoma, lung cancer and colorectal cancer. The dysfunction of reptin and / or pontine has also been demonstrated in other cancers such as chronic leukemia, mesothelioma and multiple myeloma, high grade lymphoma, Burkitt's lymphoma, brain tumors such as gliomas and tumors of the bladder.

Survivin is also known as a therapeutic target in the case of cancer. The majority of solid tumors (breast cancer, prostate, lung, kidney, ... etc.) or hematopoietic tumors (multiple myeloma, leukemia, ... etc.) express it aberrantly. It is also known that cancerous breast, lung and kidney tumor cells overexpress survivin.

Moreover, it is known that beta-elemene can be used as anti-cancer. It has, moreover, a broad antineoplastic spectrum, including the tumors resistant to anticancer drugs conventionally used. It is also known to be non-cytotoxic and well tolerated by patients. It can pass the blood-brain barrier and has immunostimulatory properties. Its anti-inflammatory activity is also known. It is also known that beta-elemene inhibits survivin. It is also known to reduce or even suppress the resistance of cancer cells to anti-cancer drugs.

It is also known that beta-elemene also reduces the resistance of cancer cells to certain drugs. Thus, it significantly inhibits MDR1, MRP and GST-JL Chen et al., (2006) (Journal of Medicinal Plants Research Vol.6 (46), pp. 5720-5729, December 3, 2012) have shown that β -Elémene inevitably increases the intracellular accumulation of AMD in U251 / AMD cells (cells that have developed resistance to AMD) of human glioblastoma, reduces the IC50 of U251 / AMD cells from 0.915 to 0.051 mg / 1.

In addition, it is also known that lupeol (also known as Fagarsterol or Clerodol), is a pharmacologically active compound with anti-inflammatory, anti-cancer properties including its antiproliferative activity, regulation cell cycle, apoptosis, angiogenesis and its effect on the epithelial-mesenchymal transition. It also stimulates the immune system of cancer patients. It should be noted that lupeol at the effective therapeutic dose shows no toxicity to normal cells and tissues. In the case of CHC, lupeol is known to inhibit BD F protein (Brain-

Derived Neurotrophic F actor-F Neurotrophic Actor Derived from the Brain). Lupeol inhibits HCCLM3 cell proliferation in CHC as a function of concentration and time through Caspase-3 activation and PARP [poly (ADP-ribose) polymerase] cleavage. However, Zhang L et al., (European Journal of Pharmacology, Volume 762, September 5, 2015, Pages 55-62) also found that lupeol-induced death of these cells was associated with a marked decrease in the expression of BDNF protein and GSK-3B serum phosphorylation (Glycogen Synthase Kinase 3 Beta), with concomitant suppression of Akt1 expression, PI3K (phosphatidyl inositol 3-kinase), B-catenin, c-Myc and Cyclin D1 mRNA. Inhibition of BDNF overexpression thus results from decreased expression of Akt and PI3k proteins, as well as reactivation of GSK-3B function.

In addition, oral administration of lupeol at a dose of 50 mg / kg for 18 consecutive days produced no mortality or systemic toxicity in rats.

It is also known that cinnamaldehydes in particular, cinnamaldehyde (CA), 2-hydroxycinnamaldehyde (HCA), and 2-benzoyloxycinnamaldehyde (BCA), a semisynthetic derivative of HCA, each have an anti-inflammatory, anti-inflammatory activity. proliferative, anti-angiogenic, anti-metastatic via inhibition of TEM (epithelio-mesenchymal transition) and pro-apoptotic on many human cancer cells such as melanoma, breast cancer, lung cancer, cancer of the lung ovarian, colon cancer, prostate cancer, myeloma and leukemia.

It is also known that HCA has an effect on HCC. Thus, Moon EY., (Eun-Yi Moon et al., (2005) European Journal of Pharmacology 530 (2006) 270-275) investigated the inhibitory effect of HCA on farnesyl transferase. In a model mouse that developed hepatocellular carcinoma following a transgenic mutation H-rasl2V and under the control of a specific promoter such as albumin. It was found that, administration of HCA / BCA for 10 weeks delayed the development of liver cancer compared with to the control group. HCA / BCA significantly reduces the number and size of liver damage. HCA / BCA increases the number of splenocytes and infiltration of lymphocytes in the liver. These data suggest that delayed onset of hepatic cancer could be caused by an immunostimulatory effect of HCA / BCA on T cells. Moreover, beta-sitosterol is known to possess anti-inflammatory, antipyretic, antineoplastic and immunomodulatory properties.

[Technical problem to be solved]

An object of the present invention is to provide a novel pharmaceutical composition that can be used as a medicament and more particularly that can be used in the treatment of cancer.

Another object of the invention is to provide a new pharmaceutical composition that can be used as a medicament and more particularly that can be used in the treatment of cancer that overcomes all or some of the disadvantages related to the compositions of the aforementioned prior art. Another object of the invention is to provide a pharmaceutical composition which is particularly advantageous in the treatment of HCC.

Another object of the present invention is to provide a pharmaceutical composition that can be used as a medicament, in particular for the therapeutic treatment of HCC, of hepatocellular insufficiency and in particular of cirrhosis of the liver. Another object of the present invention is to provide a pharmaceutical composition for use in the treatment of breast cancer and / or prostate cancer.

Another object of the present invention is to provide a pharmaceutical composition, especially as mentioned above, which has reduced toxicity and / or is well tolerated by patients. Another object of the present invention is to provide a pharmaceutical composition which makes it possible to reduce or inhibit drug resistance and in particular drug resistance to anticancer agents.

Another object of the present invention is to provide a pharmaceutical composition which acts specifically on cancer cells which overexpress at least one protein selected from the group consisting of reptin, pontine and survivin and in particular which acts on cells cancerous overexpressing reptin and pontine and possibly survivin.

Another object of the present invention is to provide a pharmaceutical composition which inhibits the role of stromal cells in the overexpression by cancer cells of at least one protein selected from the group consisting of reptin, pontine and survivin. Another object of the present invention is to provide a pharmaceutical composition which makes it possible to inhibit the overexpression of CXCR4 receptors by cancer cells, to block the formation of a new vascularization, and thus to reduce the blood supply to the growing tumors.

Another object of the present invention is to provide a pharmaceutical composition which makes it possible to inhibit the formation of tumor stroma, the metastatic process, to reduce the risk of tumor recurrence.

Another object of the present invention is to provide a pharmaceutical composition which makes it possible to inhibit inflammation, alteration of the intestinal mucosa, bacterial, viral or fungal translocation of the intestinal lumen to the bloodstream, immune hyperactivation. systemic, to induce a vigorous immune response, particularly in the lymphoid tissue associated with the digestive tract.

[Brief Description of the Invention]

To solve at least one of the above-mentioned technical problems, the present invention provides a pharmaceutical composition, which typically comprises, as active ingredient, a combination of beta-elemene, lupeol and a pharmaceutical agent. The active ingredient is cinnamaldehyde, 2-hydroxycinnamaldehyde, 2'-benzoyloxycinnalmaldehyde, beta-sitosterol, curcumin and mixtures thereof.

The Applicant has indeed found that such a pharmaceutical composition was active in the treatment of cancer, especially in the case of HCC, breast cancer and prostate.

The Applicant has also demonstrated a synergistic effect of at least two of the constituents which provides a reinforced action of the composition of the invention on at least one mechanism involved in the cancer phenomenon, namely a mechanism chosen from the formation of the tumor stroma, cell growth, apoptosis, angiogenesis, the process metastatic, activation of cellular signaling pathways involved in inflammation, lipid metabolism, carbohydrate, bacterial, viral and fungal infection.

The Applicant has also demonstrated that the composition according to the invention has an effect on cells overexpressing reptin and / or pontine, which is the case of the cancer cells of the majority of cancers, including HCC, cancer breast and prostate.

The Applicant has also demonstrated that the composition according to the invention inhibits certain axes, in particular CXCR4 / CXCL12, which play a fundamental role in proliferation, tumor growth, metastasis and the formation of an immunosuppressive microenvironment. [Detailed description]

The pharmaceutical composition according to the invention can be used as a medicament and especially for its use in the therapeutic treatment of cancer. According to a particular embodiment of the present invention, the composition of the invention may furthermore comprise a mixture of beta-sitosterol and cinnamaldehyde or a mixture of beta-sitosterol and 2-hydroxycinnamaldehyde or a mixture of beta-sitosterol and 2'-benzoyloxycinnamaldehyde or a mixture of cinnamaldehydes including cinnamaldehyde with one or more of its synthetic derivatives and / or metabolites.

Preferably, it does not include a mixture of 2-hydroxycinnamaldehyde, 2'-benzoyloxycinnalmaldehyde and beta-sitosterol.

By way of example, it may comprise, as a mass percentage of the total mass of the active ingredients, a mass percentage of lupeol substantially equal to or greater than 15% and substantially equal to or less than 55, and in particular substantially equal to or greater than 30% o and substantially equal to or less than 50%, a percentage of beta-elemene substantially equal to or greater than 10%> and substantially equal to or less than 55%, and especially substantially equal to or greater than 20% and substantially equal to or less than 40%, a percentage of cinnamaldehyde substantially equal to or greater than 10% and substantially equal to or less than 45%), and in particular substantially equal to or greater than 20% and substantially equal to or less than 40%), a percentage of 2-hydroxycinnamaldehyde substantially equal to or greater than at 10% and substantially equal to or less than 45%, and especially substantially equal to or greater than 20% and substantially equal to or less than 40%, a percentage of 2'-benzoyloxycinnalmaldéhyde substantially equal to or greater than 10% and substantially equal to or less than 45%, and especially substantially equal to or greater than 20% and substantially equal to or less than 40%, a percentage of beta-sitosterol, when said composition contains this ingredient, substantially equal or greater than 10% and substantially equal to or less than 45%, and especially substantially equal to or greater than 20% and substantially equal to or less than 40%.

When the composition comprises cinnamaldehyde and one of its derivatives, their weight percentage relative to the total mass of the active ingredients is especially equal and in particular substantially equal to 20%.

The composition according to the invention further comprises at least one pharmaceutically acceptable excipient. This excipient can be solid or liquid. It may be chosen, for example, from purified water, ethyl alcohol, propylene glycol, glycerin, vegetable oils, animal oils, hydrocarbons, silicones, sugars such as glucose, levulose, wheat starch, corn starch, potato starch, xanthan gum, gum arabic, gum tragacanth, gum Sterculia, guar gum or "guaranates", pectins, alginates carrageenates, agar agar or agar, gelatin, cellulose and its derivatives.

The composition of the invention may be administered by any suitable route, for example by oral, rectal, local (topical, for example), intraperitoneal, systemic, intravenous, intramuscular, subcutaneous or mucosal, especially sublingual or using a patch, or in encapsulated form, or immobilized on liposomes, microparticles, microcapsules, associated with nanoparticles and the like. Mention may be made, by way of non-limiting examples of excipients suitable for oral administration, talc, lactose, starch and its derivatives, cellulose and its derivatives, polyethylene glycols, polymers of acrylic acid, gelatin, magnesium stearate, animal, vegetable or synthetic fats, paraffin derivatives, glycols, stabilizers, preservatives, antioxidants, wetting agents, anti-caking agents, dispersants, emulsifiers , taste modifying agents, penetrating agents, solubilizing agents. The techniques of formulation and administration of drugs and pharmaceutical compositions are well known in the art here considered, the skilled person may in particular refer to the book Remington's Pharmaceutical Sciences, latest edition. According to the invention, the composition can advantageously be administered orally by intravenous injection.

Advantageously, the composition according to the invention is adapted to be administered orally or intravenously at a dose substantially equal to or greater than 40 mg / kg / 24h and substantially equal to or less than 200 mg / kg / 24h in one or more doses. a mammal with such a need.

By way of example, the composition of the invention may be used in the therapeutic treatment of a cancer selected from hepatocellular carcinoma, colon cancer, rectal cancer, stomach cancer, cancer of the mouth, including cancer of the tongue, prostate cancer, metastatic prostate cancer, kidney cancer, breast cancer, chemoresistant breast cancer, bladder cancer, leukemia in chronic form or acute, multiple myeloma, lymphoma, brain tumors, lung cancer, especially lung adenocarcinoma, uterine cancer, ovarian cancer, bone tumors, pancreatic cancer, gall bladder cancer and liver cancer.

The composition according to the invention can advantageously be used in patients suffering from a chronic inflammatory disease and in particular, in inflammatory bowel diseases, more particularly ulcerative colitis, in patients carrying a pathogenic agent capable of to induce inflammation, in particular, Helicobacter pylori, in patients with diabetes, dyslipidemia, osteoarticular disorders, patients with hepatocellular insufficiency, patients with bacterial, fungal or viral infections, in particular patients with hepatitis B virus and / or hepatitis C and / or acquired immunodeficiency virus (HIV). In the case of CHC, the Applicant has demonstrated that the composition according to the invention gave good results at least in vitro and showed no toxicity to the liver cells of the patient.

The mode of action of the composition of the invention is not fully understood. It is more than likely that it acts simultaneously on different mechanisms of cancer. Thus, the composition of the invention can be used in the therapeutic treatment of cancer as an agent blocking the recruitment of bone marrow stem cells to the bone marrow. tumor microenvironment and / or as an inhibitor of tumor stromal formation and / or as an inhibitor of the overexpression of at least one protein selected from the group consisting of reptin, pontine and survivin, and preferably of reptin and pontine and / or anti-inflammatory agent and / or agent causing apoptosis of cancer cells and / or angiogenesis inhibiting agent and / or anti-metastatic agent.

This mechanism would be by destructuring, a restructuring of the lipid composition of the cell membranes, and thereby inhibit the cell signaling pathways involved in metabolic diseases, the secretion of inflammatory cytokines, chemokines, cell proliferation, and cell proliferation. angiogenesis, metastasis, and in bacterial, viral or fungal infection.

The present invention also relates to a pharmaceutical preparation which comprises the composition according to the invention, and, in addition, as a mixture or separately packaged, at least one anti-cancer agent for their use in the therapeutic treatment of cancer simultaneously, sequentially or spaced in time. By way of example, the anticancer agent may be chosen from the inhibitors of

VEGF, EGFR receptor blockers, multiple tyrosine kinase receptor inhibitors that simultaneously target angiogenesis and cell proliferation, kinase inhibitors, IGF-1R receptor inhibitors, mTOR inhibitors, inhibitors of the MEK-ERK signaling pathway, paclitaxel, As4S4, tamoxifen, curcumin, vincristine, adriamycin, aclarubicin, oxaliplatin, calcium folinate, 5-fluorouracil, capecitabine, cisplatin , tetramethylpyrazine, methotrexate, daunorubicin, certain genetically modified viruses which preferentially target cancer cells and mixtures thereof, and in particular mixtures of two of said anti-cancer agents, radioactive agents which can be used in brachytherapy and / or injectable or unmanageable.

The present invention also relates to a pharmaceutical preparation which comprises in combination beta-elemene, lupeol and / or beta-sitosterol, cinnamaldehyde and / or 2-hydroxycinnamaldehyde and / or 2'-benzoyloxycinnamaldehyde and optionally curcumin. The present invention also relates to a dietary supplement which comprises, in combination, beta-elemene, lupeol and / or beta-sitosterol and a pharmaceutically active agent chosen from cinnamaldehyde, 2-hydroxycinnamaldehyde, 2'-benzoyloxycinnalmaldehyde and mixtures thereof and optionally curcumin. [Definitions]

The term "therapeutic treatment" refers to curative treatment and prophylactic treatment; within the meaning of the present invention, a therapeutic treatment makes it possible to at least partially restore, at least partially correct or at least partially modify physiological functions by exerting a pharmacological, immunological or metabolic action.

The term "patient" refers to an animal or human mammal. The composition according to the invention can also be used in veterinary medicine. For the purpose of the present invention, an "anti-cancer agent" is an element that has, at least in vitro, an action against cancer cells, regardless of its mechanism of action. For the purposes of the present invention, the term "action" means the destruction or at least partial modification of the cancer cells, which makes it possible in particular to limit the proliferation of the cancer cells and / or their propagation. The term "patients with diabetes" refers to patients with type 1 diabetes, patients with type 2 diabetes, patients with gestational diabetes, patients with diabetes insipidus and patients with renal diabetes.

The term "dyslipidemia" refers to hyperlipidemia and hypolipidemia determined according to the criteria in force. The term "patients with osteoarticular disorders" refers to patients who have at least two signs selected from inflammatory signs, fistulas and the proven presence of bacteria detected by puncture or blood culture.

The term "patients with hepatocellular insufficiency" refers to patients with hepatitis, regardless of its cause (viral, toxic, drug or ischemic) and patients with cirrhosis of the liver.

The term "viral infection" refers to a biological entity whether it is the hepatitis B virus (HBV), the hepatitis C virus (HCV), the human immunodeficiency virus (HIV), the virus herpes simplex virus (HSV), cytomegalovirus (CMV) requiring a host, usually a cell, which it uses the constituents to multiply. For the purposes of the present invention, a "food supplement" is a foodstuff whose purpose is to supplement the normal diet and which constitutes a concentrated source of nutrients or other substances having a nutritional or physiological effect alone or in combination. With regard to the anti-cancer agents mentioned, the terms used, unless otherwise indicated, include the constituent isomers, the stereoisomers of conformation, the enantiomers and the diastereoisomers of the chemical compound in question.

Regarding cinnamaldehyde in the composition according to the invention, the term encompasses, unless otherwise indicated, cinnamaldehyde derivatives, formation dimers, in this case HCA-HCA, BCA-BCA, CA-CA.

[Examples]

The percentage of the compositions below is a percentage by weight relative to the total mass of the active ingredients.

Composition 1a: beta-elemene (30%), lupeol (30%) and 2-hydroxy cinnamaldehyde (40%). Composition lb: beta-elemene (30%), lupeol (30%) and 2'-benzoyloxycinnamaldehyde (40%).

Composition 2: beta-elemene (30%), lupeol (30%), 2-hydroxy cinnamaldehyde (20%) and 2'-benzoyloxy cinnamaldehyde (20%).

Composition 3a: beta-elemene (50%), lupeol (30%), and 2-hydroxycinnamaldehyde (20%).

Composition 3b: beta-elemene (50%), lupeol (30%), and 2'-benzoyloxycinnamaldehyde (20%). Composition 4a: beta-elemene (15%), lupeol (50%), beta-sitosterol (25%) and 2-hydroxy cinnamaldehyde (10%).

Composition 4b: beta-elemene (15%), lupeol (50%), beta-sitosterol (25%) and 2'-benzoyloxy cinnamaldehyde (10%).

Composition 5a: beta-elemene (20%), lupeol (20%), beta-sitosterol (40%) and 2-hydroxycinnamaldehyde (20%).

Composition 5b: beta-elemene (20%), lupeol (20%), beta-sitosterol (40%) and benzoyloxy cinnamaldehyde (20%).

Composition 6a: beta-elemene (25%), lupeol (35%), beta-sitosterol (15%) and 2 hydroxycinnamaldehyde (25%).

Composition 6b: beta-elemene (25%), lupeol (35%), beta-sitosterol (15%) and 2'-benzoyloxycinnamaldehyde (25%).

Composition 7a: beta-elemene (40%), lupeol (20%), beta-sitosterol (20%) and 2-hydroxycinnamaldehyde (20%).

Composition 7b: beta-elemene (40%), lupeol (20%), beta-sitosterol (20%) and benzoyloxycinnamaldehyde (20%).

[Experimental results]

Different human cell lines Hep3B (Hepatocellular Carcinoma), MCF-7 (Breast Cancer), DU-145 (Prostate Cancer) were studied. They were selected on the basis of their ability to overexpress at least one protein selected from reptin, pontine and survivin. The stromal cells have also been studied in order to determine the impact of the composition according to the invention in the tumor microenvironment. These cells were maintained in DMEM, supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution (MSP), containing penicillin, streptomycin and amphotericin B under standard growth conditions. (5% CO 2, 37 ° C, humidified atmosphere). The above compositions were dissolved and diluted in DMSO.

The above cells were treated with increasing solutions for 72 h in complete cell media. All treatment and control protocols were prepared as previously described.

In monotherapy, an antiproliferative effect of lupeol, 2-hydroxycinnamaldehyde more marked on the three lines, including Hep3B, DU-145 and MCF-7 compared with β-Elemene, was observed after 72 hours exposure. The solutions combining the molecules have been studied, in particular Lupeol-β-

Elemene, 2-hydroxycinnamaldehyde-P-Elemene and 2-hydroxycinnamaldehyde-Lupeol. After 72 hours exposure, an additional, synergistic effect of the higher 2-hydroxycinnamaldehyde-Lupeol combination was observed on Hep3B, DU145 and MCF-7 cell lines compared to the other combinations. The concomitant combination of Lupeol, β-Elemene and 2-hydroxycinnamaldehyde potentiated, synergized the antiproliferative effects of each molecule monotherapy in Hep3B and DU145 cell lines. However, the antiproliferative effect of the combination was less important in MCF-7 cell lines.

The effect of this combination on the induction of Hep3B cell apoptosis was determined using annexin-V / propidium iodide after 48h of treatment. Increased staining of annexin-V in Hep3B cells was observed while minimal staining was observed in untreated control cells.

In a co-culture of cancerous and stromal cells, treatment of confluent (50% confluent) cells with any of the prepared solutions resulted in decreased adhesion associated with cancer cell death, inhibition of production by stromal cells of soluble factors found in the tumor microenvironment and involved in the metastatic process and chemoresistance.

These results as a whole suggest that this pharmaceutical composition inhibits tumor stromal formation and hence metastatic spread and chemoresistance. It can, rightly, be used in the treatment of hepatocellular carcinoma, breast cancer, prostate cancer even at advanced stages.

Claims

Claims:

1. A pharmaceutical composition, characterized in that it comprises as active ingredient a combination of beta-elemene, lupeol and a pharmaceutically active agent selected from cinnamaldehyde, 2-hydroxycinnamaldehyde and 2 ' benzoyloxycinnalmaldehyde, beta-sitosterol, curcumin and mixtures thereof.

2. Pharmaceutical composition according to claim 1, characterized in that it further comprises a mixture of beta-sitosterol and cinnamaldehyde or a mixture of beta-sitosterol and 2-hydroxycinnamaldehyde or a mixture of beta-sitosterol and 2 ' benzoyloxycinnalmaldehyde or a mixture of cinnamaldehyde with one or more of its synthetic derivatives and / or metabolites.

3. Pharmaceutical composition according to any one of the preceding claims, characterized in that it comprises a mass percentage of the total mass of active ingredients, a mass percentage of lupeol substantially equal to or greater than 15% and substantially equal to or less than 55 , and in particular substantially equal to or greater than 30% and substantially equal to or less than 50%, a percentage of beta-elemen substantially equal to or greater than 10% and substantially equal to or less than 55%, and especially substantially equal to or greater than 20% and substantially equal to or less than 40%, a percentage of cinnamaldehyde substantially equal to or greater than 10% and substantially equal to or less than 45%, and in particular substantially equal to or greater than 20% and substantially equal to or less than 40%, a percentage of 2-hy droxycinnamaldehyde substantially equal to or greater than 10% and substantially equal to or less than 45%, and in particular substantially equal to u greater than 20% and substantially equal to or less than 40%, a percentage of 2'-benzoyloxycinnalmaldehyde substantially equal to or greater than 10% and substantially equal to or less than 45%, and in particular substantially equal to or greater than 20% and substantially equal to or less than 40%, a percentage of beta-sitosterol, when said composition contains this ingredient, substantially equal to or greater than 10% and substantially equal to or less than 45%, and in particular substantially equal to or greater than 20% and substantially equal to or less than 40%.

4. Pharmaceutical composition according to any one of the preceding claims, for use in the therapeutic treatment of a cancer selected from hepatocellular carcinoma, colon cancer, rectal cancer, stomach cancer, cancer mouth, including cancer of the tongue, prostate cancer, cancer of the Metastatic prostate cancer, kidney cancer, breast cancer, chemoresistant breast cancer, bladder cancer, leukemia in chronic or acute form, multiple myeloma, lymphoma, brain tumors, lung cancer, particularly lung adenocarcinoma, uterine cancer, ovarian cancer, bone tumors, pancreatic cancer, gall bladder cancer and liver cancer.

5. Pharmaceutical composition according to any one of the preceding claims, for its use in the therapeutic treatment of cancer in patients with a chronic inflammatory disease and in particular in inflammatory bowel diseases, particularly colitis. in patients with a pathogen that may cause inflammation, in particular, Helicobacter pylori, in patients with diabetes, dyslipidemia, osteo-articular diseases, patients with hepatocellular insufficiency, patients with bacterial, fungal or viral infections, including patients with hepatitis B virus and / or hepatitis C and / or acquired immunodeficiency virus (HIV).

6. Pharmaceutical composition according to any one of the preceding claims for its use in the treatment of cancer, viral, bacterial or fungal infections, in particular in patients according to claim 5, which makes it possible to inhibit the alteration of the intestinal mucosa. , bacterial, viral or fungal translocation from the intestinal lumen to the bloodstream, systemic immune hyperactivation, to induce a vigorous immune response, particularly in the lymphoid tissue associated with the digestive tract.

7. Pharmaceutical composition according to any one of the preceding claims which destructures, restructures the lipid composition of cell membranes, and thereby inhibits the cell signaling pathways involved in metabolic diseases, the secretion of inflammatory cytokines, chemokines, in cell proliferation, angiogenesis, metastasis, and in bacterial, viral or fungal infection.

A pharmaceutical composition according to any one of the preceding claims for use in the treatment of cancer as a blocking agent for the recruitment of bone marrow stem cells to the tumor microenvironment and / or as an inhibitory agent for the treatment of cancer. tumor stroma formation and / or as an inhibitory agent for the overexpression of at least one protein selected from the group consisting of reptin, pontine and survivin and preference of reptin and pontine.

9. Pharmaceutical preparation, characterized in that it comprises the composition according to any one of the preceding claims and, in addition, in a mixture or separately packaged, at least one anti-cancer agent for their use in the therapeutic treatment of cancer in a manner simultaneous, sequenced or spaced in time.

10. Pharmaceutical preparation according to claim 9, characterized in that said anti-cancer agent is chosen from VEGF inhibitors, EGFR receptor inhibitors, inhibitors of several receptors with tyrosine kinase activity which parallel target angiogenesis and cell proliferation, kinase inhibitors, IGF-1R receptor inhibitors, mTOR inhibitors, MEK-ERK pathway inhibitors, paclitaxel, As4S4, tamoxifen, curcumin, vincristine, adriamycin, aclarubicin, oxaliplatin, calcium folinate, 5-fluorouracil, capecitabine, cisplatin, tetramethylpyrazine, methotrexate, daunorubicin, certain genetically modified viruses which preferentially target cancer cells and mixtures thereof, and especially mixtures of two of said anti-cancer agents, radioactive agents for use in brachytherapy and / or radioisotopes Injectable or unmanageable drugs.