USE OF FUCOSYLATED CHONDROITIN SULFATE , PHARMACEUTICAL COMPOSITION AND METHOD FOR PREVENTION AND/OR TREATMENT OF

CANCER METASTASIS FIELD OF THE INVENTION

The invention concerns the use of sulfated polysaccharides, specifically fucosylated chondroitin sulfates, from holothuroidea, or sea cucumbers, in the preparation of a medicament useful for prevention and/or treatment of cancer metastasis in the animal body.

BACKGROUND

Sulfated polysaccharides constitute a complex group of macromolecules known to possess a wide range of important biological properties. These anionic polymers are widespread in nature, occurring in a great variety of organisms. For instance, in marine algae the carrageenans and fucoidans are composed mainly of sulfated galactose and fucose, respectively. In the animal kingdom, sulfated glycosaminoglycans abound in vertebrate tissues. Invertebrate species are also a rich source of sulfated polysaccharides with novel structures.

Anticoagulant and antithrombotic activities are among the most widely studied properties of sulfated polysaccharides. The anticoagulant glycosaminoglycan heparin is an important therapeutic agent used in the prophylaxis and treatment of thrombosis; dermatan sulfate is also an anticoagulant, although of lower potency than heparin. A chemically sulfated xylan from beechwood, pentosan polysulfate, has been available for many years as an anticoagulant polysaccharide. Sulfated fucans from brown seaweed have anticoagulant activity due to the ability to potentiate inhibition of thrombin by antithrombin or heparin cofactor II.

More recently sulfated polysaccharides from the body wall of sea cucumbers was isolated, and it was found out that the main fraction is comprised of a chondroitin sulfate-like structure, containing large numbers of sulfated alpha-L-fucopyranose branches linked to position 3 of the b-D-glucuronic acid residues. This fucosylated chondroitin sulfate (FCS) was proven to have high anticoagulant properties { Anticoagulant Action of a Fucosylated Chondroitin Sulfate", The Journal of Biological Chemistry, vol. 271, no 39, Issue of September 27, pp. 23973-23984, 1996). In the same direction, it was shown that FCS is an anti-thrombotic agent, with oral administration ("Fucosylated chondroitin sulfate as a new oral antithrombotic agent", Thromb Haemost 2006; 96: 822- 9), and an anti-metastatic agent ("Selectin blocking activity of a fucosylated chondroitin sulfate glycosaminoglycan from sea cucumber : Effect on tumor metastasis and neutrophil recruitment" , The Journal of biological chemistry 2007, vol. 282, no20, pp. 14984-

14991) .

The prior art brings indication that FCS is active against cancer metastasis. The patent document US 5, 989,592. discloses a method for inhibiting the complement pathway in a mammal after administering an effective dose of a composition containing an active principle selected from isolated sea cucumber (phylum Echinodermata , class Holothuroidea) body wall, isolated sea cucumber epithelial layer, isolated sea cucumber flower, sea cucumber fucosylated chondroitin sulfate, combinations thereof, active derivatives thereof or combinations of active derivatives thereof.

The content of that disclosure is too broad and unspecific, and not at all enabled:

- while the daily dosage of such active principle is cited to be within a range of 0.1 to 1, 500 mg/kg of body weight, there is no indication on how this figure was attained, as the efficacy tests included in the document were only performed with cells, in vitro;

- it is merely indicated, with no technical tests to support that claim, that the most preferable daily dose of the active is 25 mg/kg of body weight, a value randomly intercalated between purportedly preferable range values of 1 and 1,000 mg/kg of body weight.

- there is no differentiation as to the administration way, orally, topically, rectally or via injection, with respect to the daily dose, or adequate formulations for them;

- there is no differentiation as to the active being the isolated sea cucumber body wall, the isolated epithelial layer, the isolated flower or fucosylated chondroitin sulfate from the body wall, with respect to the daily dose.

- there is no specific indication that any of the cited actives actually act on cancer metastasis, there is only a literature listing of 19 possible clinical implications of the release of the C5a protein on the complement pathway, so dissimilar among themselves as tissue destruction in burns, asbestos-induced inflammation, renal cortical necrosis and cancer metastasis, without any indication on how to better act on any of them.

Also importantly, example 8 of that document (or its Canadian counterpart CA 2217322), on "Inhibition of Complement By Fucosylated Chondroitin Sulfate" mentions FCS prepared according to example n° 10 - but there are only 9 examples, so the ambiguity keeps one from precisely understanding what is actually being disclosed.

The sure information contained in that document is therefore the in vitro effect of FCS on enzymes, particularly C5a, that allegedly would be involved in physiological mechanisms related to a series of diseases, among which cancer cell metastasis.

The existing prior art as a whole, then, provides the following picture: FCS is likely to have anti-metastatic effects on mammals. Despite no hard evidence in that sense, other than just unsupported allegations, it seems likely that oral administration of FCS would also cause that effect, at least to some extent - a technical person with the common knowledge in the art would be led to accept that effective oral forms (a) would have to contain larger amounts of FCS, as compared to injected amounts, (b) would have to observe the amount generally accepted as safe for sulfated polysaccharides (around 1200 mg/day or around 17mg/kg/day, see for instance HATHCOCK JN & SHAO A. 2007. Risk assessment for glucosamine and chondroitin sulfate. Regul Toxicol Pharmacol. 47 (1) : 78-83. ) .

DESCRIPTION OF THE INVENTION

It was verified that the FCS effective anti-metastatic amount for safe oral administration is surprisingly small, not foreseeable based only on prior art teaching, with all the benefits that this new knowledge brings: lesser amounts of an active compound in the body of the patient, therefore with less possibility of side effects or toxic effects, besides more economical to the patient.

The oral administration of FCS facilitates its use in a chronic way, and its mechanism of action (inhibition of the formation of microemboli, comprised of circulating tumoral cells and platelets) does not interfere with chemoterapics already in use, making it adequate as an adjuvant medicament. Its combination with other chemoterapics is apt to increase the survival and quality of life of patients once metastasis is decisive for such prognostics .

As another advantage, despite the similarity of its mechanism of action with heparin, FCS does not increase the bleeding in animal models and is not known to be associated with hemorrhagic events, increasing its potential for chronic use. Additionally, in the small doses as utilized in the present invention, chondroitin does not present side effects .

A first aspect of this invention is, therefore, the use of low oral daily doses of FCS in the prevention and/or treatment of cancer metastasis, from 0.01 to 20 mg/kg of body weight, by itself or as an adjuvant. In a particular embodiment of the invention,. FCS can be used along with another or other known anti-metastatic compounds, so as to obtain complementary effects, based on the fact that different compounds may act in the body through different mechanisms .

In another aspect, the invention concerns the use of FCS in the manufacture of a low oral dose composition useful in the prevention and/or treatment of cancer metastasis .

In another aspect, the invention concerns an oral composition and/or oral medicament comprising an effective amount of FCS in the prevention and/or treatment of cancer metastasis, from 0.1 to about 20 mg per kg of body weight of fucosylated chondroitin sulfate and pharmaceutically acceptable excipients. Typical excipients include diluents, binders or granulating agents, glidants (flow aids) and lubricants to ensure efficient tableting; desintegrantes to promote tablet break-up in the digestive tract; sweeteners or flavors to enhance taste; and pigments to- make the tablets visually attractive. A polymer coating may be applied to make the tablet smoother and easier to swallow, to enhance the tablet's appearance, to control the release rate of the active principle for instance as a powder or granules, or to make it more resistant to the environment (extending its shelf life) .

Excipients for such formulations are not per se part of the invention, as they are well-known in the art, for instance as disclosed in Remington's Pharmaceutical Sciences, 18th ed. , Mack Publishing Co., Easton, Pa (1990). Adequate oral formulations, not excluding any other, are under the form of tablets, coated tablets, pills, capsules, powders, granules, fine granules, solutions, emulsions and suspensions. Particular formulations of the invention are unit dosage forms comprising FCS - unit dosage forms, as known to one skilled in the art, are typically solid or liquid, and contain a specific dosage, for instance a minimum dosage for daily administration of the . active compound .

In a particular embodiment, the FCS is nanoencapsulated with microparticules of acrylic compounds, for example with the product commercialized by Evonik Industries under the tradename Eudragit.

In another aspect, the invention concerns a method of prevention and/or treatment of cancer metastasis comprising administering to a mammal daily oral dose of about 0.1 to about 20 mg per kg of body weight of fucosylated chondroitin sulfate, particularly 0.05 - 20 mg/kg of body weight .

As used herein, the term "sea cucumber" refers to any species of the Phylum Echinodermata, Class Holothuroidea, such as species of the genera Actinopyga (e.g., A. lacanora, L. echinites) , Cucumaria (e.g., C. frondosa, C. echinata, C. chronh. ielmi) , Eupentacta (e.g., E. quinquesemita) , Halodeima (e.g., H.

Holothuria (e.g., H. pervicax, H. atra, H. edulis, H. scabra, H. monoacaria, H. leucospilota) , Leptosynapta (e.g., L. inhaerens) , Ludwigothuria (e.g., L. grisea) , Microthele (e.g., M. nobilis) , Molpadia (e.g., M. musculus) , Parastichopus (e.g., P. nigripunctatus) , Paracaudina (e.g., P. chilensis) , Pelaaothuria , Pentacta (e.g, P. australis) , Polycheira (e.g., P. rufescens) , Psolus (e.a., P. chitonoides) , Stichopus (e.g., S. iaponicus, S. chloronoyus, S. variegatus) , Synapta (e.g., S. maculata) , Thelenota (e.g., T. ananas) or Thyone (e.g., T. briareus) .

The preponderant structure of fucosylated chondroitin sulfate from sea cucumber is given, for instance, by Borsig, L et al, on the Journal of Biological Chemistry, volume 282 number 20, May 18, 2007, page 14985.

EXAMPLES

The examples given herein are only particular embodiments of the invention, not imposing any limitation to its scope other than in the attached claims.

1. FCS extraction from Ludwigothurea grisea

An extract from the external walls of Ludwigothurea grisea is performed as described in SILV7A LC . 2006. Isolation and purification of chondroitin sulfate. Adv Pharmacol 53:21-31.). FCS is provided under the form of sterile saline solution.

2. Anti-metastatic effect of FCS administered orally

To investigate the anti-metastatic effect of FCS from sea cucumber in the tumoral progression, by oral administration, 1.7 and 0.17 mg/kg doses of FCS were administered by gavage to B57 mice one hour before the inoculation de 3 x 10 ⁵ cells/animal of B16/F10 murine melanoma cells, and daily for 15 days. After this, the mice were sacrificed and the lungs were evaluated concerning the presence of metastatic nodules.

As can be seen in the graph of figure I the oral administration of FCS drastically reduced pulmonary metastasis, as compared with animals treated with saline solution .

By way of comparison, similar tests with 50, 30 and 17 mg/kg of FCS were run with the same conditions above and the number of metastatic nodules were only slightly below the averages obtained with 1.7 and 0.17 mg/kg body weight - graph on figure 2.

Obs : PBS stands for phosphate-buffered saline

The person skilled in the art, with the aid of the teachings presented herein, may be able to use the invention in ways not expressly described, but with the same function to reach similar results - any such equivalent way is also encompassed by the attached claims.