Pharmaceutical composition for preventing or inhibiting metastasis

Abstract

The present invention relates to compositions and agents for preventing or arresting metastasis, particularly metastasis originating from epithelial cell cancers (e.g., skin cancer), lung cancer, stomach cancer, brain cancer, breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, head / neck cancer, and esophageal cancer. It has been found that the compositions of the present invention irreversibly convert cancer cells with metastatic potential into harmless cells without metastatic potential.

Description

Technical Field

[0001] The present invention relates to compositions and agents for preventing metastasis or arresting metastasis in progress, particularly metastasis originating from epithelial cancers (such as skin cancer), lung cancer, breast cancer, colorectal cancer, prostate cancer, liver cancer, stomach cancer, brain cancer, kidney cancer, gallbladder cancer, pancreatic cancer, head / neck cancer, bladder cancer, melanoma and esophageal cancer. It has been found that the compositions of the present invention can prevent or arrest metastasis. Background Art

[0002] The occurrence of cancer is widespread. These cancers are often treated with aggressive drugs, such as chemotherapy, which cause many typical negative side effects to the human body. The treatment of metastases is even worse, because these metastases are often spread throughout the body and therefore cannot usually be effectively treated, at least not without causing serious damage to the body.

[0003] Cancer is related to cells that proliferate in an uncontrolled manner and therefore, more rapidly and constantly proliferate compared to other living cells. Primary tumors are thus formed. Generally, metastasis involves spreading from the initial cancer site to different parts in the living body. These different parts can thus be referred to as metastasis. This spread is caused by cancer cells that can circulate in the body to other parts of the body. It can form secondary tumors, i.e. metastatic tumors, in the other parts. Metastasis may have a smaller volume, but it is generally widely spread in the body. Most cancers can be transferred, and some of them are particularly thorny. The cells of the transfer are generally similar to the cells of the original tumor, and therefore refer to the initial tumor when characterizing the transfer. Therefore, in principle, it can be treated in a similar manner, but because it is widely distributed, treatment may be cumbersome and harmful to the body. However, transfer can obtain new features, making it unresponsive to anticancer drugs that can effectively target primary tumors, making it even more difficult to treat. At present, primary tumors that have not been transferred can be effectively treated by surgery (usually in combination with chemotherapy and / or radiotherapy). This is why cancer patients usually do not die from their primary tumors, but die from the main reason for metastasis. Therefore, there is an unmet need for new treatments that can prevent metastasis or arrest metastasis in progress.

[0004] For the treatment of cancer metastasis, combination therapy is often used, i.e., combining two or more chemical substances. These combination therapies are often more effective. Also, such therapies usually need to be determined for each individual human being, which often involves testing therapies that are deemed ineffective after evaluation.

[0005] Reference may be made to some documents. WO 88 / 05301 describes sulfated polysaccharides having anti-metastatic and / or anti-inflammatory activity, and methods for anti-metastatic and / or anti-inflammatory treatment of animals or human patients, which comprises administering to the patient an effective amount of at least one sulfated polysaccharide that blocks or inhibits the activity of endoglycosidases (especially heparinase). Suitable sulfated polysaccharides include heparin and modified heparin, fucoidan, pentosan sulfate, polyglucose sulfate and carrageenan lambda. WO96 / 33726A1 describes the preparation and use of sulfated oligosaccharides, wherein the oligosaccharide has the general formula (I): R1-(R x ) n -R2, where R1 and R2 and each R xRepresents monosaccharide units, which may be the same or different, adjacent monosaccharide units are connected by 2, 3, 4 and / or 6 glycosidic bonds and n is an integer from 1 to 6, and its use as an anti-angiogenic, anti-metastatic and / or anti-inflammatory agent. WO 2011 / 113892 A2 describes the combined use of phospholipids and polysaccharides with sulfate groups, which are used to inhibit metastatic spread, and a medicament for inhibiting the metastatic spread of tumors, which comprises a phospholipid component and a polysaccharide component with polysaccharides containing sulfate groups. The two components can be in the same or separate preparations. Panigraphy et al., in "PPARγ as a therapeutic target of tumour angiogenesis and metastasis" in "Cancer Biology & Therapy" 4:7, 687-693, July 2005, commented on the effects of PPARγ agonists on angiogenesis and inflammation in the case of tumor formation in the form of a whole tissue process. It discusses possible reasons for the contradictory effects of PPARγ agonists against tumor development and metastasis. Therefore, sorting out the various modes of action and defining their relative contributions in the case of tumors and host tissues as heterologous targets will be key to understanding the multiple effects of PPARγ. WO 2018 / 132899 A1 describes a method for treating, preventing, reducing or delaying the onset of osteosarcoma lung metastasis in a subject, comprising: administering a therapeutically or prophylactically effective amount of a PPARγ agonist, an anti-inflammatory agent or a neutrophil depleting agent to a subject suffering from or suspected of suffering from osteosarcoma. WO 2017 / 105229 A1 describes a pharmaceutical composition for inducing beige or brown adipose tissue, comprising (i) a compound comprising at least one carbohydrate and containing at least one sulfate, and (ii) at least one non-protein or non-NA chain compound capable of activating PPAR, preferably PPARγ, different from (i), and (iii) optionally, at least one pharmaceutically acceptable carrier. Preferably, the sulfated compound comprises a monosulfide-containing agent, such as MSM, or a polysulfide-containing agent, such as pentosan, adequan. The second part of the composition is a PPARγ agonist selected from the group of thiazolidinediones, NSAIDs, sulfonylureas and indoles (e.g., indomethacin or pioglitazone).

[0006] Because therapies for treating metastasis are sparse or ineffective, there remains a need for improved drugs and treatments that overcome the deficiencies of the prior art. Summary of the invention

[0007] One object of the present invention is to provide a composition that can prevent metastasis or prevent metastasis (such as preventing ongoing metastasis or preventing further metastasis) by irreversibly blocking the metastatic ability of tumor cells. The present invention also provides a method of use of such compositions, and use thereof to prevent metastasis and prevent ongoing metastasis, especially in the case where mammals tend to develop metastasis (such as in cancer phase II or III phase), and its restriction or inhibitory effect. In fact, the present invention provides two synergistic aspects of a first compound comprising at least one carbohydrate and containing more than one sulfate and a second compound selected from a group of thiazolidinediones, NSAIDs, sulfonylureas and indoles, that is, (A) (A1) prevents ongoing metastasis and (A2) prevents metastasis, and (B) compared to the expression sum of the compound administered alone, the combination of the first compound and the second compound provides at least twice the expression of certain genes (e.g., FABP4, CD36, PLIN1). For effect (A1), breast cancer cell lines show that these cells differentiate (cell type changes) into brown / beige and white fat cells. Therefore, ongoing metastasis is prevented. As for effect (A2), it was found that cancer cells that differentiate into stem cells are then differentiated into brown / beige fat cells under the influence of the two aforementioned compounds. It is believed that two mouse cell lines as well as cancer cell lines confirm this effect. In the cancer cell lines, new cancer cells are hardly "formed", but fat cells are formed. Metastasis is thus prevented.

[0008] For effect (B), the experimental results and additional results provide clear evidence.

[0009] The present invention provides a pharmaceutical composition for preventing metastasis and stopping ongoing metastasis, which comprises a first compound comprising at least one carbohydrate and containing more than one sulfate, and a second compound different from the first compound that can activate PPAR, preferably PPARγ. The first compound preferably comprises two or more sulfates, and is more preferably a compound comprising oligosulfates or polysulfates, such as 3-20 sulfates, preferably 4-15 sulfates, more preferably 5-12 sulfates, such as 6-10 sulfates, for example 7-8 sulfates, such as (C5H6X i O 10 S2) n , where X can be selected from monovalent metal atoms, such as Na + and K + , i is 2, and is selected from divalent metal atoms such as Ca 2+ and Mg 2+, i is 1, and n is preferably in the range of 2-20, more preferably 3-12, such as 4-8. When referring to the first compound, oligosulfates and polysulfates are included, as indicated above. The molecular weight of the first compound is preferably not too large, such as <10kDa, preferably <7kDa, such as <5kDa. A compound that can activate PPAR means that the PPAR receptor activity increases by 2 to 3 times compared to the baseline situation in which the compound that activates PPAR is not present. The compound that can activate PPAR is neither a protein nor a nucleic acid (NA) chain compound. Suitable compounds that can activate PPAR include thiazolidinediones, NSAIDs, sulfonylureas and indoles. The inventors have found that the composition according to the present invention can prevent metastasis or prevent ongoing metastasis. The composition comprises a combination of a carbohydrate comprising more than one sulfate group (such as polysulfated pentosan) and a PPAR (preferably a PPARγ agonist, such as indomethacin or pioglitazone) that synergistically stimulates the formation of adipocytes with beige / light brown and / or BAT characteristics, i.e., cancer cells with metastatic potential are irreversibly transformed into harmless cells. It should be noted that white adipocytes, beige adipocytes, and brown adipocytes are clearly distinguishable from each other, such as in terms of being UCP1 negative or positive, the size of lipid droplets, secretion of adipose cytokines, etc. Therefore, it is not difficult to determine the formation of beige / light brown cells. Such transformation can be found in the mesenchymal progenitor KS483 cell line deposited with the German Collection of Microorganisms (DSMZ, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on January 13, 2016 under the deposit number DSM ACC3286, but other cell lines of similar origin can also be used, such as the 3T3-L1 cell line available from Sigma-Aldrich and epithelial cancer cell lines with metastatic potential.

[0010] Therefore, the present invention provides a pharmaceutical composition for preventing metastasis or arresting ongoing metastasis comprising a compound comprising at least one carbohydrate (saccharide) and containing at least one sulfate in combination with a second compound capable of activating PPAR, preferably PPARγ.

[0011] Thus, patients with cancer that is known to metastasize or that already has one or more metastases may benefit from the use of the pharmaceutical compositions of the present invention, as metastases may be prevented or ongoing metastases arrested.

[0012] Therefore, the present invention also relates to the use of the pharmaceutical composition as described as a medicament.

[0013] Preferably, the present invention also relates to the pharmaceutical composition of the present invention for preventing metastasis of these metastatic cancers or arresting ongoing metastasis. DETAILED DESCRIPTION

[0014] In exemplary embodiments, the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis may comprise pentosan sulfate and / or bovine blood plasma substitute, wherein the bovine blood plasma substitute is polysulfated glycosaminoglycan (PSGAG).

[0015] In exemplary embodiments, the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis may comprise indomethacin and / or pioglitazone.

[0016] In exemplary embodiments, the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis may include pentosan sulfate and / or bovine plasma substitute, and indomethacin and / or pioglitazone.

[0017] In exemplary embodiments, the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis may include pentosan sulfate and indomethacin, pentosan sulfate and pioglitazone, bovine plasma substitute and indomethacin, bovine plasma substitute and pioglitazone, and combinations thereof.

[0018] In exemplary embodiments of the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis, the first compound and the second compound may be provided in a molar ratio of 0.01:1 to 1:0.01, preferably 0.2:1 to 1:0.2, more preferably 0.33:1 to 1:0.33, even more preferably 0.45:1 to 1:0.245, such as 0.5:1 to 1:0.5, for example 0.9:1 to 1:0.9.

[0019] In an exemplary embodiment of the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis, the composition further comprises (iii) at least one pharmaceutically acceptable carrier.

[0020] In an exemplary embodiment of the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis, the active pharmaceutical ingredient is in a dosage form, preferably comprising 1-10 mg of active ingredient / kg body weight, preferably 2-5 mg of active ingredient / kg body weight, such as 2-1000 mg of active ingredient, preferably 10-500 mg of active ingredient, more preferably 20-100 mg of active ingredient.

[0021] In exemplary embodiments of the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis, the pharmaceutical composition may comprise separate dosage forms of individual pharmaceutically active ingredients, preferably 2-10 dosage forms, such as 3-5 dosage forms.

[0022] In exemplary embodiments of the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis, the composition may be in the form of a tablet, capsule, sustained release formulation, or injectable formulation.

[0023] In exemplary embodiments of the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis, the composition may be in the form of a tablet or capsule suitable for oral administration.

[0024] In an exemplary embodiment, the pharmaceutical composition of the present invention for preventing metastasis or preventing ongoing metastasis can be formulated in a form that allows targeting to specific cells, tissues or organs, such as in the form of nanoparticles, depending on its use. For example, the nanoparticle can be any type of nanodevice in which two compounds are packaged for targeted delivery. In a more preferred embodiment, targeting can be carried out by any specific molecule (such as an antibody) attached to a specific form (e.g., nanoparticle) or other molecules or chemical entities that can target specific cells, tissues or organs.

[0025] In exemplary embodiments, the dosage and formulation of the pharmaceutical compositions of the invention for preventing metastasis or arresting ongoing metastasis is such that all metastases in the body are targeted.

[0026] In an exemplary embodiment of the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis, the cancer metastasis may be caused by an epithelial cancer.

[0027] In exemplary embodiments of the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis, the cancer can be selected from skin cancer, stomach cancer, brain cancer, lung cancer, breast cancer, colorectal cancer, liver cancer, kidney cancer, gallbladder cancer, prostate cancer, bladder cancer, pancreatic cancer, head / neck cancer, and esophageal cancer.

[0028] In an exemplary embodiment, the pharmaceutical composition of the present invention for preventing metastasis or preventing ongoing metastasis can be used as a medicament, which is achieved by administering an effective amount of the medicament to epithelial cancer for a sufficient period of time. Typically, the pharmaceutical composition of the present invention can be used for a period of 1 week to 12 months, preferably 2 weeks to 6 months, such as 4 weeks to 3 months. Subsequently, the pharmaceutical composition can be used in another time period, typically with a lower dose, such as 10-50% of the initial dose, wherein the other time period can be at least one month, and typically it can be used for more than one year, such as five to ten years, or even for the remaining life of a pet or mammal.

[0029] In an exemplary embodiment of the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis, it can be administered to a pet or a mammal containing the tissue, wherein the mammal is preferably a human having metastasis.

[0030] In an exemplary embodiment of the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis, it may be used as a medicament.

[0031] More specifically, the present invention also relates to the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis.

[0032] The pharmaceutical composition according to the present invention may be a combined composition, wherein the active pharmaceutical ingredients are in one dosage form. The pharmaceutical composition may also comprise separate dosage forms of the individual pharmaceutically active ingredients.

[0033] The compound comprising at least one carbohydrate and containing at least one sulfate preferably has an anti-inflammatory effect, meaning that it reduces at least one inflammatory marker. Inflammatory markers are, for example, TNFα, IFN, cytokines, histamine, interleukins, chemokines, leukotrienes, lysosomal granules and prostaglandins.

[0034] A compound activating a PPAR, preferably PPARγ, is preferably a compound that can activate a PPAR, meaning that the PPAR receptor activity is increased at least 2-3 fold compared to the baseline situation in the absence of the PPAR activating compound.

[0035] Compounds which activate PPAR, preferably PPARγ, preferably have an anti-inflammatory effect, meaning that they reduce at least one inflammatory marker. Inflammatory markers are, for example, TNFα, IFN, cytokines, histamine, interleukins, chemokines, leukotrienes, lysosomal granules and prostaglandins.

[0036] Carbohydrates are organic compounds containing only carbon, hydrogen, and oxygen, typically with a 2:1 hydrogen:oxygen atomic ratio (such as in water); and have the empirical formula C m (H2O) n (where m may be different from n.) Structurally, carbohydrates can be more accurately viewed as polyhydroxy aldehydes and ketones.

[0037] Carbohydrates as used in the present invention do not refer to glycosylated proteins or nucleoside compounds (such as DNA, RNA, etc.).

[0038] The carbohydrate is preferably glycosaminoglycan (GAG) or keratin. Glucosaminoglycan or mucopolysaccharide is a long chain non-branched polysaccharide composed of repeating disaccharide units. The repeating unit is composed of amino sugar (N-acetylglucosamine or N-acetylgalactosamine) and uronic acid sugar (glucuronic acid or iduronic acid) or galactose.

[0039] Based on the core disaccharide structure, GAGs can be classified into four groups.

[0040] Preferred types of sulfated sugars are also known as heparins, heparan sulfate analogs, or heparinoid compounds.

[0041] Suitable compounds comprising carbohydrates and sulfate groups may be derived from natural sources, or may be at least partly prepared synthetically.

[0042] Examples of sulfated compounds include monosulfide-containing agents such as MSM (dimethyl sulfone), polydextrose sulfate, or polysulfide-containing agents such as polysulfated glycosaminoglycans (bovine plasma substitute), heparan sulfate pentosan, and the like.

[0043] Preferred sulfated sugar compounds are polysulfated glycosaminoglycans (bovine blood plasma substitute), heparin-like polysulfated pentosan (Elmiron), and the like.

[0044] The second part of the composition of the present invention is an agent that can activate PPAR, preferably PPARγ. Preferred PPAR agonists include triglitazone (TZD: troglitazone, rosiglitazone, pioglitazone, etc.) and indomethacin.

[0045] Preferably, the compound comprising at least one carbohydrate and containing at least one sulfate and the compound activating PPARγ preferably both have an anti-inflammatory effect, meaning that at least one inflammatory marker is reduced. Inflammatory markers are, for example, TNFα, IFN, cytokines, histamine, interleukins, chemokines, leukotrienes, lysosomal granules and prostaglandins.

[0046] Whether a compound is a PPAR, preferably PPAR gamma agonist, can be determined in a simple cell-based assay.

[0047] A PPAR agonist refers to any compound that increases the biological activity or expression of one or more PPARs (eg, PPARα, PPARγ, and PPARβ / δ) in a cell by at least 10% relative.

[0048] Examples of PPARy agonists include any thiazolidinedione, but especially rosiglitazone, troglitazone and pioglitazone and analogs thereof. Rosiglitazone and pioglitazone are preferred PPAR agonists. Other examples of PPARy agonists include nonsteroidal anti-inflammatory drugs such as indomethacin, ibuprofen, naproxen and fenoprofen, and antioxidants such as vitamin E, vitamin C, S-adenosylmethionine, selenium, beta-carotene, idebenone, cysteine, dithioerythritol, dithionite, dithiothreitol and pyrosulfite.

[0049] Examples of PPARα agonists include any of fibrates (eg, fenofibrate, bezafibrate, gemfibrozil, and analogs thereof), docosahexaenoic acid, and Wy4643.

[0050] Thus, for example, the combination of pentosan sulfate and one of these compounds may prevent metastasis or arrest metastasis in progress.More specifically, PPARγ agonism may be determined using a dual luciferase assay with a luciferase construct containing a PPARγ responsive element in a similar manner as described above.

[0051] This second compound is not a protein or nucleic acid based compound.

[0052] Suitable PPAR gamma agonists include thiazolidinediones, NSAIDs, sulfonylureas and indoles.

[0053] Thiazolidinediones (abbreviated as TZDs) are also known as glitazones. Suitable thiazolidinediones include rosiglitazone, pioglitazone, troglitazone and ciglitazone, which are selective ligands for the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) γ. Typical TZDs have Figure 5 The formula shown in , wherein n is 1, 2 or 3, Y and Z are independently O or NH; and E is a cyclic or bicyclic aromatic or non-aromatic ring, optionally containing heteroatoms selected from oxygen or nitrogen. Suitable TZDs are described, for example, in WO2000 / 27401.

[0054] NSAIDs are drugs that provide analgesia and antipyretic (antipyretic) effects and, at higher doses, anti-inflammatory effects. PPARγ is activated by several endogenous ligands that are produced by the metabolism of eicosatetraenoic acid and linoleic acid. Representative PPARγ ligands include the lipoxygenase products 13(S)HODE (produced by 15-LOX-1 from linoleic acid) and 15(S)HETE (produced by 15-LOX-1 and 15-LOX-2 from eicosatetraenoic acid, but 15-LOX-2 catalyzes this reaction significantly more efficiently). Induction of 15-LOX-1 activity by NSAIDs occurs independently of COX-2 inhibition.

[0055] NSAIDs can be classified based on their chemical structure or mechanism of action. Early NSAIDs were known before their mechanism of action was elucidated and are therefore classified based on chemical structure or source. Newer substances are more often classified based on mechanism of action.

[0056] Suitable NSAIDs include salicylates, propionic acid derivatives, acetic acid derivatives, enol (oxicam) derivatives and fenamic acid derivatives.

[0057] Suitable salicylates include aspirin (acetylsalicylic acid), diflunisal (Dolobid TM ), Disalcid TM ) and choline magnesium trisalicylate (Trilisate TM ).

[0058] Suitable propionic acid derivatives include ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin and loxoprofen.

[0059] Suitable acetic acid derivatives include indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac and nabumetone (the drug itself is non-acidic, but the active, major metabolite has a carboxylic acid group).

[0060] Suitable enol(oxicam) derivatives include piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam.

[0061] Suitable fenamic acid derivatives (fenamic acid esters) include mefenamic acid, meclofenamic acid, flufenamic acid and tolfenamic acid.

[0062] Sulfonylurea derivatives are antidiabetic drugs used to manage type 2 diabetes. Examples include carbutamide, acetohexamide, chlorpropamide, tolbutamide, tolazamide, glipizide, gliclazide, glibenclamide (glyburide), glibornuride, gliquidone, glisoxepide, glyclopyramide and glimepiride. It works primarily by increasing insulin release from beta cells in the pancreas. All sulfonylureas contain a central S-phenylsulfonylurea structure as well as a para-substituent (R) on the benzene ring and various groups (R2) that cap the urea N' end group (see Figure 6 ).

[0063] Indoles include indole-derived agents that can bind to PPARγ and primarily comprise sulfonyl-indoles.

[0064] Dual alpha-gamma agonists are also suitable and include glitazar.

[0065] Suitable glitazars include aleglitazar, muraglitazar and tesaglitazar.

[0066] PPARα agonists include fibrates and biguadine.

[0067] Preferably, the compound comprising at least one sugar and at least one sulfate salt may be present in the pharmaceutical composition in an amount of, for example, 0-10% by weight, based on the total weight.

[0068] Preferably, the PPARγ agonist may be present in an amount of 0-10 wt % based on the total weight.

[0069] Preferably, the relative amounts between the two compounds (sulfated compound:PPARγ agonist) are 1:1 to 1:5.

[0070] The pharmaceutically acceptable carrier may be present in an amount of 0.01% to 99.9%, preferably 0.1% to 10%, and the content thereof will depend on the formulation.

[0071] In one embodiment, the medicament is in the form of a tablet suitable for oral administration.

[0072] In another embodiment, the medicament is in a form suitable for topical administration.

[0073] The medicament may comprise the components in the form of a solid or liquid formulation.

[0074] The dosage form may be an immediate release or an extended release formulation.

[0075] The present invention also relates to the pharmaceutical composition of the present invention for use in preventing metastasis or arresting an ongoing metastasis.

[0076] More specifically, the present invention also relates to the pharmaceutical composition of the present invention for preventing metastasis or arresting ongoing metastasis.

[0077] One advantage of the present invention is that the components of the composition can be non-biological products, and several exemplary components are approved pharmaceutical agents.

[0078] Pentosan polysulfate (PPS), made from beech wood, is an FDA-approved oral medication for the treatment of interstitial cystitis (IC), also known as painful bladder syndrome. PPS is known to have anti-inflammatory and pro-chondrogenic properties. Pentosan polysulfate can be used in pill form or infused directly into the bladder.

[0079] Bovine plasma substitute, a polysulfated glycosaminoglycan, is a well-known veterinary agent for the treatment of joint pain.

[0080] Indomethacin is a nonsteroidal anti-inflammatory drug (NSAID) approved by the FDA. It is generally used to reduce fever, pain, stiffness and swelling. In addition, indomethacin is a COX inhibitor, which blocks prostaglandin production and is used as an inflammatory inhibitor. At higher doses, it can also induce adipogenesis in mesenchymal progenitor cells in vitro by activating PPARγ.

[0081] Pioglitazone is a potent PPARγ agonist and belongs to the class of thiazolidinediones or glitazones and is designed for the treatment of type II diabetes by improving insulin sensitivity.

[0082] Schematic Overview

[0083] Figure 1 Pictures showing histological staining experiments on the synergistic effect of the combination of pentosan sulfate and indomethacin.

[0084] Figure 2 Pictures showing histological staining experiments on the synergistic effect of the combination of pentosan sulfate and pioglitazone.

[0085] Figure 3 Pictures showing histological staining experiments on the synergistic effect of the combination of bovine blood plasma and indomethacin.

[0086] Figure 4 Shown are in vitro measurements of PPAR transcriptional activity in KS-483 cells transfected with a dual PPAR reporter system and exposed to various compounds, expressed as relative light units (RLU).

[0087] Figure 5 shows the schematic structure of thiazolidinedione and Figure 6 Shown is the schematic structure of sulfonylurea.

[0088] Figure 7a , 7b , 8, 9a, 9b, 10, 11a, 11b, 12, 13a, 13b and 14 show the experimental results of the claimed composition and its synergistic effect, Figures 15a-15d Show stimulus packages, Figure 16a-16c and 17a-17d show the staining results, and Fig.18a , 18b Demonstrate synergy.

[0089] Detailed description of the diagram

[0090] The invention will be illustrated by the following non-limiting examples and experiments.

[0091] Figure 1-3 The effect of using a compound containing a carbohydrate containing more than one sulfate (bovine blood plasma or pentosan sulfate) in combination with indomethacin or pioglitazone is shown. The concentration of the active compound used to stimulate the cells is provided in the picture. The active compound is added to the culture medium, and then the cells are suspended in the culture medium. After 3 days, the cells are stained with Oil red O or Nile Red stain.

[0092] Oil Red O (Solvent Red 27, Sudan Red 5B, CI26125, C26H24N4O) is a fat stain (lipid-soluble dye) diazo dye used to stain neutral triglycerides and lipids on frozen sections. The picture shows the central nucleus, increased lipid formation and droplets produced by the combined use of the composition of the present invention, and thus indicates that cancer cells with metastatic potential are transformed into harmless cells. Nile red (also known as Nile blue oxazone) is a lipophilic dye that accumulates in lipid globules within cells. Nile red fluoresces strongly when distributed in lipids, but is almost completely non-luminescent in aqueous solutions.

[0093] Figure 4The effect of exposure of KS-483 cells to a compound containing carbohydrates and sulfate (bovine plasma substitute or pentosan sulfate) and either indomethacin or pioglitazone is shown. Similar results were obtained with other cell lines.

[0094] Similar experiments were performed with other cell lines, organoids, and assays with similar results.

[0095] Examples and Experiments

[0096] After stimulation with the compound, the upregulation or downregulation of genes in KS-483 cells was evaluated. Briefly, KS-483 cells were cultured in α-MEM medium supplemented with glutamax, penicillin / streptomycin and 10% heat-inactivated fetal calf serum (FCS) under standard culture conditions. Similar experiments were performed using other cell lines, such as the 3T3-L1 cell line.

[0097] From experiments with up- and down-regulated genes and the activity of said genes, it is evident that a composition comprising (i) a compound comprising at least one carbohydrate and containing more than one sulfate in combination with (ii) indomethacin and / or pioglitazone blocks metastatic development.

[0098] Activity measurement

[0099] Activity measurements were performed using KS-483 cells. From the RLU measured after exposure to the single compounds, it is apparent that the composition comprising (i) a compound comprising at least one carbohydrate and containing more than one sulfate and indomethacin and / or pioglitazone caused a significant increase in activity and a reduction in metastatic cells in a transformed manner.

[0100] The present invention has been described with reference to certain embodiments discussed above. It will be appreciated that various modifications may be readily made to these embodiments without departing from the scope of the present invention and that alternative forms are well known to those skilled in the art. Therefore, although specific embodiments have been described, these embodiments are merely examples and do not limit the scope of the present invention as defined by the appended claims.

[0101] Experimental design Cell culture

[0102] for Figure 7a-14 , using the following conditions:

[0103]

[0104] Other experiments

[0105] background

[0106] The overall rationale for the in vitro experiments performed to test the efficacy of the combination compounds of the present invention in treating cancer was primarily based on their ability to induce definitive adipogenesis and conversion to beige / brown adipocytes under experimental conditions using a previous publication (Ishay-Ronen et al., Cancer Cell 2019, https: / / doi.org / 10.1016 / j.ccell.2018.12.00) with different compounds but similar endpoints.

[0107] result

[0108] MTΔECad cells were obtained from MMTV-Neu transgenic mice (a genetic model of irreversible EMT).MTΔECad cells were treated with the following compounds: indomethacin (Indo), pioglitazone (Pio), and pentosan polysulfate (PPS).

[0109] Processing and methods

[0110] Following the protocol established by the study of Isher-Rohn et al., the present inventors used a combination of insulin, dexamethasone, and rosiglitazone on 3T3-L1 cells (treatment A) and rosiglitazone and BMP2 on MTΔECad cells (treatment B).

[0111] For stimulation of MTΔECad cells, the following concentrations were used: PPS (5 μg / ml), indomethacin (50 μM), pioglitazone (10 μM).

[0112] Untreated cell lines were used as negative controls. As a result of adipogenesis, several methods were tested:

[0113] 1. Oil Red O staining by histochemistry

[0114] 2. Nile Red Staining by Immunofluorescence (IF)

[0115] 3.RTqPCR (gene expression analysis)

[0116] -Adiponectin (adipocyte marker)

[0117] -EpCam (epithelial cell marker)

[0118] Fig.15a -dShow stimulus package; Fig.15a -b is based on prior art and Figure 15c-15d According to the present invention.

[0119] Figure 16 shows Nile red staining of cells treated with MTΔECad. Induction of adipogenesis (indicated by red staining), epithelialization (i.e., reacquisition of epithelial features, i.e., MET; indicated by morphological changes from spindle-shaped to cuboidal cells), and induction of adipogenesis is shown under two conditions:

[0120] Control = untreated MTΔECad cells.

[0121] Treatment B( Fig.15b ) = Rosiglitazone and BMP2 (based on Ishe-Rohn et al., Cancer Cell 2019)

[0122] Processing C and D ( Fig.15c -d) =PPS and indomethacin and PPS and pioglitazone (combinations according to the invention).

[0123] Although the following images only indicate the processes caused by the different treatments and do not allow their quantitative assessment, they demonstrate the fact that both processes, adipogenesis and transformation into adipocytes, can proceed independently of each other. Interestingly, it is thought that this could prevent and even 'cure' metastasis formation.

[0124] Figure 16. Left control ( Fig.16a ), i.e., untreated MTΔECad cells, which clearly display the spindle-shaped cell characteristics of epithelial cells undergoing EMT. Fig.16b ), many MTΔECad cells were treated with B( Fig.16b ) and treatment D( Fig.16c ) differentiated into adipocytes (red staining) as a positive control. It is important to note that MTΔECad cells that were not differentiated into adipocytes by treatments B or D have a more pronounced cuboidal phenotype characteristic of epithelial cells compared to the spindle shape found in untreated control cells. Arrows indicate the presence of a large number of adipocytes.

[0125] Figure 17 shows Oil Red O staining of cells treated with MTΔECad. Under these culture conditions, adipocyte formation was found in stimulated cells after stimulation with a combination of indomethacin / PPS and pioglitazone / PPS. Pioglitazone alone can stimulate adipocyte formation at low levels. Fig.17a : control; Fig.17b : Indomethacin + Pentosan; Fig.17c : Pioglitazone; Fig.17d : Pioglitazone + pentosan.

[0126] EpCAM gene expression

[0127] The present inventors investigated the effect of treating MTΔECad cells with different compounds and combinations on the expression of EpCAM as an epithelial cell marker and adiponectin as an adipocyte marker.

[0128] qPCR

[0129] When measuring gene expression, each step was performed using real-time PCR.

[0130] First, RNA was isolated and characterized for quantity and integrity. During the two-step reaction, cDNA was first synthesized and then used as a PCR template. The results were standardized using the housekeeping gene GAPDH. Each sample was calculated using a standardization factor. Fluorescence data was divided by its standardization factor to obtain standardized data, which was then subjected to statistical analysis.

[0131] result

[0132] EpCam

[0133] Indomethacin and pioglitazone significantly stimulate EpCAM expression. PPS itself has no significant effect. However, when PPS is combined with indomethacin or pioglitazone, a synergistic effect occurs. 3T3-L1 pre-adipogenic cells that are not stimulated or stimulated for adipocytes (treatment A) are used as controls because they are mesenchymal cells and therefore do not express EpCAM, data not shown. Cells treated as positive controls (treatment B) show stimulated EpCAM gene expression.

[0134] Cells treated with MTΔECad showed a synergistic upregulation of EpCam gene expression after stimulation with a combination of indomethacin / PPS or pioglitazone / PPS. This suggests that cells treated with MTΔECad express more epithelial features, as EpCam is expressed only on epithelial cells and not on mesenchymal cells.

[0135] Adiponectin

[0136] Cells treated with MTΔECad displayed a synergistic upregulation of adiponectin gene expression after stimulation with a combination of indomethacin / PPS or pioglitazone / PPS, indicating that the treated cells expressed adipocyte characteristics since adiponectin expression was restricted to adipocytes.

[0137] 3T3-L1 pre-adipogenic cells were stimulated for adipocytes (treatment A) and used as a control, adiponectin expression was upregulated after stimulation, data not shown.

[0138] Indomethacin and pioglitazone significantly stimulated adiponectin gene expression and showed a clear synergistic effect after stimulation with a combination of PPS and indomethacin or pioglitazone. PPS itself had no effect on adiponectin gene expression.

[0139] Cells treated as a positive control (treatment B) showed stimulated adiponectin gene expression. Fig.18a , 18b .

[0140] in conclusion

[0141] Both indomethacin and pioglitazone in combination with pentosan polysulfate (PPS) enable MTΔECad cells (a type of breast cancer cell) to terminally differentiate into adipocytes, but at the same time they can induce MET (mesenchymal to epithelial transition). This is confirmed by the presence of adipocytes (such as cells that also express adiponectin) containing Nile red-positive lipid droplets in the culture as measured by qPCR. However, cuboidal non-adipocytes can be found at the same time, indicating the presence of epithelial cells, which can be confirmed by the fact that the expression of the epithelial marker EpCAM is also found using qPCR.

[0142] Figure 7a , 7b , 8, 9a, 9b, 10, 11a, 11b, 12, 13a, 13b and 14 show experimental results of the claimed compositions and their synergistic effects. Figure 7a , 7b Demonstrate the effect of compounds on perilipin-1 gene expression in 3T3-L1 cells; Figure 7a to demonstrate the effects of individual compounds, and Figure 7b The effects of compounds stimulated in the presence of insulin / dexamethasone are shown in FIG. Figure 8 The effects of compounds on perilipin-1 gene expression in KS-483 cells are shown. Figure 9a , 9b Demonstrate the effect of compounds on FABP4 gene expression in 3T3-L1 cells; Figure 9a to demonstrate the effects of individual compounds, and Figure 9b The effects of compounds stimulated in the presence of insulin / dexamethasone are shown in FIG. Fig.10 The effects of compounds on FABP4 gene expression in KS-483 cells are shown. Fig.11a , 11b Demonstrate the effect of compounds on CD-36 gene expression in 3T3-L1 cells; Fig.11a to demonstrate the effects of individual compounds, and Fig.11b The effects of compounds stimulated in the presence of insulin / dexamethasone are shown in FIG. Fig.12 The effects of compounds on CD-36 gene expression in KS-483 cells are shown. Fig.13a , 13b Demonstrate the effect of compounds on adiponectin gene expression in 3T3-L1 cells; Fig.13ato demonstrate the effects of individual compounds, and Fig.13b The effects of compounds stimulated in the presence of insulin / dexamethasone are shown in FIG. Fig.14 The effects of compounds on adiponectin gene expression in KS-483 cells are shown.

Claims

1. A pharmaceutical composition for preventing cancer metastasis or stopping ongoing cancer metastasis, comprising (i) a first compound with a molecular weight <10 kDa and (ii) a second compound, wherein the first compound is pentosan sulfate and / or bovine blood plasma; and the second compound is indomethacin and / or pioglitazone; wherein the cancer is breast cancer.

2. The pharmaceutical composition for preventing cancer metastasis or inhibiting ongoing cancer metastasis according to claim 1, wherein the first compound and the second compound are provided in a molar ratio of 0.01:1 to 1:0.

01.

3. The pharmaceutical composition for preventing cancer metastasis or inhibiting ongoing cancer metastasis according to claim 1, wherein the composition further comprises (iii) at least one pharmaceutically acceptable carrier.

4. The pharmaceutical composition for preventing cancer metastasis or inhibiting ongoing cancer metastasis according to claim 1, wherein the active ingredients are the first compound and the second compound, and the active ingredients are prepared into a dosage form.

5. The pharmaceutical composition for preventing cancer metastasis or inhibiting ongoing cancer metastasis according to claim 4, wherein the dosage form comprises 1-10 mg of active ingredient per kg body weight.

6. The pharmaceutical composition for preventing cancer metastasis or inhibiting ongoing cancer metastasis according to claim 4, wherein the dosage form comprises 2-1000 mg of the active ingredient.

7. The pharmaceutical composition for preventing cancer metastasis or inhibiting ongoing cancer metastasis according to claim 1, wherein the pharmaceutical composition comprises independent dosage forms made with independent active ingredients.

8. The pharmaceutical composition for preventing cancer metastasis or inhibiting ongoing cancer metastasis according to claim 1, which is in the form of tablets, capsules or injectables.

9. The pharmaceutical composition for preventing cancer metastasis or inhibiting ongoing cancer metastasis according to claim 1, wherein the composition is in the form of a sustained-release agent.

10. The pharmaceutical composition for preventing cancer metastasis or inhibiting ongoing cancer metastasis according to claim 1, wherein the composition is in the form of a tablet or capsule suitable for oral administration.

11. The pharmaceutical composition for preventing cancer metastasis or inhibiting ongoing cancer metastasis according to claim 1, which is formulated into a form that allows targeting to specific cells, tissues or organs, in the form of nanoparticles, depending on its use.

12. The pharmaceutical composition for preventing cancer metastasis or inhibiting ongoing cancer metastasis according to claim 1, which is used as a medicament by administering an effective amount of the medicament to cancer cells with metastatic potential for a sufficient period of time.

13. The pharmaceutical composition for preventing cancer metastasis or inhibiting ongoing cancer metastasis according to claim 12, wherein the composition is administered to a pet or a mammal, wherein the mammal is a human suffering from a cancer with metastatic potential.

14. The pharmaceutical composition according to any one of claims 1 to 13, for use as a medicament for preventing metastasis or arresting ongoing metastasis.

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