WO2025021972A1 - A marine bacterial exopolysaccharide derivative with anticancer properties and uses thereof - Google Patents

Abstract

The invention provides a 50-60 kDa over-sulfated exopolysaccharide prepared from a marine native exopolysaccharide, named infernan, excreted by a mesophilic marine bacterium from a deep-sea hydrothermal environment (an Alteromonas infernus species of the Alteromonas genus), and relates to the use of the 50-60 kDa over-sulfated exopolysaccharide for the prevention or treatment of cancer. The invention also provides a drug combination comprising the 50-60 kDa over-sulfated exopolysaccharide and an anticancer agent, such as doxorubicin, for use in the prevention or treatment of cancer.

Description

A Marine Bacterial Exopolysaccharide Derivative with Anticancer Properties and Uses Thereof

Related Application

The present application claims priority to European Patent Application No. EP 23 188 267 filed on July 27, 2024, which is incorporated herein by reference in its entirety.

Background of the Invention

Cancer is among the leading causes of mortality in developed countries. Current major treatments for cancer management include surgery, cytotoxic chemotherapy, targeted therapy, radiation therapy, endocrine therapy, and immunotherapy. Despite the endeavors and achievements made in treating cancers during the past decades, disease recurrence and progression remain a major obstacle to therapy. One of the main clinical issues is the development of drug resistance. Drug resistance exists in two forms: acquired resistance, where the drug is initially efficient but becomes ineffective over time; and intrinsic resistance, which occurs when a drug is ineffective from the beginning of treatment. Many strategies have been designed to combat drug resistance, either by combining the currently available therapies or by developing novel therapies. While the focus is shifting to the development and application of novel therapeutic agents for immunotherapy and targeted therapy, chemotherapy is still standard-of-care in the treatment of most cancers and new and effective chemotherapeutic agents are still needed.

Carbohydrates, and especially heparin or heparan sulfate, are now considered as good candidates to treat cancers. However, their therapeutic use is limited because they exhibit significant anticoagulant activity and therefore, they can induce adverse bleeding complications. Another disadvantage of heparin and heparan sulfate is their animal origin, which can result in a high risk of unknown cross-species contamination (Stevenson et al., Research, 2007, 120: S107-S 111; Velasco et al., Drug Discov. Today, 2010, 15: 553-560). Consequently, the exploration of the therapeutic potential of heparin and heparan sulfate mimetics is booming. These mimetics are often less anticoagulant than heparin and heparan sulfate but they maintain some of their benefits in suppressing cancer growth and metastasis through their weak anticoagulant activity by inhibiting thrombin generation, fibrin formation but also heparanase (enzyme involved in heparan sulfate metabolism and turnover) (Sasisekharan et al., Nat. Rev. Cancer, 2002, 2: 521-528; Bobek and Kovarik, Biomedicine & Pharmacotherapy, 2004, 58: 213-219). These mimetics via a multi-target mechanism of action have inhibitory effects on heparinase, selectins, growth factor receptor signaling with limited side effects. Sulfated oligosaccharides have been studied, such as a sulfated form of phosphomannopentaose and phosphomannotetraose named PI-88 but also a sulfated tetrasaccharide derivative named PG545 (Lanzi & Cassinelli, Molecules, 2018, 23(11): 2915; Ferro et al., Carbohydr. Res., 2001, 332: 183-189); and a sulfated form of maltohexose and sulfated maltotriose (Vismara et al., Molecules, 2012, 17: 9912-9930). Two polysaccharides extracted from Prunella vulgaris L. have also been described for their anti-lung adenocarcinoma activity (Feng et al., Molecules, 2010, 15: 5096-5103).

In recent years, there has been a growing interest in the isolation and identification of new microbial polysaccharides that might have new applications in diverse industries. They compete with polysaccharides from other sources such as seaweeds, crustaceans, animals or plants. Interest in mass culture of microorganisms from the marine environment has increased considerably, representing an innovative approach to the biotechnological use of under-exploited resources. When they are sulfated, polysaccharides from different sources can share some biological properties with glycosaminoglycans (GAGs), and especially heparan sulfate or heparin, without exhibiting the same bleeding risks and with a low risk of contamination by a non- conventional transmissible agent such as prions or emerging viruses due to a large “species-barrier” (DeAngelis, Appl. Microbiol. Biotechnol., 2012, 94: 295-305).

Marine bacteria associated with deep-sea hydrothermal conditions have demonstrated their ability to produce, in an aerobic carbohydrate-based medium, unusual extracellular polymers. They present original structural features that can be modified to design bioactive compounds and improve their specificity (Rehm et al. , Rev. Microbiol., 2010, 8: 578-592; Colliec-Jouault et al., Handbook of Exp. Pharmacol., 2012, 423-449). In particular, with the aim of promoting biological activities, chemical modifications (depolymerization and substitution reactions) of an exopolysaccharide (GY785 EPS) and recently named infeman (Colliec-Jouault et al., Biomacromolecule, 2023, 24: 462-470) produced by a deep-sea hydrothermal bacterial named A Iteromonas infernus have been undertaken. The structure of the native infeman EPS has been previously described as a monosulfated nonasaccharide (Roger et al., Carbohydr. Res., 2004, 339: 2371-2380) and recently a slight variation of the infernan repeating unit was described as a disulfated octasaccharide presenting roughly the same monosaccharide composition and assembly (Colliec-Jouault et al., Biomacromolecule, 2023, 24: 462- 470), suggestion that during its biosynthesis two types of sugar assembly can occur. A low molecular weight (LMW) over-sulfated exopolysaccharide (OS-EPS) of 24 kDa has been isolated after chemical modifications of this native infernan EPS. This LMW derivative was found to be less efficient (10 fold) than heparin in clotting assays. In activated partial thromboplastin time, the same anticoagulant effect was obtained with a concentration of 10 p.g/ml of 24 kDa OS-EPS and with a concentration of 1.5 p.g/ml of heparin, respectively (Colliec-Jouault et al., Biochim. Biophys. Acta, 2001, 1528: 141- 151). Another LMW OS-EPS of 15-20 kDa (called MAPI) obtained after modification of the marine native EPS from the A Iteromonas infernus strain was found to exhibit anti- metastatic properties (WO 2017/055310).

Summary of the Invention

The present Inventors have shown that a 50-60 kDa over-sulfated polysaccharide, called MAP4 and derived from a marine native exopolysaccharide (EPS), named infeman, excreted by the Alteromonas infernus strain, efficiently reduces cancer cell viability and significantly inhibits cancer cell proliferation in a large variety of human cancer cell lines, including osteosarcoma, lung cancer, breast cancer, melanoma, prostatic carcinoma, and colon cancer cell lines. In in vivo mouse models, MAP4 strongly inhibits tumor growth with no apparent toxicity. At the doses tested, the biological effects of MAP4 were found to be significantly greater than those of MAPI, a 15-20 kDa over-sulfated infernan exopolysaccharide derivative with the same degree of sulfate-group substitution (40%) as MAP4. In addition, the present Inventors have shown that MAP4 in combination with doxorubicin inhibits the viability of cells from osteosarcoma, lung cancer, and colon cancer cell lines to a greater extent than MAP4 alone, while the MAPI /doxorubicin combination does not exhibit such an additive effect.

Accordingly, in a first aspect, the present invention relates to a 50-60 kDa oversulfated exopolysaccharide (EPS) for use in the prevention or treatment of cancer in a subject, wherein said 50-60 kDa over-sulfated EPS is obtained by a method comprising:

(a) a step consisting of free-radical depolymerization of a marine native EPS, named infernan, excreted by a strain of the Alteromonas genus, named Alteromonas infer mis, so as to obtain a depolymerized EPS having a molecular weight of 5,000 to 100,000 g/mol;

(b) a subsequent step consisting of sulfation of the depolymerized EPS to obtain an over- sulfated depolymerized EPS, comprising adding to the depolymerized EPS at least one sulfation agent in an amount sufficient to obtain an oversulfated exopolysaccharide having a degree of sulfate-group substitution of between 35% and 45% by weight relative to the total weight of the over-sulfated depolymerized EPS, preferably about 40% by weight relative to the total weight of the over- sulfated depolymerized EPS; and

(c) a subsequent step consisting of isolating the 50-60 kDa over-sulfated EPS from the over-sulfated depolymerized EPS, wherein the 50-60 kDa over-sulfated EPS has a molecular weight of about 60,000 g/mol, and a polydispersity index lower than 5, preferably comprised between 1,5 and 4, more preferably lower than 2.

In certain embodiments, the step of isolating the 50-60 kDa over-sulfated EPS from the over- sulfated depolymerized EPS is carried out by fractionation, in particular fractionation performed by size exclusion chromatography.

In certain particular embodiments, the 50-60 kDa over-sulfated EPS is a 50-60 kDa over-sulfated EPS having a molecular weight of about 60,000 g/mol, a polydispersity index lower than 2, and a degree of sulfate-group substitution of 40% by weight relative to the total weight of the over-sulfated depolymerized EPS.

In certain preferred embodiments, the subject is a cancer patient. A cancer patient may be suffering from a cancer or may have previously undergone therapy for cancer. When the cancer patient is suffering from a cancer, the cancer patient may be undergoing therapy for cancer.

The cancer of the patient may belong to the group consisting of carcinoma, lymphoma, blastoma, sarcoma, and leukemia. For example, the cancer may belong to the group consisting of bone cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, carcinoma of the sexual and reproductive organs (e.g., prostate cancer, uterine cancer, etc.), Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma.

In certain embodiments, the cancer is a solid malignant tumor. In particular, the solid cancer may be selected from the group consisting of osteosarcoma, non-small cell lung cancer, melanoma, colon cancer and breast cancer. In certain embodiments, the solid cancer is osteosarcoma, non-small cell lung cancer, melanoma or colon cancer.

In certain embodiments, the cancer is a metastatic cancer.

In the same first aspect, the present invention relates to a method for treating cancer in a subject, the method comprising a step of administering to said subject in need thereof a therapeutically effective amount of a 50-60 kDa over-sulfated EPS as defined herein. In particular, the 50-60 kDa over-sulfated EPS may be a 50-60 kDa over-sulfated EPS having a molecular weight of about 60,000 g/mol, a polydispersity index lower than 2, and a degree of sulfate-group substitution of 40% by weight relative to the total weight of the over-sulfated depolymerized EPS. In certain preferred embodiments of the method of prevention or treatment of the invention, the subject is a cancer patient, as described above. In certain preferred embodiments, the cancer to be treated is as described above.

In another aspect, the present invention relates to a drug combination for use in the prevention or treatment of a cancer in a subject, wherein the drug combination comprises a 50-60 kDa over-sulfated EPS as defined herein and an anticancer agent. In particular, the 50-60 kDa over-sulfated EPS may be a 50-60 kDa over-sulfated EPS having a molecular weight of about 60,000 g/mol, a polydispersity index lower than 2, and a degree of sulfate-group substitution of 40% by weight relative to the total weight of the over-sulfated depolymerized EPS. In certain embodiments, the anticancer agent is a topoisomerase II inhibitor, in particular a topoisomerase II poison, for example an intercalating topoisomerase II poison or a non-intercalating topoisomerase II poison. In certain embodiments, the anticancer agent is DNA intercalating agent. In certain preferred embodiments, the DNA intercalating agent belongs to the anthracycline family. In certain most preferred embodiments, the DNA intercalating agent is doxorubicin. The subject to be treated with a drug combination described herein is a cancer patient, as described above. In certain preferred embodiments, the cancer to be prevented or treated is as described above. In particular, the cancer may be a solid malignant tumor selected from the group consisting of osteosarcoma, non-small cell lung cancer, colon cancer and breast cancer.

In yet another embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of a 50-60 kDa over-sulfated EPS as defined herein, or of a drug combination as defined herein, and at least one pharmaceutically acceptable carrier or excipient for use in the prevention or treatment of cancer in a subject. In particular, the 50-60 kDa over-sulfated EPS may be a 50-60 kDa over-sulfated EPS having a molecular weight of about 60,000 g/mol, a polydispersity index lower than 2, and a degree of sulfate-group substitution of 40% by weight relative to the total weight of the over- sulfated depolymerized EPS. In certain preferred embodiments, the subject to be treated with a pharmaceutical composition described herein is a cancer patient, as described above. In certain preferred embodiments, the cancer to be prevented or treated is as described above.

These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments. Brief Description of the Figures

Figure 1: Results of MTT tests on the proliferation of the MNNG-HOS human osteosarcoma cell line cultured in the presence of different infeman derivatives and control compounds.

Figure 2. Results of MTT tests on the proliferation of the A549 human non-small cell lung cancer cell line cultured in the presence of different infeman derivatives and control compounds.

Figure 3. Results of MTT tests on the proliferation of the MDA-MB-231 human breast carcinoma cell line cultured in the presence of different infernan derivatives and control compounds.

Figure 4. Results of MTT tests on the proliferation of the A375 human melanoma cell line cultured in the presence of different infeman derivatives and control compounds.

Figure 5. Results of MTT tests on the proliferation of the CaCO2 human colon cancer cell line cultured in the presence of different infernan derivatives and control compounds.

Figure 6. Results of MTT tests on the proliferation of cells of the DU145 human prostatic cancer cell line cultured in the presence of different infeman derivatives and control compounds.

Figure 7. Effect of different infeman derivatives and of control compounds on the proliferation of the A375 human melanoma cell line by xCELLigence.

Figure 8. Effects of Heparin, MAPI and MAP4, alone or in combination with doxorubicin on the viability of cells from (A) the MNGG-HOS human osteosarcoma cell line, and (B) the Caco2 human colon cancer cell line.

Figure 9. (A) Concentration of MAP4 in the plasma of male mice following i.v. administration of the infeman derivative at a dose of 10 mg/kg. (B) Concentration of MAP4 in the plasma of male mice following s.c. administration of the infernan derivative at a dose of 30 mg/kg. (C) Mean plasma anti-Xa (Ul/ml) activity after i.v. injection of MAP4 at 10 mg/kg in mice. (D) Mean plasma anti-Xa (UVml) activity after s.c. injection of MAP4 at 30 mg/kg in mice (error bar represents SD). Figure 10. Effects of MAPI and MAP4 on tumor growth of (A) B16/F10 murine melanoma cells and (B) CMT167 murine lung carcinoma cells, injected subcutaneously in C57/BL 6 mice (n=6).

Figure 11. Genes expressed by cells of the A549 human non-small cell lung cancer cell line and modulated by (A) MAPI and (B) MAP4. Total of 794 genes.

Figure 12. Main functional pathways related to the genes most significantly modulated by (A) MAPI and (B) MAP4.

Figure 13. Monitoring of body weight of mice treated with doxorubicin, MAP4 and a combination thereof compared to a vehicle.

Figure 14. Therapeutic efficacy of MAP4 on A549 human lung cancer injected subcutaneously in nude mouse model.

Definitions

As used herein, the term ' subject " refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like), that can develop a cancer, but may or may not be suffering from the disease. Non-human subjects may be transgenic or otherwise modified animals. In many embodiments of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an "individual " or a "patient ". These terms do not denote a particular age, and thus encompass new-borns, children, teenagers, and adults. The term ‘"patient” more specifically refers to an individual suffering from a disease. Thus, the term “cancer patient” refers to an individual suffering from a cancer. A cancer patient may or may not have been diagnosed with cancer. The term also includes individuals who have previously undergone therapy for cancer.

As used herein, the term “cancer” refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth, lack of differentiation and ability to invade local tissues and metastasize. Cancer can develop in any tissue of any organ. Examples of cancers include, but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularly, examples of such cancers include bone cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, rectal cancer, stomach cancer, colon cancer, breast cancer, carcinoma of the sexual and reproductive organs (uterine cancer, ovarian cancer, prostate cancer, etc.), Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma.

The terms ""aggressive” and "invasive” are used herein interchangeably. When used herein to characterize a cancer, they refer to the proclivity of a tumor for expanding beyond its boundaries into adjacent tissue. Invasive cancer can be contrasted with organ- confined cancer wherein the tumor is confined to a particular organ. The invasive property of a tumor is often accompanied by the elaboration of proteolytic enzymes, such as collagenases, which degrade matrix material and basement membrane material to enable the tumor to expand beyond the confines of the capsule, and beyond confines of the particular tissue in which that tumor is located.

The term ' metastasis ' , as used herein, refers to the spread of tumor cells from one organ or tissue to another location. The term also refers to tumor tissue that forms in a new location as a result of metastasis. A “metastatic cancer” is a cancer that spreads from its original, or primary, location, and may also be referred to as a “secondary cancer” or “secondary tumor”. Generally, metastatic tumors are named for the tissue of the primary tumor from which they originate. The process of tumor metastasis is a multistage event involving local invasion and destruction of intercellular matrix, intravasation into blood vessels, lymphatics or other channels of transport, survival in the circulation, extravasation out of the vessels in the secondary site and growth in the new location.

As used herein, the term "inhibit " means to prevent something from happening, to delay occurrence of something happening, and/or to reduce the extent or likelihood of something happening. Thus, the terms “inhibiting metastasis”, “inhibiting metastases” and “inhibiting the formation of metastases”, which are used herein interchangeably, are intended to encompass preventing, delaying, and/or reducing the likelihood of occurrence of metastases as well as reducing the number, growth rate, size, etc. of metastases.

The term ' treatment " is used herein to characterize a method or process that is aimed at (1) delaying or preventing the onset of a disease or condition (here a cancer); (2) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease or condition; (3) bringing about amelioration of the symptoms of the disease or condition; or (4) curing the disease or condition. A treatment may be administered after initiation of the disease or condition, for a therapeutic action. Alternatively, a treatment may be administered prior to the onset of the disease or condition, for a prophylactic or preventive action. In this case, the term "prevention " may be used.

A “pharmaceutical composition” is defined herein as comprising an effective amount of a 50-60 kDa over-sulfated infeman derivative described herein, and at least one pharmaceutically acceptable carrier or excipient.

As used herein, the term “effective amount” refers to any amount of a compound, agent, or composition that is sufficient to fulfil its intended purpose(s), e.g., a desired biological or medicinal response in a cell, tissue, system or subject.

The term “pharmaceutically acceptable carrier or excipient” refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredient(s), and which is not excessively toxic to the host at the concentration at which it is administered. The term includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and adsorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art (see for example “Remington ’s Pharmaceutical Sciences”, E.W. Martin, 18^th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety).

The terms “approximately” and “about”, as used herein in reference to a number, generally include numbers that fall within a range of 10% in either direction of the number (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Detailed Description of Certain Preferred Embodiments

As mentioned above, the present invention provides 50-60 kDa over-sulfated derivatives derived from the native infeman EPS, that exhibit anticancer properties, and the use of the 50-60 kDa over-sulfated infernan derivatives in the prevention or treatment of cancer.

I - 50-60 kDa Over-Sulfated Infernan Derivatives

The over-sulfated exopolysaccharide (EPS) derivatives used in the present invention are derived from a native EPS, which is excreted by the Alteromonas inf emus strain, a mesophilic marine bacterium from a deep-sea hydrothermal environment of the Alteromonas genus. In recent years, there has been a growing interest in the isolation and identification of new polysaccharides of marine origin. Marine bacterial EPSs and derivatives thereof have some great advantages as therapeutic compounds because they can be produced at viable economic cost, in controlled conditions in agreement with Good Manufacturing Practices and they exhibit a very low risk for patients to be infected by a non-conventional transmissible agent, such as prions or emerging viruses, due to a large “species-barrier”.

Marine bacteria from deep-sea hydrothermal vent environments, belonging to three main genera (Vibrio, Alteromonas and Pseudoalteromonasf have demonstrated their ability to produce unusual extracellular polymers in an aerobic carbohydrate- supplemented medium. For example, the strain GY785 named A Iteromonas inf emus, was isolated from a sample of fluid collected among a dense population of Riftia pachyptila, in the vicinity of an active hydrothermal vent of the Southern depression of the Guaymas basin (Gulf of California) (Raguenes et al., J. Appl. Microbiol., 1997, 82(4): 422-430); and the native EPS excreted by the strain GY785 has been described (Guezennec et al., Carbohydr. Polym., 1998, 37: 19-24).

Processes for obtaining lower-molecular-weight over-sulfated polysaccharide derivatives from the marine native EPS according to the invention are fully described in the international application WO 2006/003290, and also by Colliec Jouault S. et al. in Biochim. Biophys. Acta 2001, 1528(2-3): p.141-151, and by Guezennec J. et al. in Carbohydrate Polymers 1998, 37: 19-24. In the practice of the present invention, a 50-60 kDa over-sulfated EPS is prepared using a method comprising:

(a) a step consisting of free-radical depolymerization of a marine native EPS, named infernan, excreted by a strain of the Alteromonas genus, named Alteromonas infer mis, so as to obtain a depolymerized EPS having a molecular weight of 5,000 to 100,000 g/mol;

(b) a subsequent step consisting of sulfation of the depolymerized EPS to obtain an over-sulfated depolymerized EPS, comprising adding to the depolymerized EPS at least one sulfation agent in an amount sufficient to obtain an over-sulfated polysaccharide having a degree of sulfate-group substitution of between 35% and 45% by weight relative to the total weight of the over- sulfated depolymerized EPS, preferably about 40% by weight relative to the total weight of the over-sulfated depolymerized EPS; and

(c) a subsequent step consisting of isolating the 50-60 kDa over-sulfated EPS from the over-sulfated depolymerized EPS, wherein the 50-60 kDa over-sulfated EPS has a molecular weight of about 60,000 g/mol, and a poly dispersity index lower than 5, preferably comprised between 1,5 and 4, more preferably lower than 2.

In certain embodiments, the depolymerized EPS obtained at the end of step (a) is lyophilized.

In certain embodiments, step (b) of the process is followed by a dialysis step.

During the free-radical depolymerization step (step a)), the native infernan EPS can be used in a liquid form, i.e. , as it is excreted by the bacteria into the culture medium. Preferably, the culture medium is centrifuged and only the supernatant containing the native infernan EPS and that is free of bacterial debris is collected. The native infernan EPS can be collected by any suitable technique known to those skilled in the art, such as for example membrane ultrafiltration. Optionally, the native infernan EPS can then optionally be lyophilized as is or in the form of an addition salt.

The step consisting of free -radical depolymerization (step (a)) of the native infernan EPS is preferably carried out by adding a solution of an oxidizing agent to a reaction mixture comprising the native infernan EPS, preferably in the presence of a metal catalyst. The oxidizing agent is preferably chosen from peroxides, in particular hydrogen peroxide, and peracids, especially peracetic acid and 3 -chloroperbenzoic acid. The addition is preferably carried out continuously and with stirring for a period of between 30 minutes and 10 hours. The reaction mixture is preferably maintained at a pH of between 6 and 8, for example by addition of a basifying agent such as sodium hydroxide, and at a temperature of between approximately 30°C and 70°C throughout the duration of the free-radical depolymerization reaction.

According to a specific embodiment of the present invention, in this step, the native inf eman EPS is present in the reaction mixture at a concentration of between about 2 mg/ml and about 10 mg/ml of reaction mixture.

In preferred embodiments, the oxidizing agent is a solution of hydrogen peroxide (H2O2) preferably having a concentration of between about 0.1% and about 0.5% by weight, preferably of the order of 0.1% to 0.2% by weight, and is added at a flow rate of VI/1000 to VI/10 mL/minute, preferably VI/50 and VI/500 mL/minute, and more preferably of the order of VI/100 mL/minute, wherein VI is the volume of the reaction medium containing a marine exopolysaccharide (EPS) to which a solution of hydrogen peroxide is added.

The metal catalysts that can be used during the free-radical depolymerization step are preferably chosen from Cu²⁺, Fe²⁺ and Cr³⁺ions and the CnO?²’ anion, as described in particular in patent application EP 0 221 977. According to a specific embodiment, the metal catalyst is present in the reaction mixture at a concentration of between about 10'³ M and about 10'¹ M, and preferably at a concentration of between about 0.001 M and about 0.05 M.

The free -radical depolymerization process according to the invention and as described above makes it possible to obtain, in a single step and with a good yield, homogeneous, lower-molecular-weight polysaccharide derivatives (5,000 to 100,000 g/mol). In the context of the present invention, the term “homogeneous derivatives” is intended to mean derivatives which, when assessed using high performance size exclusion chromatography, exhibit a single main peak representing a predominant population of polysaccharide chains that are homogeneous with respect to size, characterized by a polydispersity index (I) < 5. The polydispersity index (I), which is a measure of the molecular mass of the derivatives, is calculated as the weight average molecular weight (Mw) divided by the number average molecular weight (Mn).

In certain embodiments, when the depolymerization reaction is over, the depolymerized EPSs obtained are reduced using a reducing agent, so as to stabilize the chains, the reducing ends of which are very reactive, and in particular to avoid chain hydrolysis by the “peeling” reaction. The nature of the reducing agents that can be used to this effect is not essential. In particular, the reducing agent may be sodium borohydride.

The metal catalyst used in the free -radical depolymerization step can be eliminated at the end of the depolymerization reaction, using any suitable method, for example by ion exchange chromatography, preferably a weak cation exchange resin passivated beforehand, or by treatment with EDTA (ethylenediaminetetraacetic acid).

The depolymerized EPSs resulting from the free-radical depolymerization and/or from the reduction step can, if necessary, be recovered using any suitable technique well known to those skilled in the art, such as, for example, by membrane ultrafiltration or dialysis. Then, they are lyophilized and fractionated by size exclusion chromatography to increase their purity, which is required to improve the subsequent sulfation step. Finally, the purified depolymerized EPSs are conditioned in salt form by addition of a weak or strong base that may be chosen, for example, from pyridine, triethylamine, tributylamine, tetrabutylammonium hydroxide and sodium hydroxide. This lyophilized salt may be prepared, for example, by elution of an aqueous solution of the polysaccharide derivatives at a concentration of between 1 and 8 mg/ml on an ion exchange resin column such as, for example, those sold under the name DOWEX® by the company Dow Chemical. The eluate is collected as long as the pH remains acid, for example less than 5, then the pH is subsequently adjusted to approximately 6.5 with the desired base as defined above. The EPS derivatives in the form of a salt are then ultrafiltered and lyophilized.

The lyophilized EPS derivatives, possibly in the form of an addition salt, are preferably dissolved in an anhydrous solvent at the beginning of the sulfation step (step (b)). The solvent is preferably chosen from dimethylformamide (DMF), dimethyl sulfoxide (DMSO) formamide, and mixtures thereof. The concentration of EPS derivatives present in the anhydrous solvent may be between approximately 1 and 10 mg/mL, preferably between about 1 mg/mL and about 5 mg/mL, and even more preferably this amount is about 2.5 mg/mL. The dissolution of the EPS in the anhydrous solvent is preferably conducted, with stirring, at ambient temperature for about 1 hour to about 2 hours and then at a temperature of between 40°C and 50°C, preferably at a temperature of about 45 °C for about 2 hours under argon or azote with molecular sieves.

The one or more chemical sulfation agents used during the sulfation step can be added to the depolymerized and/or reduced EPSs that are in lyophilized form or in the form of a solution.

The sulfation agents are preferably chosen from complexes of pyridine sulfate (free or coupled to a polymer), of dimethylformamide sulfate, triethylamine sulfate and of trimethylamine sulfate. The one or more chemical sulfation agents are added to the solution of EPS derivatives in a weight amount preferably representing from about 4 to about 6 times, and even more preferably about 5 times, the mass of EPS derivatives in solution. The chemical sulfation reaction is then preferably carried out with stirring for a period of between 2 and 24 hours depending on the desired degree of sulfation. When the desired degree of sulfation is reached, the sulfation reaction is stopped after cooling of the reaction medium: either by precipitation in the presence of sodium chloride/saturated acetone or of methanol, and then dissolution of the precipitate in water; or, preferably, by addition of water in a proportion preferably equal to 1/10 of the reaction volume and adjustment of the pH of the reaction medium to 9 with a basifying agent such as, for example, sodium hydroxide (3 M).

In the context of the present invention, the chemical sulfation reaction is continued until the degree of sulfation (or degree of sulfate-group substitution) reaches a value comprised between 35% and 45% by weight relative to the total weight of the oversulfated depolymerized EPS, for example about 35%, or about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45% by weight relative to the total weight of the over-sulfated depolymerized EPS. In certain embodiments, the chemical sulfation reaction is continued until the degree of sulfation reaches a value of about 40% by weight relative to the total weight of the over- sulfated depolymerized EPS, i.e., about 38%, about 39%, about 40%, about 41%, or about 42% by weight relative to the total weight of the over-sulfated depolymerized EPS. In certain embodiments, the chemical sulfation reaction is continued until the degree of sulfation reaches a value of 40% by weight relative to the total weight of the oversulfated depolymerized EPS.

According to certain embodiments, the solution of over-sulfated EPS derivatives is preferably dialyzed in order to remove the various salts, and then lyophilized. The final product (the 60kDa over-sulfated EPS), typically with an accurate molecular weight and a low poly dispersity index, is obtained by isolation from the over- sulfated depolymerized EPS obtained in step (b). Isolation (step (c)) may be performed by any suitable method known in the art. Preferably, isolation is carried out by fractionation performed by size exclusion chromatography.

The 50-60 kDa over-sulfated EPS (called MAP4) obtained after isolation (step (c)), has a low polydispersity index of less than 5, preferably a polydispersity index comprised betweenl.5 to 4, more preferably a polydispersity index of less than 2. The term “50-60 kDa”, as used herein to characterize an over-sulfated EPS, refers to the molecular weight of the over-sulfated EPS being comprised between about 50,000 g/mol (i.e., 50 kDa) and about 60,000 g/mol i.e., 60 kDa). Thus, for example, the molecular weight of a 50-60 kDa over-sulfated EPS may be about 50,000 g/mol, about 51,000 g/mol, about 52,000 g/mol, about 53,000 g/mol, about 54,000 g/mol, about 55,000 g/mol, about 56,000 g/mol, about 57,000 g/mol, about 58,000 g/mol, about 59,000 g/mol, or about 60,000 g/mol. In certain particular embodiments, the 50-60 kDa over-sulfated EPS is an over-sulfated EPS, as defined herein, having a molecular weight of about 60,000 g/mol, a polydispersity index lower than 2, and a degree of sulfate-group substitution of 40% by weight relative to the total weight of the over-sulfated depolymerized EPS.

II - Drug Combinations

In another aspect, the present invention relates to a drug combination for use in the prevention or treatment of a cancer in a subject, wherein the drug combination comprises, or consists of, a 50-60 kDa over-sulfated EPS as described herein and an anticancer agent. In certain preferred embodiments, the 50-60 kDa over-sulfated EPS present in a drug combination according to the invention is an over- sulfated EPS, as defined herein, having a molecular weight of about 60,000 g/mol, a polydispersity index lower than 2, and a degree of sulfate-group substitution of 40% by weight relative to the total weight of the over-sulfated depolymerized EPS.

As used herein, the term ' anticancer agent " refers to a molecule capable of inhibiting cancer cell function. The agent may inhibit proliferation or may be cytotoxic to cells. A variety of anticancer agents can be used and include those that inhibit protein synthesis and those that inhibit expression of certain genes essential for cellular growth or survival. Anticancer agents include those that result in cell death and those that inhibit cell growth, proliferation and/or differentiation.

In certain embodiments, the anticancer agent in a drug combination according to the present invention is doxorubicin. Doxorubicin is an antibiotic derived from the Streptomyces peucetius bacterium. It is sold under the brand name ADRIAMYCIN® among others. It is part of the anthracycline group of chemotherapeutic agents. Doxorubicin may be used to treat soft tissue and bone sarcomas and cancers of the breast, ovary, bladder, and thyroid. It is also used to treat acute lymphoblastic leukemia, acute myeloblastic leukemia, Hodgkin lymphoma, and small cell lung cancer. The primary mechanism of action of doxorubicin involves the drug’s ability to intercalate within DNA base pairs, causing breakage of DNA strands and inhibition of both DNA and RNA synthesis. Doxorubicin inhibits the enzyme topoisomerase II, causing DNA damage and induction of apoptosis.

In certain embodiments, the anticancer agent in a drug combination according to the present invention is, like doxorubicin, an anthracycline. Anthracyclines are a class of chemotherapy drugs that are extracted from Streptomyces bacterium. Examples of anthracyclines include, but are not limited to, daunorubicin (a doxorubicin precursor), epirubicin (a doxorubicin stereoisomer), and idarubicin (a daunorubicin derivative), which are, with doxorubicin, the most clinically important anthracyclines. Other clinically used drugs in the anthracycline group are pirarubicin and aclarubicin (also known as aclacinomycin A).

In certain embodiments, the anticancer agent in a drug combination according to the present invention is, like doxorubicin, a DNA intercalating agent. Such agents interact with DNA through intercalation, which can be defined as the process by which compounds containing planar aromatic or heteroaromatic ring systems are inserted between adjacent base pairs perpendicularly to the axis of the helix and without disturbing the overall stacking pattern due to Watson-Crick hydrogen bonding. Examples of DNA intercalators used in the treatment of cancer include anthracyclines (see above), mitoxantrone, and dactinomycin. Mitoxantrone is an anthracycline derivative that is mainly used in the treatment of acute myeloid leukemia. Dactinomycin (or actinomycin D) is a member of the actinomycin family of compounds, which was isolated from several Streptomyces strains. It is used in the treatment of Wilms tumor, rhabdomyosarcoma, Ewing’s sarcoma, trophoblastic neoplasm, testicular cancer, and certain types of ovarian cancer.

In certain embodiments, the anticancer agent in a drug combination according to the present invention is, like doxorubicin, a topoisomerase II inhibitor. Topoisomerase II forms a homodimer that functions by cleaving double stranded DNA, winding a second DNA duplex through the gap, and re-ligating the strands. Topoisomerase II is necessary for cell proliferation and is abundant in cancer cells, which make topoisomerase II inhibitors effective anti-cancer treatments. Topoisomerase II inhibitors are divided into two main classes: poisons and catalytic inhibitors. An anticancer agent in a drug combination according to the present invention is preferably, like doxorubicin, a topoisomerase II poison. Topoisomerase II poisons are characterized by their ability to create covalent bonds with DNA. Topoisomerase II poisons are divided into two groups: intercalating and non-intercalating poisons. Thus, the anticancer agent in a drug combination according to the present invention may be, an intercalating topoisomerase II poison or a non-intercalating topoisomerase II poison. Intercalating topoisomerase II poisons are mainly found in the anthracycline family (see above). Examples of nonintercalating topoisomerase II poisons include etoposide (which is used in the treatment of small cell lung cancer, testicular carcinoma and malignant lymphoma, non-Hodgkin’s lymphoma) and teniposide (which is used in leukemia).

In a drug combination according to the present invention, the 50-60 kDa oversulfated EPS and the anticancer agent, as defined above, are generally present in a molar or mass ratio which are sufficient to achieve the desired goal (z.e., a preventive or therapeutic action). One skilled in the art knows how to determine such molar or mass ratios. Thus, for example, a drug combination according to the present invention may comprise the 50-60 kDa over-sulfated EPS and doxorubicin in a mass ratio ranging from 100:0.0001 (w/w) to 100:10 (w/w), for example about 100:0.0001 (w/w), or about 100:0.001 (w/w), or about 100:0.01 (w/w) or about 100:0.01 (w/w) or about 100:1 (w/w) or about 100:10 (w/w).

Ill - Uses of the 50-60 kDa Over-Sulfated Infernan Derivatives

A. Indications

Due to its ability to efficiently reduce cancer cell viability and to significantly inhibit cancer cell proliferation in a large variety of human cancer cell lines, a 50-60 kDa over-sulfated infernan derivative according to the present invention, either alone or in a drug combination described herein, may be used in the prevention or treatment of a cancer in a subject.

A prevention or treatment of a cancer in a subject according to the present invention may be accomplished using a 50-60 kDa over-sulfated EPS or a drug combination thereof, as described herein, or a pharmaceutical composition thereof. These methods generally comprise administration of an effective amount of a 50-60 kDa over-sulfated EPS, as described herein, or of a drug combination thereof or of a pharmaceutical composition thereof, to a subject in need thereof. Administration may be performed using any of the methods known to one skilled in the art. In particular, a 50-60 kDa over-sulfated EPS, as described herein, or a drug combination or pharmaceutical composition thereof, may be administered by any of various routes including, but not limited to, aerosol, parenteral, oral or topical route.

Generally, the subject is a cancer patient, preferably a human cancer patient. The cancer patient may be suffering from a cancer or having previously undergone therapy of cancer. The cancer patient suffering from a cancer may be undergoing therapy for cancer.

In the practice of the present invention, the cancer may be any cancer developed in any tissue of any organ. Thus, the cancer may be a carcinoma, lymphoma, blastoma, sarcoma, or leukemia. Examples of cancers include, but are not limited to, bone cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, rectal cancer, stomach cancer, colon cancer, breast cancer, carcinoma of the sexual and reproductive organs (uterine cancer, ovarian cancer, prostate cancer, etc.), Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma.

In certain embodiments, the cancer is a solid malignant tumor. In particular, the solid cancer may be selected from the group consisting of osteosarcoma, non-small cell lung cancer, melanoma, colon cancer and breast cancer.

In certain embodiments, the cancer is metastatic cancer.

In certain embodiments, a 50-60 kDa over-sulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, as described herein, is administered to a patient with advanced cancer and presenting thromboembolic episodes often increased by chemotherapy and by the use of a central venous catheter.

In general, a 50-60 kDa over-sulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, as described herein, will be administered in an effective amount, i.e., an amount that is sufficient to fulfill its intended purpose. The exact amount to be administered will vary from subject to subject, depending on the age, sex, weight and general health condition of the subject to be treated, the desired biological or medical response and the like. In certain embodiments, an effective amount is one that delays or prevents the onset of cancer, and/or one that slows down or stops the progression, aggravation, or deterioration of the symptoms of cancer, and/or one that brings about amelioration of the symptoms of cancer, and/or one that prevents, delays and/or reduces the likelihood of occurrence of metastases formation and/or one that reduces the number, growth rate, size, etc. of metastases if metastases are already present in the subject. The effects of a treatment according to the invention may be monitored using any of the diagnostic assays, tests and procedures known in the art.

In certain embodiments, a 50-60 kDa over-sulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, as described herein, is administered alone in a method of prevention or treatment according to the present invention. In other embodiments, the 50-60 kDa over-sulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, as described herein, is administered in combination with at least one additional therapeutic agent or therapeutic procedure. The 50-60 kDa over-sulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, as described herein, may be administered prior to the therapeutic procedure or administration of the therapeutic agent, concurrently with the therapeutic agent or procedure, and/or following the therapeutic procedure or administration of the therapeutic agent.

Therapeutic agents that may be administered in combination with the 50-60 kDa over-sulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, may be selected among a large variety of biologically active compounds that are known to have a beneficial effect in the treatment of cancer or to a patient in general (e.g., anticancer agents, anti-inflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, antibacterial agents, antibiotics, antioxidants, antiseptic agents, and combinations thereof). Therapeutic procedures that may be performed in combination with administration of the 50-60 kDa over-sulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, include, but are not limited to, surgery, radiotherapy, and the like.

Anticancer agents that may be administered in combination with the 50-60 kDa over-sulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, include drugs conventionally classified in one of the following group: alkylating agents, purine antagonists, pyrimidine antagonists, plant alkaloids, intercalating antibiotics, aromatase inhibitors, anti-metabolites, mitotic inhibitors, growth factor inhibitors, cell cycle inhibitors, topoisomerase inhibitors, biological response modifiers, anti-hormones and anti-androgens. Examples of such anticancer agents include, but are not limited to, BCNU, cisplatin, gemcitabine, hydroxyurea, paclitaxel, temozolomide, topotecan, fluorouracil, vincristine, vinblastine, procarbazine, decarbazine, altretamine, methotrexate, mercaptopurine, thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine, azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin, daunorubicin, dactinomycin, idarubicin, plicamycin, mitomycin, bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminogluthimide, anastrozole, amsacrine, asparaginase, mitoxantrone, mitotane and amifostine.

Other examples of such anticancer agents include therapeutic antibodies used in the treatment of cancer, including, but are not limited to, anti-CD52 antibodies such as alemtuzumab (CAMPATH™), which is used in the treatment of chronic lymphocytic leukemia; anti-VEGF antibodies including bevacizumab (AVASTIN™) which is used in the treatment of colorectal cancer and breast cancer; anti-CD33 antibodies, including gemtuzumab ozogamicin (MYLOTARG™) which is used in the treatment of acute myeloid leukemia; anti-CD20 antibodies including ibritumomab (ZEVALIN™) which is used in the treatment of lymphoma, rituximab (RITUXAN™) which is used in the treatment of Hodgkin lymphoma, tositumomab (BEXXAR™) which is used in the treatment of Hodgkin lymphoma and of ofatumumab (ARZERRA™) which is used in the treatment of chronic lymphocytic leukemia; anti-EGFR antibodies such as cetuximab (ERBITUX™) which is used in the treatment of colorectal cancer, head and neck cancer, and squamous cell carcinoma, and panitumumab (VECTIBEX™) which is used in the treatment of colorectal cancer; anti-Her2 antibodies, including trastuzumab (HERCEPTIN™) which is used in the treatment of breast cancer and stomach cancer; anti-CTLA4 antibodies including Ipilimumab (YERVOY™) which is used in the treatment of melanoma; adnectins; and domain antibodies. Active fragments and fusions of these antibodies will also find use herein.

B. Administration

A 50-60 kDa over-sulfated EPS, or a drug combination thereof, (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients), in a desired dosage can be administered to a subject in need thereof by any suitable route. Various delivery systems are known and can be used in the practice of the present invention, including tablets, capsules, injectable solutions, encapsulation in liposomes, microparticles, microcapsules, etc. Methods of administration include, but are not limited to, dermal, intradermal, intramuscular, intraperitoneal, intralesional, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular, and oral routes. A 50-60 kDa over-sulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, may be administered by any convenient or other appropriate route, for example, by infusion or bolus injection, by adsorption through epithelial or mucocutaneous linings (e.g., oral, mucosa, rectal and intestinal mucosa, etc.). Administration can be systemic or local. Parenteral administration may be directed to a given tissue of the patient, such as by catheterization. As will be appreciated by those of ordinary skill in the art, in embodiments where a 50-60 kDa over-sulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, is administered along with an additional therapeutic agent, the 50-60 kDa over- sulfated EPS, or drug combination thereof, or pharmaceutical composition thereof, and the therapeutic agent may be administered by the same route (e.g., intravenously) or by different routes (e.g., orally, intranasally and intravenously).

C. Dosage

Administration of a 50-60 kDa over-sulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, according to the present invention will be in a dosage such that the amount delivered is effective for the intended purpose. The route of administration, formulation and dosage administered will depend upon the preventive or therapeutic effect desired, the severity of the disorder being treated, the presence of any infection, the age, sex, weight and general health condition of the patient as well as upon the potency, bioavailability and in vivo half-life of the active ingredient, the use (or not) of concomitant therapies, and other clinical factors. These factors are readily determinable by the attending physician in the course of the therapy. Alternatively, or additionally, the dosage to be administered can be determined from studies using animal models. Adjusting the dose to achieve maximal efficacy based on these or other methods is well known in the art and is within the capabilities of trained physicians. As studies are conducted using 50-60 kDa over-sulfated infernan derivatives, further information will emerge regarding the appropriate dosage levels and duration of treatment.

A (preventive or therapeutic) treatment according to the present invention may consist of a single dose or multiple doses. Thus, administration of a 50-60 kDa oversulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, as described herein may be constant for a certain period of time or periodic and at specific intervals, e.g., hourly, daily, weekly (or at some other multiple day interval); monthly, yearly (e.g., in a time release form). Alternatively, the administration may occur at multiple times during a given time period, e.g., two or more times per week, two or more times per month, and the like. The delivery may be continuous delivery for a period of time, e.g., intravenous delivery.

IV - Pharmaceutical Compositions

As mentioned above, the 50-60 kDa over-sulfated EPS, or a drug combination thereof described herein, may be administered per se or as a pharmaceutical composition. Accordingly, the present invention provides pharmaceutical compositions comprising an effective amount of a 50-60 kDa over-sulfated EPS or a drug combination thereof and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the composition further comprises one or more additional biologically active agents.

A 50-60 kDa over-sulfated EPS, or a drug combination thereof, or a pharmaceutical composition thereof, may be administered in any amount and using any route of administration effective for achieving the desired prophylactic or therapeutic effect. The optimal pharmaceutical formulation can be varied depending upon the route of administration and desired dosage. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered active ingredient.

The pharmaceutical compositions of the present invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression “unit dosage form”, as used herein, refers to a physically discrete unit suited as unitary dosages for the patient to be treated. It will be understood, however, that the total daily dosage of the compositions will be decided by the attending physician within the scope of sound medical judgement.

A. Formulation

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 2,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solution or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid may also be used in the preparation of injectable formulations. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Liquid pharmaceutical compositions which are sterile solutions or suspensions can be administered by, for example, intravenous, intramuscular, intraperitoneal or subcutaneous injection. Injection may be via single push or by gradual infusion. Where necessary or desired, the composition may include a local anesthetic to ease pain at the site of injection.

In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the ingredient from subcutaneous or intramuscular injection. Delaying absorption of a parenterally administered active ingredient may be accomplished by dissolving or suspending the ingredient in an oil vehicle. Injectable depot forms are made by forming micro-encapsulated matrices of the active ingredient in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active ingredient to polymer and the nature of the particular polymer employed, the rate of ingredient release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the active ingredient in liposomes or microemulsions which are compatible with body tissues.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, and pressurized compositions. In addition to the 50-60 kDa over-sulfated EPS, or drug combination thereof, the liquid dosage form may contain inert diluents commonly used in the art such as, for example, water or other solvent, solubilising agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cotton seed, ground nut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, suspending agents, preservatives, sweetening, flavouring, and perfuming agents, thickening agents, colors, viscosity regulators, stabilizes or osmo-regulators. Examples of suitable liquid carriers for oral administration include water (potentially containing additives as above, e.g., cellulose derivatives, such as sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols such as glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For pressurized compositions, the liquid carrier can be halogenated hydrocarbon or other pharmaceutically acceptable propellant. For intranasal drug delivery, concentrated solution or spray or liposomal formulation per nostril administration can be used. In such liquid forms, the 50-60 kDa over-sulfated EPS described herein can be associated to absorption enhancers (e.g., nonionic surfactants such as alkylglycosides). For pulmonary administration, the 50-60 kDa over-sulfated EPS described herein can be formulated as aerosol particles or droplets or nano vectors.

Solid dosage forms for oral administration include, for example, capsules, tablets, pills, powders, and granules. In such solid dosage forms, the 50-60 kDa over-sulfated EPS described herein, or a drug combination thereof, may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and one or more of: fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; binders such as, for example, carboxymethylcellulose, alginates, gelatine, polyvinylpyrrolidone, sucrose, and acacia; humectants such as glycerol; disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; wetting agents such as, for example, cetyl alcohol and glycerol monostearate; absorbents such as kaolin and bentonite clay; and lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. Other excipients suitable for solid formulations include surface modifying agents such as non-ionic and anionic surface modifying agents. Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatine capsules using such excipients as lactose or milk sugar as well as high-molecular-weight polyethylene glycols and the like. The solid dosage forms of tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition such that they release the active ingredient(s) only, or preferably, in a certain part of the intestinal tract, optionally, in a delaying manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

In certain embodiments, it may be desirable to administer an inventive composition locally to a specific area. This may be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, by injection, by means of a catheter, by means of suppository, or by means of a skin patch or stent or another implant.

For topical administration, the composition is preferably formulated as a gel, an ointment, a lotion, or a cream which can include carriers such as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, and mineral oil. Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylenemonolaurat (5%) in water, or sodium lauryl sulfate (5%) in water. Other materials such as antioxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.

In addition, in certain instances, it is expected that the inventive compositions may be disposed within transdermal devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the active ingredient(s) by either passive or active release mechanisms. Transdermal administrations include all administration across the surface of the body and the inner linings of bodily passage including epithelial and mucosal tissues. Such administrations may be carried out using the present compositions in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).

Transdermal administration may be accomplished through the use of a transdermal patch containing an active ingredient (i.e., a 50-60 kDa over-sulfated EPS or a drug combination thereof, as described herein) and a carrier that is non-toxic to the skin, and allows the delivery of the ingredient(s) for systemic absorption into the bloodstream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquids or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may be suitable. A variety of occlusive devices may be used to release the active ingredient into the bloodstream such as a semi-permeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient.

Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository’s melting point, and glycerine. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used.

Materials and methods for producing various formulations are known in the art and may be adapted for practicing the subject invention. Suitable formulations can be found, for example, in “Remington’ s Pharmaceutical Sciences”, E.W. Martin, 18^th Ed., 1990, Mack Publishing Co.: Easton, PA.

B. Additional Biologically Active Agents

In certain embodiments, a 50-60 kDa over-sulfated EPS, or a drug combination thereof, is the only active ingredient in a pharmaceutical composition of the present invention. In other embodiments, the pharmaceutical composition further comprises one or more additional biologically active agents. Examples of suitable biologically active agents include, but are not limited to, anticancer agents, anti-inflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, antibacterial agents, antibiotics, antioxidants, antiseptic agents, and combinations thereof. Examples of specific anticancer agents, including anticancer antibody agents, have been listed above.

In such pharmaceutical compositions, the 50-60 kDa over-sulfated EPS, or the drug combination thereof, and the at least one additional biologically active agent may be combined in one or more preparations for simultaneous, separate or sequential administration of the 50-60 kDa over-sulfated EPS, or the drug combination thereof, and the biologically active agent(s). More specifically, an inventive composition may be formulated in such a way that the 50-60 kDa over-sulfated EPS, or the drug combination thereof, and the biologically active agent(s) can be administered together or independently from each other. For example, a 50-60 kDa over-sulfated EPS, or drug combination thereof, and a biologically active agent can be formulated together in a single composition. Alternatively, they may be maintained (e.g., in different compositions and/or containers) and administered separately.

C. Pharmaceutical Packs or Kits

In another aspect, the present invention provides a pharmaceutical pack or kit comprising one or more containers (e.g., vials, ampoules, test tubes, flasks or bottles) containing one or more ingredients of an inventive pharmaceutical composition, allowing administration of a 50-60 kDa over-sulfated EPS, or a drug combination thereof, of the present invention.

Different ingredients of a pharmaceutical pack or kit may be supplied in a solid (e.g., lyophilized) or liquid form. Each ingredient will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Packs or kits according to the invention may include media for the reconstitution of lyophilized ingredients. Individual containers of the kits will preferably be maintained in close confinement for commercial sale.

In certain embodiments, a pack or kit includes one or more additional therapeutic agent(s). Optionally, associated with the container(s) can be a notice or package insert in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The notice of package insert may contain instructions for use of a pharmaceutical composition according to methods of prevention and treatment disclosed herein.

An identifier, e.g., a bar code, radio frequency, ID tags, etc., may be present in or on the kit. The identifier can be used, for example, to uniquely identify the kit for purposes of quality control, inventory control, tracking movement between workstations, etc.

Further aspects and advantages of this invention will be disclosed in the figures and following examples, which should be regarded as illustrative and not limiting the scope of this application.

Examples

The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually performed or that data were actually obtained.

The aim of the study was to analyze the effects of different infernan derivatives on the viability and proliferation of cells of various tumor cell lines and in in vivo murine models. The effects of different infernan derivatives combined to doxorubicin on the tumor cell lines were also investigated, as were the cytotoxicity and pharmacokinetic properties of the exopolysaccharides.

Materials and Methods

Chemicals

The bacterial GY785 exopolysaccharide (EPS), named infernan, was produced, purified and characterized as previously described (Guezennec et al., Carbohydr. Polym., 1998, 37: 19-24). The preparation, purification and characterization of low (LMW) and middle molecular weight (MMW) over-sulfated (OS) EPS derivatives were performed as previously reported (Ruiz Velasco et al., Glycobiology, 2011, 21: 781- 795; WO 2006/003290). Briefly, native high molecular weight (HMW) infernan EPS was depolymerized first using a free-radical depolymerisation process to obtain LMW and MMW derivatives with different molecular weights. LMW and MMW inf eman derivatives, in a pyridinium salt form, were then sulfated in dimethylformamide (DMF) using pyridine sulfate as sulfating agent leading to LMW and MMW OS-EPS derivatives. Molecular weights (Mws) before and after sulfation were determined by HPSEC-MALS and sulfur content (wt% S) by HPAEC chromatography. ATR-FTIR and NMR spectroscopy were used to assess the efficiency of sulfation reaction. Heparin and Dalteparin were purchased from Sigma Aldrich (Saint- Quentin Fallavier, France).

Table 1. GY785 derivatives and control compounds used in the study.

DR : radical depolymerization step. S : chemical sulfation step. Mw: weight average molecular weight. Mn : number average molecular weight. I : polydispersity.

Doxorubicin was purchased from Sigma Aldrich (Saint- Quentin Fallavier, France).

Tumor Cell Lines and Culturing Media

All human tumor cell lines used in the present study were obtained from the American Tissue Cell Collection (ATCC, Molsheim, France). Cells from the MNNG/HOS osteosarcoma cell line, A375 melanoma cell line, and CaCO2 colon cancer cell line were cultured with DMEM 4.5 g/E high glucose, pyruvate, non-glutamine from Gibco (Thermo-Fisher), supplemented with glutamine (Thermo-Fisher) and 5% of fetal bovine serum (FBS, Thermo-Fisher). Cells from the A549 non-small cell lung cancer cell line were cultured with DEMEM/F12 (Sigma Aldrich), supplemented with glutamine (Thermo-Fisher) and 5% of fetal bovine serum (FBS, Thermo-Fisher). Cells from the MDA-MB-231 breast carcinoma cell line were cultured with E-15 media from Gibco (Thermo-Fisher), supplemented with glutamine (Thermo-Fisher) and 5% of fetal bovine serum (FBS, Thermo-Fisher). All experiments were conducted at 37 °C in a humidity- saturated controlled atmosphere and 5% CO2.

MTT Cell Viability Test

Cell viability essay was performed by seeding, in triplicate, 3,000 cells of the indicated cell type par well (25 pL) with 25 p L of culture medium for 4 hours in a 96- well Tissue Culture Plate Flat bottom (Falcon) before adding 50 pL of a GY785 EPS derivative at concentrations of 1, 5, 10, 50, 100, 500 pg/mL and 1 mg/mL.

Each plate was incubated at 37°C in a humidity- saturated controlled atmosphere and 5% CO2 for 72 hours. At three days of treatment, a volume of 10 pL of 5 mg/mL MTT (Sigma- Aldrich) was added and incubated for at least 3 hours at 37 °C and 5% CO2. After that time, the liquid was removed and 200 pL of DMSO were added to each well to dissolve the formed formazan crystals before proceeding to the colorimetric quantification using a multi- well spectrophotometer (Victor 3x from PerkinElmer) at the wavelength of 500-600 nm.

Proliferation Assay

Cell proliferation was analyzed by xCELLigence technology (Agilent). Background was measured by adding 50 pL of corresponding media into an E-Plate view 96 (Chem Agilent). Before the beginning of cell treatment, cells were seeded in triplicate at 5,000 cells per well (50 pL) for 4 hours. Then, 100 pL of the infernan derivatives were added at the concentrations of 100 and 500 pg/mL for MNNG/HOS, A549 and MDA-MB-231 cells, and 25 ad 50 pg/mL for A375 and Caco2 cells. The choice of these concentrations for each particular cell line was determined as a function of the IC50 established by the MTT assay. Proliferation curves were normalized with respect to the time point of drug incorporation. The plate was monitored for 100 hours (for MNNG/HOS, A549 and MDA-MB-231 cells) or 120 hours (for A375 and Caco2 cells) using a RTCA instruments (Agilent and ACEA).

Results

Effects of Different Over-Sulfated Infernan Derivatives on the Viability of Different

Tumor Cell Lines by MTT An MTT essay was used to determine the effect of the four infeman derivatives and two control compounds on the viability of cells of five tumor cell lines. The four infeman derivatives studied were MAPI (Mw: 20 kDa, 40% sulfation), MAP2 (Mw: 60 kDa, 10% sulfation), MAP3 (Mw: 60 kDa, 20% sulfation), and MAP4 (Mw: 60 kDa, 40% sulfation); the two control compounds were: heparin (Mw: 15 kDa, 30% sulfation) and dalteparin (Mw: 5 kDa, 30% sulfation); and the six human tumor cell lines were: MNNG-HOS (osteosarcoma), A549 (lung cancer), MDA MB 231 (breast cancer), A375 (melanoma), Caco2 (colon cancer) and DU145 (prostatic carcinoma). A range of concentrations from 1 pg/mL to 1 mg/mL was evaluated for each molecule.

The results are presented on Figure 1 (for the MNNG-HOS human osteosarcoma cell line), Figure 2 (for the A549 human non-small cell lung cancer cell line), Figure 3 (for the MDA-MB-231 human breast carcinoma cell line), Figure 4 (for the A375 human melanoma cell line), Figure 5 (for the Caco2 human colon cancer cell line), and Figure 6 (for the DU145 human prostatic carcinoma cell line). The results show that MAP4 (a 50-60 kDa over-sulfated EPS according to the present invention) inhibits cell viability in a dose-dependent matter. At equal concentrations, its effects are greater than those of other infeman derivatives and of the controls: heparin and dalteparin.

Effects of Different Over-Sulfated Infeman Derivatives on the Proliferation of Different Tumor Cell Lines by xCELLigence

The xCELLigence technology allows the impact of compounds on cellular proliferation to be monitored dynamically (z.e., in real time).

The tests were conducted using two different concentrations: 25 pg/mL and 50 pg/mL of the four infeman derivatives (MAP2, MAP3, and MAP4). Four human tumor cell lines have been studied: the A549 (lung cancer), A375 (melanoma), Caco2 (colon cancer), and DU145 (prostatic carcinoma) cell lines. The results obtained for the A375 human cell line are presented on Figure 7. For each curve, the data were normalized (normalized cell index) with respect to the time of EPSs treatment.

The results obtained demonstrate the ability of the infeman derivatives to inhibit the proliferation of tumor cell lines. They confirm certain observations made with the MTT method, namely that, at equal concentrations, MAP4 (a 50-60 kDa over-sulfated EPS according to the present invention) inhibits tumor cell proliferation more efficiently than the other EPS derivatives and of the controls: heparin and dalteparin for all the tumor cell lines tested except for the MDA-MD-231 breast cancer cell line for which MAPI, at the highest concentration, was found to reduce proliferation whereas MAP2, MAP3 and MAP4 only have little effect.

Effects of Different Over-Sulfated Infernan Derivatives in Combination with Doxorubicin on the Viability of Different Tumor Cell Lines

An MTT essay was used to evaluate the effect of two infernan derivatives (MAPI and MAP4) and two control compounds (heparin and dalteparin) on the viability of cells of five tumor cell lines (MNNG-HOS (osteosarcoma), A549 (lung cancer), MDA MB 231 (breast cancer), A375 (melanoma) et Caco2 (colon cancer). Each of the infernan derivatives and control compounds, used at a concentration of 100 g/mL, was studied alone and in combination with doxorubicin at different concentrations (10 pg/mL, 1 pg/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL and 0,1 ng/mL).

The results obtained for two cell lines (MNNG-HOS and Caco2) are presented on Figure 8.

With the exception of the A375 human melanoma cell line, the results obtained show that the MAP4/doxorubicin combination inhibits the viability of cells from all the other tumor cell lines tested to a greater extent than the MAP4 or MAPI alone. In contrast, the MAPI /doxorubicin does not exhibit such an additive effect.

Toxicological Studies

The toxicity studies conducted on the infernan derivatives, MAPI and MAP4, using a mouse model demonstrated that the two EPS derivatives show no toxicity at doses of 10 mg/kg and 30 mg/kg. Histopathological analyses confirmed these observations. At very high doses (100 mg/kg), MAPI caused in total, the death of 2 male mice: one on the 5^th day and another on the 6^th day of the study (group of 8 mice). No mortality was observed with MAP4, even at the very high dose of 100 mg/kg.

Pharmacokinetic Study of MAP4

A study to obtain pharmacokinetic data on MAP4 was carried out. Pharmacokinetic data are essential to enable clinical development of a drug candidate. This study was part of a service conducted by an external company, the “Glyco-mix' glycan platform of the University of Paris Est Creteil Vai de Marne.

In brief, the quantification of MAP4 was carried out using a colorimetric assay specific for sulfated molecules of the glycosaminoglycan type. The EPS derivative is detectable and quantifiable with good linearity in an aqueous solution at concentrations from 12.5 to 100 pg/mL and in plasma at concentrations from 6.3 to 100 pg/mL.

Male mice were injected intravenously with a solution of MAP4 at a concentration of 10 mg/kg (30 treated mice + 5 “control” mice). Blood was then collected at different times following the injection: at 15, 60, 120, 180 and 240 minutes (groups of 5 mice per time point), and the concentration of MAP4 in mice plasmas was determined. The results obtained are presented on Figure 9(A). Like heparin, dextran sulfate and other heparinoids, thanks to its high solubility, over-sulfated MAP4 was found to be rapidly eliminated (less than 4 hours) from the body. MAP4 exhibits an anticoagulant activity which can be detected in plasma. Its anticoagulant activity corresponds to its concentration in plasma, and it is no longer detected 4 hours after the i.v. injection.

A pharmacokinetic study was also conducted by s.c. injection of MAP4 at a dose of 30 mg/kg. Blood was collected at different times after the injection: 30, 60, 90, 120, 180, 240, 480 and 1440 minutes (groups of 5 mice per time point + 5 “control” mice). The concentration of MAP4 in the plasma was evaluated at the different time points. Tissues from different organs were also collected and stored at -180°C: liver, spleen, kidney, heart, lung, stomach, muscle and brain. The results obtained are presented on Figure 9(B). Starting at time point 30 minutes, the MAP4 concentration in the plasma increases to reach a maximum at 90 minutes. Then, the MAP4 concentration decreases to reach, at 1440 minutes (24 hours), the concentration observed in the control group suggesting its complete elimination in the blood. The dosage of the anticoagulant activity also makes possible the monitoring and quantification of MAP4, giving an almost identical profile (see Figure 9(C-D)). The conversion of Xa factor activity to lU/ml was performed by considering that 0,19 IU is equivalent to 1 pg of factor Xa. This conversion was performed based on the instructions provided by Diagnostica Stago laboratories that furnished the product used. The pharmacokinetic parameters of MAP4 were calculated for the two different modes of injection tested (i.v. and s.c.) and are presented in Table 2.

Table 2. Pharmacokinetic parameters of MAP4.

Ke: elimination rate constate. Cmax: maximum concentration of the molecule. Tmax: time of the maximum concentration. Tl/2: half-life. AUC: area under the curve corresponding to total drug exposure. AUMC: area under the first-order moment curve of concentration/time. CL: clearance. Vd: volume of distribution. MRT: AUMC/AUC average presence time. F: absolute bioavailability.

For the two modes of injection i.v. and s.c., the maximum concentration (Cmax) of MAP4 in plasma was determined to be 32.8 pg/mL and 30.1 g/mL, respectively. MAP4 was found to quickly reach the Cmax value and is eliminated with a short halflife. MAP4 has a poor tissue distribution, and its clearance is indicative of a poor extraction. It exhibits a high solubility with a high affinity for plasma proteins, which could explain its high bioavailability after s.c. administration in mice.

In vivo Studies of the Effects of MAPI and MAP4 in Murine Models

The effects of MAPI and MAP4 were analyzed in two complementary mouse models. These models consisted in inoculating murine melanoma cells B16F10 (melanoma) and CMT167 (lung carcinoma) subcutaneously in immunocompetent C57/BL6 mice and monitoring the evolution of the tumor volume with a caliper every two days. The weight of the 8 week-old animals was monitored twice a week. MAPI and MAP4 were injected at a dose of 10 mg/kg per day, five times per week and compared with a solution of NaCl. The results, which are presented on Figure 11, show an inhibitory effect of the two infernan derivatives on tumor growth in both models studied, thus demonstrating an effect of the two compounds on the primary tumor. No apparent toxicity was observed and no impact on animal weight was reported. MAP4 was found to show a significantly greater effect than MAPI at the dose tested.

In vitro Functional Analyses

Complementary functional studies of MAPI and MAP4 were carried out in vitro. Cells from the A549 human lung adenocarcinoma cell line were treated or not with 100 .g/mL of MAPI or MAP4 for 24 hours. Cell messenger RNAs were extracted and a differential analysis of 814 genes (NH_Hs_TumorSig_vl.O) was performed by nanostring technology. The experiments were conducted in triplicate.

Out of 794 genes, 25 were significantly modulated by MAPI (see Figure 11(A)) and 37 by MAP4 (see Figure 11(B)).

The bioinformatics analyses (Kegg enrichment) showed that the proteoglycans and the PI3K-Akt signaling pathway are among the most modulated by MAPI and M14 (see Figure 12). These results demonstrate a direct activity of MAPI and MAP4 on the cancer cells studied.

Effects of MAP4, Doxorubicin and Combination Thereof in Human Lung Tumor (A549)-Bearing Nude Mice

After one week of quarantine, 10⁶ A549 cells were injected per female NMRI nude mouse in the right flank via s.c. (50 p.L PBS + 50 p.L matrigel). Mice were then distributed randomly in 4 groups:

Control group (8 mice): NaCl, s.c., 5 times per week;

Doxorubicin group (8 mice): 3 mg/kg, i.v., 3 consecutive days;

Combined group (8 mice): Doxorubicin 3 consecutive days + MAP4 5 times per week;

MAP4 group (8 mice): 4 mg/kg; s.c., 5 times per week.

Three days later, treatment was started, and weight and tumor size of each mouse was evaluated twice per week using a caliper. The experiment was stopped after 1.5 months as ulcers started to appear in control tumors.

Results. The monitoring of mouse body weight did not reveal any side effect of doxorubicin, of MAP4, and of the combination thereof (Figure 13). Interestingly, individual treatment with doxorubicin (P<0.001) and MAP4 (P<0001) resulted in a reduction of proliferation of tumor compared to control group. MAP4 exhibited similar anti-tumor activity compared to doxorubicin. The very significant effect of MAP4 on 4549 tumor progression did not allow to show any significant additive effect of the combined treatment (Figure 14). However, the combined treatment did not exhibit any critical toxicity in treated mice.

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

1. A 50-60 kDa over-sulfated exopolysaccharide derivative for use in the prevention or treatment of cancer in a subject, wherein said 50-60 kDa oversulfated exopolysaccharide derivative is obtained by a method comprising:

(a) a step consisting of free-radical depolymerization of a marine native exopolysaccharide (EPS) from the strain GY785 of the Alteromonas genus so as to obtain a depolymerized EPS having a molecular weight of 5,000 to 100,000 g/mol;

(b) a subsequent step consisting of sulfation of the depolymerized EPS to obtain an over- sulfated depolymerized EPS, comprising adding to the depolymerized EPS at least one sulfation agent in an amount sufficient to obtain an over-sulfated polysaccharide having a degree of sulfategroup substitution of between 35% and 45% by weight relative to the total weight of the over- sulfated depolymerized EPS, preferably about 40% by weight relative to the total weight of the over-sulfated depolymerized EPS; and

(c) a subsequent step consisting of isolating the 50-60 kDa over-sulfated exopolysaccharide from the over-sulfated depolymerized EPS, wherein the 50-60 kDa over-sulfated EPS has a molecular weight of about 60,000 g/mol, and a poly dispersity index lower than 5, preferably comprised between 1,5 and 4, more preferably lower than

2.

2. The 50-60 kDa over-sulfated exopolysaccharide derivative for the use according to claim 1, wherein step (c) is carried out by fractionation, in particular fractionation performed by size exclusion chromatography.

3. The 50-60 kDa over-sulfated exopolysaccharide derivative for the use according to claim 1 or claim 2, wherein the 50-60 kDa over-sulfated exopolysaccharide derivative is a 50-60 kDa over-sulfated EPS having a molecular weight of about 60,000 g/mol, a polydispersity index lower than 2, and a degree of sulfate-group substitution of 40% by weight relative to the total weight of the over-sulfated depolymerized EPS.

4. The 50-60 kDa over-sulfated exopolysaccharide derivative for use according to any one of claims 1 to 3, wherein the subject is a cancer patient.

5. The 50-60 kDa over-sulfated exopolysaccharide derivative for use according to claim 4, wherein the cancer patient is suffering from cancer, or has previously undergone therapy for cancer.

6. The 50-60 kDa over-sulfated exopolysaccharide derivative for use according to claim 5, wherein the cancer patient suffering from a cancer has a thromboembolic disease.

7. The 50-60 kDa over-sulfated exopolysaccharide derivative for use according to any one of claims 1 to 6, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

8. The 50-60 kDa over-sulfated exopolysaccharide derivative for use according to any one of claims 1 to 6, wherein the cancer is bone cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, carcinoma of the sexual and reproductive organs, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, or pituitary adenoma.

9. The 50-60 kDa over-sulfated exopolysaccharide derivative for use according to any one of claims 1 to 6, wherein the cancer is osteosarcoma, non-small cell lung cancer, melanoma, colon cancer or breast cancer.

10. The 50-60 kDa over-sulfated exopolysaccharide derivative for use according to any one of claims 1 to 9, wherein the cancer is metastatic cancer.

11. A drug combination for use in the prevention or treatment of cancer in a subject, wherein the drug combination comprises a 50-60 kDa over-sulfated exopolysaccharide derivative as defined in any one of claims 1 to 3 and an anticancer agent.

12. The drug combination for the use according to claim 11, wherein the anticancer agent is a topoisomerase II inhibitor, in particular a topoisomerase II poison, such as an intercalating topoisomerase II poison or a non-intercalating topoisomerase II poison.

13. The drug combination for the use according to claim 12, wherein the intercalating topoisomerase II poison is a DNA intercalating agent of the anthracycline family.

14. The drug combination for the use according to claim 13, wherein the DNA intercalating agent of the anthracycline family is doxorubicin.

15. A pharmaceutical composition for use in the prevention or treatment of a cancer in a subject, the pharmaceutical composition comprising an effective amount of a 50-60 kDa over-sulfated exopolysaccharide as defined in any one of claims 1 to 3 or of a drug combination as defined in any one of claims 11 to 14, and at least one pharmaceutically acceptable carrier or excipient.