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Title:
PALYTOXYN, ITS MEDICAL USE AND PROCESS FOR ITS ISOLATION
Document Type and Number:
WIPO Patent Application WO/2015/090591
Kind Code:
A1
Abstract:
The invention relates to a pharmaceutical composition intended for use in the prevention and/or treatment of leukaemia in a subject, said composition comprising palytoxin (PLTX) and a pharmaceutically acceptable carrier. The application also relates to other medical uses of palytoxin and a method for its extraction from Palythoa clavata polyps. It is also described a method for isolating the symbiodinium dinoflagellate from Palythoa clavata.

Inventors:
DETOURNAY, Olivier (4 chemin de Mordeau, Allauch, Allauch, F-13190, FR)
LORQUIN, Jean (1195, chemin de la Fontaine de cinq sous, le Beausset, F-83330, FR)
GAULT, Frederic (Chemin de la Pierre Mouraou, Allée du Ganeou, Le Beausset, F-83330, FR)
Application Number:
EP2014/003411
Publication Date:
June 25, 2015
Filing Date:
December 17, 2014
Export Citation:
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Assignee:
CORAL BIOME (Marseille 9 - Zone Luminy Entreprises Case 922, 163 Avenue de Luminy, Marseille, F-13009, FR)
INSTITUT DE RECHERCHE POUR LE DEVELOPPEMENT (IRD) (Le sextant 44, Bd de Dunkerque CS 90 009, Marseille Cedex 02, F-13572, FR)
International Classes:
C12N1/12; A61K31/357; A61P17/00; A61P31/00; A61P33/00; A61P35/02; C07D493/08
Other References:
JAMES D REIMER ET AL: "Molecular identification of symbiotic dinoflagellates (Symbiodinium spp.) from Palythoa spp. (Anthozoa: Hexacorallia) in Japan", CORAL REEFS ; JOURNAL OF THE INTERNATIONAL SOCIETY FOR REEF STUDIES, SPRINGER, BERLIN, DE, vol. 25, no. 4, 14 September 2006 (2006-09-14), pages 521 - 527, XP019459567, ISSN: 1432-0975, DOI: 10.1007/S00338-006-0151-4
R. J. QUINN ET AL: "Anticancer activity of zoanthids and the associated toxin, palytoxin, against ehrlich ascites tumor and P-388 lymphocytic leukemia in mice", JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 63, no. 2, 1 February 1974 (1974-02-01), pages 257 - 260, XP055113357, ISSN: 0022-3549, DOI: 10.1002/jps.2600630217
JAMES DAVIS REIMER ET AL: "Species Diversity of Shallow Water Zoanthids (Cnidaria: Anthozoa: Hexacorallia) in Florida", JOURNAL OF MARINE BIOLOGY, vol. 10, no. 5114, 1 January 2012 (2012-01-01), pages 57 - 14, XP055113398, ISSN: 1687-9481, DOI: 10.1046/j.1365-294X.2002.01652.x
HIDESHI NAKAMURA ET AL: "Studies on Polyketide Metabolites of a Symbiotic Dinoflagellate, Symbiodinium sp", BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN, vol. 71, no. 4, 1 January 1998 (1998-01-01), pages 781, XP055113408, ISSN: 0009-2673
BERESS L ET AL: "A method for the isolation of the caribbean palytoxin (C-PTX) from the coelenterate (zooanthid) Palythoa caribaeorum", TOXICON, ELMSFORD, NY, US, vol. 21, no. 2, 1 January 1983 (1983-01-01), pages 285 - 290, XP025508816, ISSN: 0041-0101, [retrieved on 19830101], DOI: 10.1016/0041-0101(83)90013-2
NAKAMURA H ET AL: "Isolation of zooxanthellatoxins, novel vasoconstrictive substances from the zooxanthella Symbiodinium sp", TOXICON, ELMSFORD, NY, US, vol. 31, no. 4, 1 April 1993 (1993-04-01), pages 371 - 376, XP025828057, ISSN: 0041-0101, [retrieved on 19930401], DOI: 10.1016/0041-0101(93)90172-F
JONATHAN R. DEEDS ET AL: "Palytoxin Found in Palythoa sp. Zoanthids (Anthozoa, Hexacorallia) Sold in the Home Aquarium Trade", PLOS ONE, vol. 6, no. 4, 1 January 2011 (2011-01-01), pages e18235, XP055113680, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0018235
Attorney, Agent or Firm:
BARBOT, Willy (SIMODORO, 1 Place des prêcheurs, Aix-en-Provence, 13100, FR)
Download PDF:
Claims:
CLAIMS

1. A pharmaceutical composition intended for use in the prevention and/or treatment of leukaemia in a subject, said composition comprising palytoxin (PLTX) and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein said subject is a human. 3. The pharmaceutical composition of any one of claim 1 or 2, wherein the route of administration of said composition is preferably parenteral; as used herein.

4. The pharmaceutical composition of claim 3, wherein intravenous and/or subcutaneous administration are preferred.

5. The pharmaceutical composition of any one of claim 1 to 4, wherein said leukaemia is a lymphoblastic or a myelogenous leukaemia.

6. The pharmaceutical composition of claim 5, wherein said leukaemia is a myelogenous leukaemia.

7. The pharmaceutical composition of any one of claim 1 to 7, wherein said PLTX is administrated between 10 ng/kg and 2 μg/kg. 8. The pharmaceutical composition of claim 7, wherein said PLTX is administrated between 20 ng/kg and 1 μg/kg;

9. The pharmaceutical composition of claim 8, wherein said PLTX is administrated between 50 ng/kg and 0.5 μg kg.

10. The pharmaceutical composition of any one of claim 1 to 9, wherein said PLTX is obtained from Palythoa aff. clavata polyps.

11. The pharmaceutical composition of claim 10, wherein the Palythoa aff. clavata comprises the Symbiodinium dinoflagellate.

12. A method for obtaining palytoxin comprising an extaction step of palytoxin from Palythoa aff. clavata polyps.

13. A method according to Claim 12, wherein the Palythoa aff. clavata comprises the Symbiodinium dinoflagellate.

14. A method according to Claim 12 or 13, comprising the following steps: (i) immersion of Palythoa aff. clavata polyps in a polar solvent, under stirring, (ii) decantation of the mixture obtained from step (i),

(iii) filtration and/or centrifugation of the liquid obtained from step (ii),

(iv) evaporation of the solvent, and

(v) purification of the pellet obtained from step (iv).

15. A method according to Claims 12 to 14, wherein the step (i) is carried out at a temperature ranging from 4 to 10°C.

16. A method according to Claim 15, wherein the stirring is maintained during 5 to 15 hours.

17. A method according to Claims 12 to 16, wherein step (i) is carried out in the dark.

18. A method according to Claims 12 to 17, wherein the solvent of step (i) is a mixture of alcohol and water.

19. A method according to Claim 18, wherein the solvent of step (i) is a methanol/water mixture, preferably in a 80/20 ratio.

20. A method according to Claims 12 to 19, wherein the filtration of step (iii) is carried out on a PTFE membrane, preferably having a pore size ranging from 0.45 to 3.0 μιη.

21. A method according to Claims 12 to 20, wherein the purification step (v) is performed by reversed phase liquid chromatography.

22. A method according to Claim 21 , wherein the purification step (v) is performed on a mini-RP18 solid phase, preferably on a Lichroprep® RP 18 column.

23. A method according to Claim 12 to 22, said method comprising a preliminary treatment of the Palythoa aff. clavata by liquid nitrogen.

24. A method for isolating the Symbiodinium dinoflagellate from Palythoa aff. clavata polyps, said method comprising an extraction step of the Symbiodinium dinoflagellate from Palythoa aff. clavata polyps.

25. A method according to Claim 24, said method comprising the following steps:

(i') suspension of Palythoa aff. clavata polyps in an extraction buffer, preferably a phosphate buffer, (ϋ') centrifugation of the mixture obtained from step (i'),

(iii') washing of the pellet obtained from step (ϋ') in an extraction buffer, preferably a phosphate buffer,

(iv') grounding the pellet obtained from step (iii') with liquid nitrogen,

(ν') resuspension of the powder obtained from step (iv') in an extraction buffer, preferably a phosphate buffer,

(vi') filtration of the liquid obtained from step (ν'), and

(νϋ') isolation of the Symbiodinium colonies.

26. A method according to Claim 24, said method comprising the following steps:

(i") incubation of Palythoa aff. clavata polyps in a strong base, and

(ii") centrifugation of the mixture obtained from step (i"),

(iii") washing of the pellet obtained from step (ii"), preferably with water,

(iv") neutralization of the mixture obtained from step (iii") with a strong acid, preferably HC1,

(v") resuspension of the pellet obtained from step (iv"), preferably in water or in a medium containing seawater, and

(vi") isolation of the Symbiodinium colonies.

27. A method according to Claim 26, wherein the strong base of step (i") is NaOH, preferably at a concentration ranging from 0.25 to 10 mol.L 1.

28. A method according to Claim 26 or Claim 27, wherein step (i") is carried out at a temperature ranging from 20 to 40°C, during 1 to 4 h.

29. A method for producing palytoxin from the isolated Symbiodinium dinoflagellate, said method comprising the following steps:

(i'") cultivation of the Symbiodinium dinoflagellate, preferably in a F/2 culture medium, and

(ii'") irradiation of the Symbiodinium dinoflagellate culture of step (i' ") under a

2 1

photon flux ranging from 50 to 200 μηιοΙ.π .s" .

30. A method according to Claim 29, wherein the incubation step (i'") is implemented at pH 8.2 and at a temperature of 26.0 ± 0.1 °C.

31. A method according to Claim 29 or Claim 30, wherein step (ii'") is implemented at a photon flux of 100 during 12 h.

32. Palytoxin for its use for the prevention and/or the treatment of diseases and/or disorders chosen amongst nosocomial diseases, parasitic diseases {Plasmodium falciparum, Leishmanii), Human Imminodeficiency Virus (HIV) disease, Ebola Virus Disease (EVD), skin diseases (psoriasis, Propionibacterium acnes).

Description:
PALYTOXYN, ITS MEDICAL USE AND PROCESS FOR ITS ISOLATION

The present international patent application claims the priority of the European patent application n°l 3197854.6 filed on December 17, 2014, which is herein incorporated by reference.

Domain of the invention

The invention relates to a method for obtaining palytoxin (PLTX) from Palythoa aff. clavata, to methods for isolating the Symbiodinium dinoflagellate from Palythoa aff. clavata and to a method for producing PLTX from the Symbiodinium dinoflagellate. The invention also relates to the use of PLTX for the prevention and/or the treatment of cancers at infinitesimal doses, i.e. 10 " mol.L " , and for the prevention and/or the treatment of diseases and/or disorders chosen amongst nosocomial diseases, parasitic diseases (Plasmodium falciparum, Leishmanii), Human Immunodeficiency Virus (HIV) disease, Ebola Virus Disease (EVD), and skin diseases (psoriasis, Propionibacterium acnes).

Background of the invention

Leukaemia is a cancer of the blood cells.

Clinically and pathologically, leukaemia is subdivided into a variety of large groups. The first division is between its acute and chronic forms:

• Acute leukaemia is characterized by a rapid increase in the number of immature blood cells. Crowding due to such cells makes the bone marrow unable to produce healthy blood cells. Immediate treatment is required in acute leukaemia due to the rapid progression and accumulation of the malignant cells, which then spill over into the bloodstream and spread to other organs of the body. Acute forms of leukaemia are the most common forms of leukaemia in children.

• Chronic leukaemia is characterized by the excessive buildup of relatively mature, but still abnormal, white blood cells. Typically taking months or years to progress, the cells are produced at a much higher rate than normal, resulting in many abnormal white blood cells. Whereas acute leukaemia must be treated immediately, chronic forms are sometimes monitored for some time before treatment to ensure maximum effectiveness of therapy. Chronic leukaemia mostly occurs in older people, but can theoretically occur in any age group.

Additionally, the diseases are subdivided according to which kind of blood cell is affected. This split divides leukaemias into lymphoblastic or lymphocytic leukaemias and myeloid or myelogenous leukaemias:

• In lymphoblastic or lymphocytic leukaemias, the cancerous change takes place in a type of marrow cell that normally goes on to form lymphocytes, which are infection- fighting immune system cells. Most lymphocytic leukaemias involve a specific subtype of lymphocyte, the B cell.

• In myeloid or myelogenous leukaemias, the cancerous change takes place in a type of marrow cell that normally goes on to form red blood cells, some other types of white cells, and platelets.

Combining these two classifications provides a total of four main categories.

Now, new therapeutic treatment are still needed for treating such leukaemia.

Summary of the invention

Palytoxin, also called PLTX, is a nonprotein toxin of marine origin (Wiles et al.,

1974).

Scheme 1: PLTX structure Even though PLTX was originally found in soft corals from tropical areas of the Pacific Ocean, i.e. in Hawaii from the tropical soft coral Palythoa sp., a zoanthid (Moore and Scheuer, 1971), its occurrence was then observed in numerous other marine organisms from the same ecological region. The literature emphasizes the sodium/potassium pump (the Na + /K + -ATPase) as the privileged target for PLTX when exerting its cytotoxic/toxic effects. Recently several analogs of PLTX were discovered in various species of the dinoflagellate genus Ostreopsis (Ramos and Vasconcelos, 2010). Indeed, sporadic occurrence of PLTX in algae, crabs and fish alike indicates that microorganisms could represent the primary source of these toxins. Table 1 below gives a non exhaustive summary of the PLTX analogues and their respective marine sources.

Table 1: Summary of the known molecules in the PLTX family in zoantharians,

cyanobacteria, algae and dinoflagellates

Compounds Organism Sample IDMW Concentration References

Zoantharians

PLTX Palythoa toxica 2679 275 wet z Moore and Scheuer, 1971

PLTX Palythoa tuberculosa 2679 13.6 μξ g wet z Kimura and Hashimoto, 1973

PLTX Palythoa caribaeorum 2679 30 dry z Beress et al., 1983

PLTX Palythoa heliodiscus VAZOA 2679 613 wet z Deeds et al., 2011

PLTX Palythoa heliodiscus 305.11.2 2679 515 μg/g wet z Deeds et al., 2011

PLTX Palythoa heliodiscus 306.39.2 2679 116 μg/g wet z Deeds et al., 2011

PLTX Palythoa heliodiscus 306.39.3 2679 1037 wet z Deeds et al., 201 1

PLTX Palythoa vestitus 2674 ND Quinn et al., 1974

PLTX Palythoa aff. margaritae 2674 ND Oka et al., 2004

PLTX Zoanthus solanderi 2674 ND Gleibs ei al., 1995

PLTX Zoanthus sociatus 2674 ND Gleibs et al., 1995

PLTX-b Palythoa tuberculosa 2720 minor Rossi et al., 2010

Homo-PLTX Palythoa tuberculosa 2692 minor Uemura al., 1985

Bishomo-PLTX Palythoa tuberculosa 2706 minor Uemura et al., 1985

Neo-PLTX Palythoa tuberculosa 2661 minor Uemura a/., 1985

Deoxy-PLTX Palythoa tuberculosa 2662 minor Uemura et al., 1985

Deoxy-PLTX Palythoa heliodiscus 306.37.3 2679 3515 glg wet z* Deeds et al., 201 1

42-Hydroxy-PLTX Palythoa tuberculosa 2694 minor Ciminiello et al., 2009

42-Hydroxy-PLTX Palythoa toxica 2694 minor Ciminiello et al., 2009

Cyanobacteria

42-Hydroxy-PLTX Trichodesmium spp. 2694 minor Kerbrat ei 2011

Algae

CA-I Chondria armata ND Yasumoto and Murata, 1990

CA-II Chondria armata ND Yasumoto and Murata, 1990

Dinoflagellates

Mascarenotoxin-a Ostreopsis mascarenensis 2588 minor Lenoir et al., 2004

Mascarenotoxin-a Ostreopsis ovata 2588 minor Rossi et al., 2010

Mascarenotoxin-b Ostreopsis mascarenensis 2606 minor Lenoir et al., 2004

Mascarenotoxin-c Ostreopsis ovata 2628 minor Rossi et al., 2010

Ostreocin-d Ostreopsis siamensis 2634 ND Ukena et al., 2001 Ovatoxin-a Ostreopsis ovata 2646 minor Ciminiello et al., 2008 Ovatoxin-b Ostreopsis ovata 2662 minor Rossi et al., 2010

Ovatoxin-c Ostreopsis ovata 2690 minor Rossi et al., 2010

Ovatoxin-d Ostreopsis ovata 2706 minor Rossi et al., 2010

ND, not determined; z, zoanthid.*, position of the deoxygenation was not determined for this analogue.

In most cases, the yield of extraction/isolation is low, pointing out the need to develop solutions to obtain higher level of toxin. Indeed, large quantity of such a molecule is required for its functional characterization applied, for instance, to cancer research. Nowadays, Wako Pure Chemical Industries in Japan remains the unique PLTX supplier in the world, their toxin being purified from Palythoa caribaeorum (Beress et al., 1983; see Table 1).

PLTX has a very complex structure (Scheme 1) with a long polyhydroxylated and partially unsaturated aliphatic backbone containing 64 chiral centers (Kan et al., 2001). It is heat-stable, not inactivated by boiling, and stable in neutral aqueous solutions for prolonged periods. In contrast, a rapid degradation occurs under acid or alkaline conditions, leading to loss of its toxicity (Katikou, 2007). Resembling to mycotoxins, other polyketide-type molecules, no pharmacological treatment have been developed to fight and/or destroy PLTX in the human or animal body.

Most of the data published in the literature about PLTX toxicity relates to PLTX mediated disruption of the actin cytoskeleton (Louzao et al., 2008 and 201 1). The widely accepted molecular action is blockade of the Na + /K + -ATPase pump (NaK) (Hilgeman, 2003; Rodrigues et al., 2008; Charlson et al., 2009; Rossini and Bigiani, 201 1 ; Wattenberg, 2011). PLTX seems to bind to the extracellular part of the NaK and thereby inhibits the active transport of Na + and K + across the cell membrane by transforming the pump into a nonspecific permanently open ion channel (Ramos and Vasconcelos, 2010). The membrane depolarization generated and the massive increase of Ca 2+ in the cytosol (Satoh et al., 2003) interferes with some vital functions of cells. It has recently been demonstrated that PLTX acts through voltage-dependent channels and the Na + /Ca 2+ exchanger (reverse mode) (Del Favero et al., 2012), emphasizing at best that the NaK would not be its only cellular target. PLTX has also been identified as a carcinogenic agent (Wattenberg, 201 1). Thus, the identification of PLTX as a tumor promoter, together with the recognition that the Na + /K + -ATPase is its receptor, suggest that PLTX triggers the modulation of signal transduction pathways. In fact, mitogen activated protein (MAP) kinases are mediators of PLTX-stimulated signaling and relay a variety of signals to the cellular machinery that regulates cell fate and function (Wattenberg, 201 1). If a cytotoxic effect for PLTX was identified in different articles and for different tumor cell lines, said activity was very dependent upon the cancer target cells as disclosed in BELLOCI et al. (201 1). As an example, QUINN et al. (1974) in addition to established an antitumoral activity on mammary adenocarcinoma, also show that PLTX has only a marginal activity on leukaemia.

Now, the inventors have surprisingly established 40 years later that PLTX has finally a strong a reproducible antitumoral activity on all tested leukaemia cell lines, including both lymphoblastic and myelogenous leukaemia.

The P-388 cell lines identified by DA WE & POTTER (1957) is a murine cell lines corresponding to one of the first model of leukaemia. Now, the correlation between drugs active against P388 leukaemia and solid experimental tumor models has not been good. One common observation is that some drugs that are active against experimental solid tumors are inactive against P388 leukaemia. For example, 15% of 84 agents that were inactive against P388 leukaemia were active against at least one of eight solid tumors tested (STAQUET et al, 1983).

Following the results obtained by the inventors on human leukaemia cell lines, it seems finally that the results of QUINN et al. (1974) with PLTX on murine P388 cell lines are not representative of the mechanism of PLTX on leukaemia.

Thus, a first object of the invention is directed to a pharmaceutical composition intended for use in the prevention and/or treatment of leukaemia in a subject, said composition comprising palytoxin (PLTX) and a pharmaceutically acceptable carrier.

The Inventors have also identified a new PLTX producer giving the highest yield ever found in the nature. The Inventors have actually found that the extraction from invertebrate Palythoa aff. clavata species which live in symbiosis with a Symbiodinium dinoflagellate from the C clade allows to obtain a pure PLTX with excellent yields, thanks to a simple and economic process (limited number of operating steps).

Therefore, a second subject of the invention is directed to a method for obtaining pure PLTX from Palythoa aff. clavata polyps, said polyps comprising the Symbiodinium dinoflagellate and possibly other symbiotic microorganisms. The method according to the invention for obtaining pure PLTX may comprise the following steps:

(i) immersion of Palythoa aff. clavata polyps in a polar solvent, under stirring,

(ii) decantation of the mixture obtained from step (i),

(iii) filtration and/or centrifugation of the liquid obtained from step (ii), and (iv) evaporation of the solvent, preferably by rotary evaporation under reduced air pressure,

(v) purification of the pellet obtained from step (iv), preferably by reversed phase liquid chromatography, and more preferably on a column Lichroprep ® RP18,

(vi) evaporation to dryness of the pellet obtained from step (v), preferably by rotary evaporation and then under reduced N 2 pressure.

Brief description of the figures

Figure 1 (A) and (B) are pictures of a Zoanthid colony collected in Indo- Pacific and cultivated at Coral Biome aquarium,

Figure 2 represents the HPLC chromatogram of the purified PLTX from Palythoa aff. clavata at 263 nm. The insert graph is the UV spectrum of PLTX eluted at 14.8 min in the gradient conditions used,

Figure 3 represents the MALDI-ToF mass spectrum of PLTX purified from Palythoa aff. clavata,

Figure 4 shows the maximum likelihood rooted tree for the dataset based on 34 ITS-rDNA sequences of Zoanthidae species (937 nucleotide positions). One hundred heuristic replicates were performed using the Kimura's 2-parameters model with estimation of gamma parameter shape distribution and proportion of invariant sites (K2 + G5 + I). Bootstrap values are shown at nodes with values > 75%. Black arrowhead indicates the sequence belonging to the Palythoa species used for PLTX extraction. Sequences/species names from previous studies in regular font with GenBank Accession Numbers,

Figure 5 shows the maximum likelihood rooted tree for the dataset based on 30 COI sequences of Zoanthidae species (462 nucleotide positions). One hundred heuristic replicates were performed using the Kimura's 2-parameters model (K2). Bootstrap values are shown at nodes with values > 50%. Black arrowhead indicates the sequence belonging to the Palythoa species used for PLTX extraction. Sequences/species names from previous studies in regular font with GenBank Accession Numbers,

Figure 6 shows the maximum likelihood unrooted tree based on the internal transcribed spacer 2 of ribosomal DNA (ITS2-rDNA) sequences for Symbiodinium from various corals and molluscs, including the Palythoa specimen from this study (332 nucleotide positions). One hundred replicates were performed using the Kimura's 2-parameters model with estimation of gamma parameter shape distribution (K2 + G5). Values at branches represent ML bootstrap values. New sequence from this study in bold. Sequences/species names from previous studies in regular font with GenBank Accession Numbers, species- associated isolates from previous studies with species name and location, as well as GenBank Accession Number. On the right, Symbiodinium clades confirmed to be in symbiosis with various metazoans species designated with shaded boxes,

- Figure 7 represents the MTT colorimetric assay results visualized by the remaining viable cells versus PLTX concentration. (A) Experiences performed with the cancer and the normal but transformed cells, HBL-100. (B) With the normal and human cell line MSC. (C) With the normal and human cell line NHDF,

Figure 8 represents the videomicroscopy experiences on human Hs683 oligodendroglioma cells (apoptose-sensitive) in presence of 0.01 or 1 nM PLTX during 22 h,

Figure 9 represents the videomicroscopy experiences on human U373n glioblastoma cells (apoptose-sensitive) in presence of 0.01 or 1 nM PLTX during 22 h.

Detailed description of the invention

In a first object, the invention is directed to a pharmaceutical composition intended for use in the prevention and/or treatment of leukaemia in a subject, said composition comprising palytoxin (PLTX) and a pharmaceutically acceptable carrier.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine or a primate, and most preferably a human.

The expression "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar undesirable reactions, such as gastric upset, dizziness and the like when administered to a human. Preferably, as used herein, the expression "pharmaceutically acceptable" means approvable by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term "carrier" refers to a solvent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

The route of administration is preferably parenteral; as used herein, the term

"parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal or intraperitoneal administration. Thus, the pharmaceutical composition contains vehicles which are pharmaceutically acceptable for a formulation intended to be injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Of these, intravenous and subcutaneous administration are most preferred.

PLTX may be solubilized in a buffer or water or incorporated in emulsions, microemulsions, hydrogels (e.g. PLGA-PEG-PLGA triblock copolymers-based hydrogels), in microspheres, in nanospheres, in microparticles, in nanoparticles (e.g. poly(lactic-co-glycolic acid) microparticles (e.g. poly lactic acid (PLA) ; poly (lactide-co-glycolic acid) (PLGA) ; polyglutamate microspheres, nanospheres, microparticles or nanoparticles), in liposomes, or other galenic formulations. In all cases, the formulation must be sterile and fluid to the extent of acceptable syringability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate, gelatin, polyols, half-life enhancing covalent and non covalent formulations.

In the context of the invention, the term "treatment", as used herein, means reversing, alleviating, inhibiting the progress of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

The term "treatment of leukaemia" as used herein means the inhibition of the growth of leukaemia cells. Preferably such treatment also leads to the regression of tumor growth, i.e., the decrease in size of a measurable tumor. Most preferably, such treatment leads to the complete regression of the tumor. As used herein, the term "leukaemia" refers to lymphoblastic and/or myelogenous leukaemia. The term "leukaemia" refers to acute and/or chronic leukaemia.

An "effective amount" of palytoxin is an amount which is sufficient to induce the regression of tumor growth. The doses used for the administration can be adapted as a function of various parameters, in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment. Naturally, the form of the pharmaceutical composition, the route of administration, the dosage and the regimen naturally depend on the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc. The ranges of effective doses provided below are not intended to limit the invention and represent preferred dose ranges. However, the preferred dose can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation.

As an illustration, an effective amount of PLTX is comprised between 10 ng/kg and 2 μg/kg, preferably between 20 ng/kg and 1 μg/kg, and most preferably between 50 ng/kg and 0.5 μg/kg. Other dosages are viable, since the molecular weight and the activity of the conjugate thereof may influence it. The skilled artisan is readily credited with determining a suitable dosage that falls within the ranges, or if necessary, outside of the ranges.

Still preferably, said effective amount corresponds to a daily effective amount.

The used PLTX may be obtained from different sources. As an example, such PLTX may be obtained from Palythoa aff. clavata polyps, or obtained from the culture of the Symbiodinium dinoflagellate isolated from Palythoa aff. Clavata. Such PLTX may be obtained by the method described hereafter.

In a second object, the invention is directed to a method for obtaining pure PLTX from Palythoa aff. clavata polyps, said polyps comprising the Symbiodinium dinoflagellate and possibly other symbiotic microorganisms. The method according to the invention for obtaining pure PLTX may comprise the following steps:

(i) immersion of Palythoa aff. clavata polyps in a polar solvent, under stirring,

(ii) decantation of the mixture obtained from step (i),

(iii) filtration and/or centrifugation of the liquid obtained from step (ii), and

(iv) evaporation of the solvent, preferably by rotary evaporation under reduced air pressure,

• (v) purification of the pellet obtained from step (iv), preferably by reversed phase liquid chromatography, and more preferably on a column Lichroprep ® RP18, (vi) evaporation to dryness of the pellet obtained from step (v), preferably by rotary evaporation and then under reduced N 2 pressure.

In the sense of the invention, a polyp is a coral that has typically a hollow cylindrical body closed and attached at one end and opening at the other by a central mouth surrounded by tentacles armed with nematocysts.

Step (i) is preferably carried out at a temperature ranging from 4 to 10°C, more preferably from 4 to 6°C, under stirring. The stirring can be maintained during 5 to 15 hours, and preferably during 10 hours. According to a preferred embodiment, step (i) is implemented in the dark.

The polar solvent of step (i) may be a mixture of alcohol and water, and is preferably a mixture of methanol or ethanol with water. More preferably said solvent is a methanol/water mixture, more preferably in a ratio 80/20.

According to a preferred embodiment, step (iii) is a filtration step carried out on a PTFE membrane, preferably having a pore size ranging from 0.45 to 3.0 μπι, or carried out by centrifugation (8000 g, 20 min). For a large-scale production, filtration would be preferred.

After homogenisation of the pellet with a mortar, the resulting powder may be diluted in cold milli-Q water and purified, preferably on a mini-RP18 solid phase, and more preferably on a Lichroprep ® RPi column.

According to another embodiment, the method according to the invention for obtaining PLTX may comprise a subsequent liquid-liquid extraction step between the evaporation step (iv) and the purification step (v), preferably with hexane or dichloromethane.

The method according to the invention for obtaining pure PLTX then comprise a purification step (v), preferably by reversed phase liquid chromatography. The purification step (v) may be implemented on a mini-RP18 solid phase, preferably on a Lichroprep ® RP 18 column.

The method according to the invention for obtaining pure PLTX may also comprise a preliminary treatment of the Palythoa aff. clavata by liquid nitrogen directly pooled on the dried out (paper) and chopped (scalpel) polyps.

Symbiodinium is a highly diverse taxon of dinoflagellates (or zooxanthellae) with nine clades (A-I) described to date and with the phylogenetic distance between clades at the order or family level (Coffroth and Santos, 2005; Pochon and Gates, 2010). In addition to the well- known symbiotic clades A-D, microbial ecologists are now describing clades that are likely only free-living and not in symbiosis with host animals (Apprill and Gates, 2007; Pochon and Gates, 2010). Patterns of host-symbiont specificity are highly complex and influenced by a broad array of factors including biogeography, environment, stress, and host ontogeny (Baker, 2003; Coffroth and Santos, 2005). Symbiodinium types have highly varying biochemistry, physiology, and responses to environmental stress (Iglesias-Prieto and Trench, 1997; Warner et al, 1999; Rowan, 2004; Tchernov et al, 2004; Stat et al, 2008). These differential biochemical and physiological qualities have direct impacts on the performance and health of the holobiont (or partnership). An obvious example is the differential performance of stress- sensitive clade C versus stress-resistant clade D symbionts to elevated temperature in corals. Whereas clade C symbionts are the overwhelmingly dominant taxon in healthy IndoPacific corals, they are at least temporarily displaced after bleaching events by clade D types in many coral species (Berkelmans and van Oppen, 2006; Stat and Gates, 201 1).

PLTX biosynthesis by the symbiotic Symbiodinium dinoflagellate represents a reproducible way to increase the yield of production at lower cost, as already suggested (Taniyama et al, 2003; Katikou, 2007). The Inventors have shown that the Symbiodinium ITS2-rDNA sequence isolated from the Palythoa aff. clavata was unambiguously placed within the generalist clade C, within a large group including many subclades of C previously observed in a variety of hosts.

Several techniques have been developed to isolate the symbiotic algae from the host. They include stripping coral tissues from the skeleton with a fine jet of seawater using a WaterPik (Johannes and Wiebe, 1970), grinding the whole coral (Santiago- Vazquez et al, 2006) or gorgonian tissue (Forcioli et al., 201 1 ; Pey et al., 201 1) in liquid nitrogen, manual tissue fractionation and potter homogenization (Richier et al., 2003). Although these techniques are effective, the Symbiodinium isolation is complicated, and the dinoflagellate not purified. Furthermore, the molecular analysis leads to DNA extracts containing both cnidarian and algal genomes. These techniques however remain valuable and could be used as comparative techniques. As an improved method, a fast and efficient protocol without host contaminant and developed on three cnidarian species has been recently validated (Zamoum and Furla, 2012).

Therefore, another object of the invention is directed to the production of PLTX from the culture of symbiotic dinoflagellate. The Inventors have now found new methods for isolating the Symbiodinium dinoflagellate from Palythoa aff. clavata polyps, as well as a method for producing PLTX by culture of the isolated Symbiodinium dinoflagellate, said methods simplifying the culture and the PLTX extraction, and being time-saving.

The Inventors have thus developed a method for isolating the Symbiodinium dinoflagellate from Palythoa aff. clavata polyps, comprising the following steps: (ΐ') suspension of Palythoa aff. clavata polyps in an extraction buffer, preferably a phosphate buffer,

(ϋ') centrifugation of the mixture obtained from step (i'),

(iii') washing the pellet obtained from step (ϋ') in an extraction buffer, preferably a phosphate buffer,

(iv') grounding the pellet obtained from step (iii') with liquid nitrogen,

(ν') resuspension of the powder obtained from step (iv') in an extraction buffer, preferably a phosphate buffer,

(vi') filtration of the liquid obtained from step (ν'), and

(νϋ') isolation of the Symbiodinium colonies.

The Palythoa aff. clavata polyps may be dried out and chopped into pieces before being suspended in the extraction buffer in step (i'). The suspension of step (i') may be homogenized, for example by sonication or with a syringe.

Preferably, the extraction buffer is a phosphate buffer of pH 7.8 having a concentration ranging from 25 to 75 mM, and preferably of 50 mM. Said phosphate buffer may be mixed with sorbitol to prevent the Symbiodinium dinoflagellate from a membrane disruption.

The Symbiodinium dinoflagellate in the mixture of step (i') is separated by centrifugation during a step (ϋ').

Then, the pellet obtained from step (ϋ') is washed with an extraction buffer, and may be centrifiiged again, and resuspended in the same extraction buffer. The extraction buffer is preferably a phosphate buffer. The pellet obtained from step (iii') is then ground, preferably in a mortar, with liquid nitrogen (step (iv')).

The powder obtained from step (iv') is then resuspended in an extraction buffer.

The liquid obtained from step (ν') is then filtered during step (vi'), preferably through a nylon mesh (100 μη ) in order to eliminate as many skeleton residues as possible.

The Symbiodinium colonies are isolated during a step (νϋ'), preferably on agar Petri dishes, and more preferably on 2.0% agar Petri dishes. Another method for isolating the Symbiodinium dinoflagellate from Palythoa aff. clavata has also been finalized by the Inventors, said method comprising the following steps:

(i") incubation of Palythoa aff. clavata polyps in a strong base, preferably NaOH, and

(ii") centrifugation of the mixture obtained from step (i"),

(iii' ') washing of the pellet obtained from step (ii"), preferably with water, (iv") neutralization of the mixture obtained from step (iii") with a strong acid, preferably HC1,

(v") resuspension of the pellet obtained from step (iv"), preferably in water or in a medium containing seawater, and

(vi") isolation of the Symbiodinium colonies.

According to a preferred embodiment, the strong base implemented in step (i") has a concentration ranging from 0.25 to 10 mol.L "1 , preferably a concentration of 1 M.

The incubation step (i") may be carried out at a temperature ranging from 20 to 40°C, preferably at 37°C. The incubation step (i") may be carried out during 1 to 4 h, preferably 1 h.

The samples can be shaken vigorously every 15 min during the step (i").

The incubated Palythoa aff. clavata polyps are then separated during a centrifugation step (ii").

The pellet obtained from step (ii") is then washed with water during step (iii"), and preferably washed twice with milli-Q water.

The mixture obtained from step (iii") is then neutralized with a strong acid during a step (iv"), preferably HC1, and more preferably with a solution of 1 M HC1.

The pellet obtained from step (iv") is resuspended, for example in water (sterilized milli-Q water) or in a medium containing seawater such as a F/2 culture medium.

The F/2 culture medium is a common and widely used general enriched seawater medium designed for growing coastal marine algae, especially diatoms (Guillard and Ryther, 1962). For one Liter of natural seawater, the composition of the medium comprises (final concentration): NaN0 3 882 μΜ, NaH 2 P0 4 H 2 0 36 μΜ, and 1 mL of trace metal solution. The solution is further autoclaved at 120°C for 20 min, and after reaching ambient temperature, 0.5 mL of vitamin solution is added under sterile conditions. The trace metal solution comprises (for one Liter of milli-Q water): FeCl 3 6H 2 0 3.15 g, Na 2 EDTA 2H 2 0 4.36 g, CuS0 4 5H 2 0 9.8 mg, Na 2 Mo0 4 2H 2 0 6.3 mg, ZnS0 4 7H 2 0 22.0 mg, CoCl 2 6H 2 0 10.0 mg, MnCl 2 4H 2 0 180 mg. The trace metal solution is then autoclaved at 120°C for 20 min and stored at 4°C. The vitamin solution comprises (for 1 Liter of milli-Q water): thiamine-HCl (vit. Bi) 200 mg, biotin (vit. H) 1 mg. The vitamin solution is then sterilized by passing on a 0.22 μιη pore size filter (from Millipore) and stored at 4°C.

The Symbiodinium colonies are then isolated during a step (vi"), preferably on agar Petri dishes, and more preferably on 2.0% agar Petri dishes in the F/2 culture medium. In this case, 2.0% (w/v) of agar are added in the F/2 culture medium comprising NaNQ 3 882 μΜ, NaH 2 P0 4 H 0 36 μΜ, and 1 mL of trace metal solution. The solution is further autoclaved at 120°C for 20 min, and the vitamin solution is added on the Petri dishes at ambient temperature.

During the isolation step (vi"), the Petri dishes are also incubated, preferably at 26°C under a 12 h light/12 h dark photoperiod, until visualization of the Symbiodinium colonies.

Another object of the invention is a method for producing PLTX from a culture of the isolated Symbiodinium dinoflagellate, said method comprising the following steps:

(i" ') cultivation of the Symbiodinium dinoflagellate in a F/2 culture medium, and (ii'") irradiation of the Symbiodinium dinoflagellate culture of step (i'") under a

2 1

photon flux ranging from 50 to 200 μιηοΐ.ηι " .s " .

According to a preferred embodiment, the incubation step (i'") is implemented at pH 8.2 and at a temperature of 26.0 ± 0.1 °C. The composition of the F/2 culture medium of step (i'") is the same as described above.

According to another preferred embodiment, the irradiation step (ii"') is implemented at a photon flux of 100 μηιοΐ.ιη "2 ^ "1 , during 12 h. Preferably, the irradiation step (ii"') is performed with a Sylvania Gro-Lux bulb.

In a subsequent step (iii"'), the irradiated Symbiodinium dinoflagellate culture obtained from step (i'") is centrifuged, preferably at 3000-5000 g, and more preferably at 3000 g. Preferably the centrifugation step (iii"') is performed at a temperature ranging from 4 to 10°C, and more preferably during 5 to 20 min.

During an optional step (iv'"), the pellet obtained from step (iii'") is resuspended with water, preferably ice-cold distilled water, and eventually further homogenized by sonication.

Then, the mixture obtained from step (iv'") may be separated during a centrifugation step (ν"'), preferably at 10000-12000 g, and more preferably at 12000 g during 5 to 20 min. Alternatively, the mixture from step (iv'") may be filtered on a nylon mesh (1 μπι pore size) so as to separate the debris from the PLTX.

The PLTX present in the filtrate can also be purified during a purification step (vi'"), preferably by reversed phase liquid chromatography. The purification step (vi'") may be implemented on a Lichroprep ® RP 18 column.

The Inventors have also demonstrated that the PLTX obtained from Palythoa aff. clavata polyps is very efficient against cancer cell lines, with an IC 50 of 0.545 ± 0.05 pM, thus at lower doses than those known from the prior art (Quinn et al., 1974; Valverde et al., 2008; Gorogh et al, 2013; Pelin et al., 2013). Therefore, the invention also concerns the use of pure PLTX obtained from Palythoa aff. clavata polyps, or obtained from the culture of the Symbiodinium dinoflagellate isolated from Palythoa aff. clavata, for its use for preventing

12 1 and/or treating cancer at infinitesimal doses, i.e. at a dose equal or less than 10 " mol.L " .

Another objet of the invention is also the use of PLTX for the prevention and/or the treatment of diseases and/or disorders chosen amongst nosocomial diseases, parasitic diseases {Plasmodium falciparum, Leishmanii), Human Imminodeficiency Virus (HIV) disease, Ebola Virus Disease (EVD), skin diseases (psoriasis, Propionibacterium acnes).

In the following, the invention is described in more detail with reference to amino acid sequences, nucleic acid sequences and examples. However, no limitation of the invention is intended by the details of the examples. Rather, the invention pertains to any embodiment which comprises details which are not explicitly mentioned in the examples herein, but which the skilled person finds without undue effort.

EXAMPLES:

1) Materials & Methods

Palythoa culture:

Palythoa spp. including Palythoa aff. clavata are maintained in a culture system comprising six tanks (70 Liters each) plugged to a biological filter. The whole system is fed by artificial seawater (Instant Oceansalts, from Seachem Salinity) maintained at 26°C and

2 1 exposed to a 12 h light/12 h dark photoperiod with an irradiance of 70 mol. quanta m " .s " . A skimmer has been set up to remove fatty acids and proteins from the system, and to avoid the accumulation of nitrogenous compounds that are toxic for marine invertebrates. The evaporation is compensated automatically by addition of Dl-water. Palythoa spp. are fed on a daily basis with pellets made of fish meat (Formula One, from Ocean Nutrition) to maximize the growth rate.

Symbiodinium dinoflagellate isolation:

Two extraction protocols have been implemented:

Protocol (A): Extractions from Palythoa aff. clavata is performed as described by Richier et al, 2003 and Forcioli et al., 201 1. One gram of Palythoa aff. clavata is quickly dried out on paper to remove excess seawater, chopped into several pieces with a scalpel, suspended in a 50 mM phosphate buffer pH 7.8, 0.4 M sorbitol, and disrupted by syringe homogenization. The Symbiodinium dinoflagellate is separated from the extract by centrifugation at 3000 g for 5 min. The pellet was washed twice with the same extraction buffer, centrifuged at 3000 g for 3 min and then grounded in a mortar with liquid nitrogen. The resulting powder is resuspended in the same extraction buffer, and the solution filtered through a nylon mesh (100 μιτι) in order to eliminate as many skeleton residues as possible. The filtrate containing the Symbiodinium dinoflagellate is further applied on Petri dishes and cultured as described in the paragraph "Symbiodinium dinoflagellate culture".

Protocol (B): NaOH solution is used to extract the Symbiodinium dinoflagellate from

Palythoa aff. clavata. Extracts are obtained by incubating 0.5 g of fresh animal previously dried out on paper and chopped into several pieces with a scalpel, in 500 μΐ. of 1 M NaOH solution, at 37°C and during 1 h. The samples are vigorously shaken every 15 min during the NaOH treatment and extracts are centrifuged at 5000 g for 3 min. The Symbiodinium dinoflagellate pellets are washed twice with mill-Q water, neutralized with a 1 M HC1, resuspended in a F/2 culture medium, applied on Petri dishes and cultured as described in the paragraph "Symbiodinium dinoflagellate culture".

Symbiodinium dinoflagellate culture:

The Symbiodinium dinoflagellate is routinely cultured in sterile conditions, in a one- Liter Erlenmeyer flask stoppered with a cotton wool, and containing a F/2 culture medium at pH 8.2 and incubated at 26.0 ± 0.1 °C. The irradiance is about 100 μηιοΐ photons, m 2 . s "1 (Sylvania Gro-Lux, Loessnitz, Germany), on a 12 h light/12 h dark photoperiod. A low stirring is performed with a magnetic stirrer.

The F/2 culture medium is prepared as follows:

For one Liter of natural seawater, the composition of the F/2 culture medium comprises (final concentration): NaN0 3 882 μΜ, NaH 2 P0 4 ¾0 36 μΜ, and 1 mL of trace metal solution. The solution is further autoclaved at 120°C for 20 min, and after reaching ambient temperature, 0.5 mL of vitamin solution is added under sterile conditions.

The trace metal solution is as following (for 1 Liter of milli-Q water): FeCl 3 6H 2 0 3.15 g, Na 2 EDTA 2H 2 0 4.36 g, CuS0 4 5H 2 0 9.8 mg, Na 2 Mo0 4 2H 2 0 6.3 mg, ZnS0 4 7H 2 0 22.0 mg, CoCl 2 6H 2 0 10.0 mg, MnCl 2 4H 2 0 180 mg. The mixture is then autoclaved at 120°C for 20 min and stored at 4°C.

The vitamin solution is as following (for 1 Liter of milli-Q water): thiamine-HCl (vit. Bi) 200 mg, biotin (vit. H) 1 mg. Then, the solution is sterilized by passing on a 0.22 μιη pore size filter (from Millipore) and stored at 4°C.

The Symbiodinium colonies are then isolated on Petri dish plates by adding 2.0% (w/v) of agar (from Difco, France) in the F/2 culture medium containing the trace elements, and autoclaved at 120°C during 20 min. The vitamin solution is added on the Petri dish plates at ambient temperature. The final extract containing the isolated Symbiodinium (see procedures described in the paragraph "Symbiodinium dinoflagellate isolation") is applied on the Petri dish plates which are further incubated at 26°C under a 12 h light/12 h dark photoperiod, until visualization of the colonies.

Cell lines and culture media used for 2rowth inhibitory effects:

The human cancer cell lines used that are sensitive to pro-apoptotic stimuli include the human Hs683 oligodendroglioma (Branle et al., 2002; Ingrassia et al, 2009) and the mouse B16F10 melanoma (Van Goietsenoven et al., 2010) cell lines. The human cancer cell lines displaying various levels of resistance to pro-apoptotic stimuli include the human A549 non- small-cell lung (NSCLC) cancer (Mijatovic et al., 2006) and the human U373n glioblastoma (Branle et al., 2002) cell lines. The rodent cancer cell lines used include the mouse B16F10 melanoma (Van Goietsenoven et al., 2010) and the 9L gliosarcoma (Lefranc et al., 2002) cell lines. PLTX target preferentially the Na pump which in rodents displays double mutation in the alpha- 1 subunit with 100-1000 times decreases in sensitivity to NaK inhibitors like the cardenolide UNBS1450, when compared to human cells (Mijatovic et al., 2007a and b; Lefranc et al., 2008). Rodent cancer cell lines have thus been assayed to assess PLTX in vitro anticancer activity. The human HBL-100 epithelial cell line is also included. It is reported as a normal cell line, while it has been transformed by means of several oncogenes to render it immortal. Thus, from a biological point of view, this cell line cannot be considered as actually normal.

The cells are cultured at 37°C in sealed (airtight) Falcon plastic dishes (Gibco, Nunc, France) containing the medium, as indicated in Table 2. All media are supplemented with a mixture of glutamine (0.6 mg.mL "1 final concentration; Gibco), penicillin (200 IU.mL "1 final concentration; Gibco), streptomycin (200 IU.mL "1 final concentration; Gibco), and 0.1 mg.mL "1 gentamycin (Gibco). The FBS and FCS are previously heated for 1 h at 56 °C.

Table 2: Cell lines and media

Cell lines Histological type Cell culture media (Co) Origin

Hs683 Oligodendroglioma (human) RPMI + 10% FBS (LTI) ATCC code HTB- 138

U373n Glioblastoma (human) RPMI + 10% FBS (LTI) ECACC 08061901

9L Gliosarcoma (rat) RPMI + 10% FCS (LTI) ATCC code CRL-2200

B16F10 Melanoma (mouse) RPMI + 10% FBS (LTI) ATCC code CRL-6475

A549 Lung carcinoma (human) RPMI + 10% FBS (LTI) DSMZ code ACC107

HBL100 Human epithelial transformed cells RPMI + 10% FBS (LTI) CLS code 330178

NHDF Normal human dermal fibroblast MEM + 10% FBS (Gibco) PC code c- 12300

(juvenile foreskin)

MSC Normal human fibroblast primocultures FGM-2 Bullet it(Lonza) Gift from

Prof J. Dubois (*) ATCC, American Type Culture Collection (Manassas, USA); ECACC, European Collection of Cell Cultures (Salisbury, UK); DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany); CLS, Cell Line Services (Eppelheim, Germany); PC, PromoCell (Europe). RPMI, Roswell Park Memorial Institute (from LTI, Life Technologies- Invitrogen); MEM, Modified Eagle Medium (from Gibco); FBS, Fetal Bovine Serum; FCS, Fetal Calf Serum.

* Address: Laboratoire de Chimie Anal yti que, Toxicologic et Chimie Physique Appliquee, Faculte de Pharmacie, Universite Libre de Bruxelles (Belgium).

Leukaemia cell lines and culture Histiocytic lymphoma U937, chronic myelogenous leukaemia K562, T-cell leukaemia Jurkat were purchased from DSMZ (Braunschweig, Germany). MOLM14 was provided by .... Cells were cultured in RPMI 1640 or DMEM medium (Lonza, Verviers, Belgium) supplemented with 10 % (v/v) fetal calf serum (FCS; Lonza, Verviers, Belgium) and 1 % (v/v) antibiotic-antimycotic (BioWhittaker, Verviers, Belgium) at 37 °C and 5 % of C02. Experiments were performed in culture medium containing 10% of FCS with cells in exponential growth phase. Cells were routinely controlled to exclude mycoplasma contamination. Non-adherent cells were seeded in fresh complete medium at concentrations of 300,000 cells/ml. After 1 h of recovery in the incubator, they were treated at the indicated concentrations. PLTX purification from Palythoa aff. clavata polyps:

One gram of fresh Palythoa aff. clavata polyps is gently detached from the stone are dried out on paper, chopped with a scalpel into several pieces and further placed in a 50 mL- Falcon tube containing 20 mL of 80% methanol in milli-Q water. After agitation during 10 h at 4°C with a magnetic stirrer and decantation, the liquid is removed with a glass pipette and is then centrifuged (8000 g, 20 min) or filtrated on a 0.45 μηι PTFE membrane. The pellet is rinsed with water, centrifuged and the supernatants pooled. Methanol from the solution is then evaporated (rotavapor Buchi) and the resulting aqueous phase extracted twice with hexane or dichloromethane for removing pigments mainly composed of carotenoids. The aqueous phase is rapidly evaporated and deposited onto a two centimeters diameter glass column filled with 10 cm 3 of Ci 8 reversed phase powder (Lichroprep ® RPig, from MERCK, code 1.09303.0100, France). The column is washed with acidified water (0.2% v/v formic acid), then with 50% MeOH in the same acidified water, the PLTX is finally eluted with 75% MeOH in acidified water. The yield of the purified PLTX is first evaluated by HPLC in this liquid fraction or by weighing the tube after evaporation to dryness of this fraction. Results are means of three determinations. Routinely, preparations containing 100 μg dry PLTX are kept at -20°C in sealed glass flasks. For in vitro growth inhibitory assays and videomicroscopy experiences, a stock solution of 0.01 M in dimethylsulfoxide is prepared, PLTX remaining stable over one month in this solution.

PLTX purification from S mbiodinium dinoflagellate cells:

The Symbiodinium dinoflagellate culture is centrifuged at 3000 g at 4°C for 10 min. The supernatant is removed, the pellet resuspended with ice-cold milli-Q water and the solution further homogenized by sonication (ultrasonic Vibra-Cell equipment). Cell disruption is assessed by microscopy. After centrifugation of the extract at 12000 g for 10 min, the PLTX present in the supernatant is further purified by reverse phase chromatography on a Lichroprep ® RPi , as described previously.

Compounds and treatment

All compounds were from SIGMA-ALDRICH (Bornem, Belgium) unless otherwise stated. Compounds were solubilized in dimethyl sulfoxide (DMSO) and stored at -20°C. Cycloheximide (CHX) was diluted in sterile water at 1 mg/ml and stored at -20°C. Cells were pre-treated before adding 10 μ©¾η1 CHX. MG132 was diluted in DMSO at a stock concentration of 5 mM. For the proteasome activity assay, U937 cells were incubated at indicated concentrations for 2 h before the measurement (see section below). For protein stability assays, U937 cells were incubated for 6h before adding 5μΜ MG132. Samples were collected for further analysis at the indicated times. For the analysis of apoptosis parameters, cells were treated before adding 5 μΜ MG132 and samples were collected after 6 h. The pan- caspase inhibitor I z-VAD (OMe)-FMK (zVAD; Calbiochem, Leuven, Belgium) was diluted in DMSO at a concentration of 50 mM and added 1 h before treatment at the final concentration of 50 μΜ.

Molecular methods and phylogenetic analysis: DNA extraction:

In order to amplify both Palythoa and Symbiodinium genes, tissue sample collected for DNA extraction consisted of five tentacles joined by a small piece of polyp oral disc. Because symbiotic cells are much less abundant than coral cells, such a strategy results in a high concentration of Symbiodinium DNA compared to coral DNA, thus increasing the successful amplification of Symbiodinium genes. Genomic DNA is extracted using the Gentra Puregene Tissue Kit (Qiagen) according to the following modified protocol:

Day 1

1. Cut 5 mm fresh tissue and place it into a 1.5 mL microcentrifuge tube,

2. Add 300 \iL cell lysis solution,

3. Add 4 iL proteinase K and mix by inverting,

4. Incubate for 45 min at 65°C,

5. Incubate at 56°C overnight.

Day 2

6. If tissue is not totally digested, add 4 μΙ_ proteinase K and incubate for 30 min at 65°C,

7. Add 100 μΐ ^ protein precipitation solution and vortex for 20 s at high speed,

8. Incubate for 15 min at 4°C,

9. Centrifuge for 5 min at 13000 rpm,

10. Pipet 300 isopropanol into a clean 1.5 mL microcentrifuge tube and add the supernatant from the previous step by pouring carefully,

11. Mix by inverting 5-6 times,

12. Centrifuge for 5 min at 13000 rpm,

13. Carefully discard the supernatant, and drain the tube by inverting on a clean piece of absorbent paper, taking care that the pellet remains in the tube,

14. Add 300 μΐ, of 70% ethanol and mix by inverting carefully,

15. Centrifuge for 5 min at 13000 rpm,

16. Carefully discard the supernatant, and drain the tube by inverting on a clean piece of absorbent paper, taking care that the pellet remains in the tube. Allow to air dry for up to l h,

17. Add 50 μΐ. DNA hydration solution and mix by inverting,

18. Incubate at room temperature for 1 h to dissolve the DNA,

19. Mix by inverting and incubate at 4°C overnight,

20. Store at -20°C. After extraction, DNA solution is diluted to a concentration of 40 ng^L "1 before the polymerase chain reaction (PCR).

Gene amplification:

Two traditional DNA barcoding markers are used for genotyping the Palythoa species, one nuclear (ITS-rDNA) and the other mitochondrial (COI). ITS-rDNA sequence of Palythoa (18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; 28S ribosomal RNA gene, partial sequence) of approximately 750-850 base pairs is amplified using the primers Zoanf- ITS (5' - CTT GAT CAT TTA GAG GGA GT - 3 '; SEQ ID No: 1) and Zoanr-ITS (5' - CGG AGA TTT CAA ATT TGA GCT - 3'; SEQ ID No: 2). The PCR program is carried out according to the following parameters : an initial denaturing step at 94°C for 3 min, followed by 35 cycles of 1 min denature at 94°C, 1 min annealing at 50°C, 2 min extension at 72°C followed by 10 min extension at 72°C.

The portion of the mitochondrial COI gene of approximately 650 base pairs was also amplified for the Palythoa, with the following primers: HC02198 (5' - TAA ACT TCA GGG TGA CCA AAA AAT CA - 3'; SEQ ID No: 3) and LCO1490 (5' - GGT CAA CAA ATC ATA AAG ATA TTG G - 3'; SEQ ID No: 4). PCR amplification is performed with the following parameters: an initial denaturing step at 94°C for 2 min followed by 5 cycles of 15 s denature at 92°C, 45 s annealing at 48°C and increasing of 24°C in 1 min, 1 min 30 extension at 72°C, followed by 30 cycles of 15 s denature at 92°C, 45 s annealing at 52°C, 45 s extension at 72°C, followed by 7 min extension at 72°C.

For genotyping the Symbiodinium clade living in the Palythoa tissues, ITS2-rDNA sequence (5.8S ribosomal RNA, partial sequence; internal transcribed spacer 2, complete sequence; 28S ribosomal RNA gene, partial sequence) of approximately 250-300 base pairs is amplified using the following specific primers ITS2-F1 (5' - GAA TTG CAG AAC TCC GTG - 3'; SEQ ID No: 5) and ITS2-R2 (5' - ATA TGC TTA AAT TCA GCG GGT - 3'; SEQ ID No: 6). PCR amplification is performed under stringent conditions for targeting the Symbiodinium genes instead of coral ones: an initial denaturing step at 94°C for 3 min followed by 12 cycles of 45 s denature at 94°C, 45 s annealing at 58°C and decreasing 0.5°C every cycle, 1 min extension at 72°C, followed by 20 cycles of 45 s denature at 94°C, 45 s annealing at 52°C and 1 min extension at 72°C followed by 7 min extension at 72°C.

PCR products are visualized by denaturing gradient gel electrophoresis. All fragments are sequenced in both directions using the amplicon primers. Phylogenetic analyses and DNA bar coding:

All sequences are first checked using NCBI BLAST then inspected by eye and aligned with orthologous sequences available in public Databases using CLUSTALW implemented in BioEdit 7.1.9 (http://www.mbio.ncsu.edu/bioedit/bioedit.html). Final alignments for phylogenetic reconstructions based on the Symbiodinium ITS2-rDNA sequences are done using Muscle (Edgar, 2004). For analyses of coral sequences, data from Terrazoanthus, Hydrozaonthus and Parazoanthidae genera are used as outgroups. Phylogenetic reconstructions based on Symbiodinium sequences are unrooted. Final alignments for both coral genes (ITS-rDNA and COI) and the single Symbiodinium gene (ITS2-rDNA) are 937, 462 and 332 nucleotides long respectively. Each data set is exported in Mega 5.1 (Tamura et al, 2011) and the best-fit models of DNA evolution are searched using MODELTEST. The evolutionary model with the lowest Bayesian Information Criterion (BIC) is selected. Alignments are subjected to phylogenetic analyses with the maximum likelihood method (ML). Phylogenetic reconstructions are performed using 100 replicates with partial deletion of 75%. For Symbiodinium ITS2-rDNA data set, the distances are calculated using a Kimura's 2- parameters model incorporating a discrete gamma distribution with five categories (K2 + G5). For coral COI data set, a Kimura's 2-parameters model (K2) is used while for ITS-rDNA data set, a Kimura's 2-parameters model incorporating invariable sites with a discrete gamma distribution is used (K2 + G5 + I).

HPLC control and quantification of the toxin:

To control the purity and quantify the purified PLTX, 2 μg of the sample diluted in milli-Q water are injected and analyzed by reverse-phase HPLC with a Waters equipment composed of a 1525 binary pump, a 2996 diode array detector, a Rheodyne injector (7725i model) fitted with a 20-μ1 loop, and a temperature control system. Files are acquired by the Empower software. Separations are carried out on a Symmetry C 18 reversed phase column (4.6 x 100 mm, ODS2, 5 μιη) from Waters and protected with a guard cartridge. The elution is performed at 30°C at a flow rate of 0.8 ml/min and using a linear gradient of methanol (A) in acidified water with 0.2% (v/v) acetic acid (B), from 5 to 100% A during 20 min. PLTX is visualized at 263 nm and total spectra analyzed from 200 to 600 nm by using the Empower software (from Waters) in order to control the purity. For quantification, a standard calibration is established with commercial PLTX (from Wako Pure Chemical Industries, Japan). HPLC data are averages from two determinations. Mass spectrometry identification and control:

Matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) analysis are performed on a Microflex II mass spectrometer (Bruker, Germany). A 10 mg/mL solution of 2,5-dihydroxybenzoic (DHB) acid in 70/30 acetonitrile/water 0.1 % trifluoroacetic acid is used as matrix (Paz et al., 2011). From a solution of 1 mg/mL of purified toxin in milli-Q water, 0.8 are mixed to 0.8 μL of the matrix solution. Mixtures are deposited as adroplet and allowed to dry at room temperature. Data are acquired in a positive reflectron mode, range is set from 600 to 5000 Da and pulsed ion extraction fixed to 150 ns. Ions formed upon irradiation by a smartbeam laser using a frequency of 200 Hz. The laser irradiance is set to 45-50% (relative scale 0-100) arbitrary units according to the corresponding threshold required for the applied matrix system. External mass calibration is done just before the acquisition of the sample using peptides calibration standard (Bruker Daltonics). Mass spectra are treated with the Flex Analysis software (Bruker, Germany) and no smoothing or baseline subtraction is done.

Purified PLTX is also analysed and controlled by ESI-MS/MS on a Q-ToF Synapt Gl High Definition mass spectrometer (Waters, UK) mounted with a nano spray ionization source and a V-mono-reflectron, in the positive mode. The following source settings are used: capillary voltage 3.2 kV, sampling cone 40 V, extraction cone 4 V, source temperature 120°C, desolvation gas flow 200 L.h "1 (N2) at a temperature of 150°C, trap collision energy 38 eV. The calibration is performed with a solution of Csl 1 mg.mL "1 and used in the 100-3200 mass range with a precision of +/- 3 ppm. A solution of purified PLTX (1 mg.mL "1 ) in MeOH/HiO (1 : 1) is diluted 25 times (15 μΜ final concentration) and injected via a syringe pump in the nano-source at a flow rate of 3 μί.ηιίη "1 .

NMR control:

The complete chemical shift assignment ofΉ signals is compared to that of Kan et al. (2001). Deuterated methanol (CD 3 OD) are chosen for it gave much sharper signals than in deuterated water (D 2 0), as already observed (Kan et al., 2001).

MTT colorimetric assays for in vitro growth inhibitory effects:

The colorimetric MTT assay measures the number of metabolically active, living cells that are able to transform the yellow product 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into the blue formazan dye via mitochondrial reduction. The amount of formazan obtained at the end of the experiment, measured via spectrophotometry, is directly proportional to the number of living cells. Therefore, determination of the optical density enables a quantitative measurement of the effect of the investigated compound when compared with the control condition (untreated cells). The cell lines and culture media used are described in Table 2. To perform the assay, cells (5000 to 8000 cells/well depending on the cell line examined) are grown in a 96-well, flat-bottomed plate in 100 μΐ, of media. Each cell line is seeded in its appropriate culture medium. Cells seeded are further treated for 72 h with different concentrations of purified PLTX ranging from 1 pM to 1 μΜ, with semilog concentration increasing. At the end of the incubation, blue formazan is solubilized with dimethylsulfoxide, and absorbance at 570 nm which is directly proportional to the number of living cells, is determined according to the method of Frederick et al. (2012). The IC 50 concentrations are calculated from 96-well plates in which each experimental condition has been carried out in six replicates (one column of 6 wells). The control condition has been run two times in six replicates (two columns in the 96-well plates).

Quantitative videomicroscopy:

Direct visualization of PLTX-induced effects on the cell proliferation and morphology of human Hs683 and U373n glioma cells is performed by means of time-lapse computer- assisted phase contrast microscopy, i.e. quantitative videomicroscopy. Specialized software packages are developed and implemented on computers connected to phase-contrast videomicroscopy systems placed in 37°C-heated incubators, as detailed by Debeir et al. (2008). Digitized images are acquired under a phase-contrast microscope (Olympus model 1X50, with a 10: 1 magnification ratio) using a digital 3.0M-PIXEL-USB 2.0 camera (model CG-032, Sharp Vision, China) driven by in-house-developed time-lapse software (Debeir et al., 2008) with a resolution of 1024x768 pixels. An image is acquired every 4 min over a period of several hours. Movies are generated from these digitized images to enable a rapid viewing of the cell behavior for the duration of the experiments in control versus treated experimental conditions. Experiments are carried out in human Hs683 and U373n glioma cell lines in presence of PLTX at 0.01 and 1 nM. Each image represents the first image of each film that has been set up thanks to this approach for observation periods lasting from 3 to 22 h. One image is digitized every four minutes during the period of observation (thus 15 digitized images are recorded each hour) and the experiments are carried out in tetraplicate.

2) Results Morphological description of the PLTX-producing Palythoa:

After the finding of a highly toxic Palythoa sp., the zoanthid and the toxin are identified. As seen in Figure 1 (A) and (B), the Palythoa is small (1-2 cm wide), greenish solitary, anemone-like hexacorals with white stripes. The button-like body comprises a broad oral disc which is brown with green or blue overtones of 10-15 mm in diameter and a light center. It is surrounded by a short fringe of tentacles distributed in two distinct rows. The body and tentacules are punctuated with white dots. This zoanthid could correspond morphologically and genetically to a specimen recently described in Reimer et al. (2012). Phylogenetic analysis showed that it corresponds to Palythoa aff. clavata belonging to the family Sphenopidae from the Zoantharia order.

Toxin purification and quantification:

The purification process performed to obtain pure PLTX from this Palythoa aff. clavata is described above in the section Materials & Methods.

Three experiments are done for evaluating the PLTX production yield by this zoanthidea. Palythoa aff. clavata is found to produce 2.81 ± 0.45 mg of PLTX/g wet polyp, as determined by HPLC. The high standard deviation recorded is probably due to the variable weight of the wet Palythoa aff. clavata. In any case, the recorded PLTX value (0.28% w/w) is the highest in regard to the literature (see Table 1) and corresponds to ten times more than the first value of 0.027% recorded by Moore and Sheuer (1971) for Palythoa toxica. In Palythoa heliodiscus, it has been found 1164 μg/g wet zoanthid for PLTX, and 3500 μg/g wet zoanthid for an analogue of PLTX, deoxy-PLTX (Deeds et al., 2011). Chemical structure determination of the palytoxin:

The purity of the obtained PLTX is controlled by DAD-HPLC all along the UV- visible range from 200 to 600 nm, no pigments or other compounds are detected even at low wavelengths. The purified toxin displayed two characteristic UV bands at 233 and 263 nm, as observed by Katikou (2007). In this case, PLTX is eluted as a symmetrical peak at within 80% methanol on our HPLC column (Figure 2), and its purity reached approximately 99% in regard to its mass spectra (Figure 3, and also Deeds et al. (2011); Ciminiello et al. (2011) that gave identical fragmentations) and 1H-NMR data (data not shown, see Kan et al. (2001)). By using the optimized matrix of Paz et al., 2011, composed of 2,5-DHB in a 0.1% TFA/ACN mixture which gives better resolution than with a 2,5-DHB or HCCA (a-cyano-4- hydroxycinnamic acid) matrix, MALDI-ToF mass spectrometry in the positive mode shows single charged molecules containing mainly Na + ions, and with a weak presence of K + ions. The purified toxin shows a clear ion profile with a prominent ion at m/z 2701.421 [M + Na] + , thus identically to Paz et al., and corresponding to a molecular mass of 2678.43 (Figure 3). Together with this main molecular ion, minor adducts at m/z 2683.359 [M + Na - H 2 0] + , 2665.262 [M + Na - 2H 2 0] + , and 2717.396 [M + ] + are noted (Figure 3), confirming the high degree of purity of this toxin. ESI-tandem mass spectrometry analyses on a Q-ToF instrument are also performed to confirm the chemical structure of the purified PLTX, revealing a very complex ion pattern with multiply charged states (not shown). The cleavage between carbon 8 and 9 of the PLTX is demonstrated by the loss of the A moiety (see Scheme 1) which is recovered as a single fragment ion [M + H] + m/z 327.126 in the full MS spectra, and corresponding to a C 16 H 27 N 2 0 5 part formula generally observed in PLTX (data not shown). Other characteristic tri- and bi-charged ions are notably pointed at m/z 906.495 [M + 2H + K] 3+ , 1351.750 [M + H + Na] 2+ , and 1359.741 [M + H + K] 2+ (data not shown). Further, MS/MS assignations confirmed the presence of a unique molecule and the chemical structure of the PLTX (Ciminiello et al., 2011; Deeds et al., 2011). The calculation based on these tri- and bi-charged ions, together with the MALDI-ToF data, attributed to the PLTX of Palythoa aff. clavata a molecular weight of 2678.48, thus similar to that of PLTX of Palythoa tuberculosa.

Palythoa species identification (ITS-rDNA and COI genes):

The Palythoa genus is paraphyletic in the tree based on the ITS-rDNA data set and only poorly recovered as monophyletic (bootstrap value, bv = 58) within that based on the COI mitochondrial gene. According to the ITS-rDNA phylogenetic reconstruction (Figure 4), two paraphyletic lineages of Palythoa are recovered with high support values, one contained Palythoa grandis, Palythoa variabilis and Palythoa heliodiscus (bv = 100) while the second consisted in a clade formed by Palythoa aff. clavata, Palythoa caribaeorum, Palythoa mutuki, Palythoa caesia, Palythoa tuberculosa and Palythoa grandiflora (bv = 100). The new sequence obtained from our specimen of Palythoa (PtOl) grouped within the latter as the sister group to Palythoa aff. clavata with high support value (bv = 100).

The COI gene analysis (Figure 5) provided a consensus tree less accurate than with the ITS-rDNA sequences. The monophyly of Palythoa is recovered but with low support (bv = 58) and only one of the two lineages highlighted in the ITS-rDNA is supported with high bootstrap value. Indeed, Palythoa heliodiscus, Palythoa variabilis and Palythoa grandis still show highly supported phylogenetic relationships (bv = 91), but the second Palythoa lineage is not supported because all the species belonging to this second clade appear in a paraphyletic assemblage. This concerns Palythoa aff. clavata, Palythoa mutuki, Palythoa grandiflora, Palythoa tuberculosa, Palythoa caribaeorum and PtOl .

Symbiodinium clade typing (ITS2-rDNA gene):

Internal transcribed spacer 2 (ITS2-rDNA) typing results from Symbiodinium sp. are shown in Figure 6. After the sequencing step, the ITS2-rDNA sequence displayed clear chromatograms in both forward and reverse directions with no 'double-peaks' so no cloning step is performed to investigate a putative intragenomic variability and/or the presence of several Symbiodinium species.

Maximum likelihood analysis of ITS2-rDNA sequences isolated from various zooxanthellate metazoans including sponges, foraminiferans, molluscs and cnidarians as well as free-living dinoflagellates produced a well-resolved consensus tree in which the main Symbiodinium clades previously described in Cnidarians received high support values (bv = 93, 100, 87, 100, 100 and 95 for clades A-G respectively). The sequences from clade F, which is only described in foraminiferans, are not included. Symbiodinium ITS2-rDNA sequence isolated from the Palythoa specimen used in this study (ex PtOl) is unambiguously placed within the generalist clade C (bv = 87), within a large group that included many subclades of C previously observed in a variety of hosts, including isolates from other cnidarians such as scleractinians, octocorallians and zoanthids. Growth inhibitory effects in vitro and ICsn determination:

The MTT colorimetric assay-related data obtained are detailed in the Table 3 and Figure 7. The data reveal that the normal cells (the MSC and NHDF fibroblast cell lines) displayed ~10 6 lower in sensitivity in term of PLTX-mediated in vitro growth inhibition than cancer cells. The data also reveal that the HBL-100 transformed cell line behaved as a cancer, not as a normal cell line. The difference of ~10 6 lower sensitivity of normal cells in terms of PLTX-mediated in vitro growth inhibition when compared to cancer cells suggest that the main cellular target of PLTX is not the NaK if one refers to the data from Mijatovic et al. (2007a) and Lefranc et al. (2008). The data further reveal that the rodent cell lines (surrounded in grey, see Table 3) display similar sensitivity to PLTX-mediated in vitro growth inhibition when compared to human cells, a feature that is once more in favor of the fact that the NaK would not be the primary/main cellular target of PLTX when exerting its in vitro growth inhibitory effects. Indeed, the IC 50 data given in Table 3 must be compared to those obtained by Mijatovic et al. (2007a and b). The data obtained by means of the MTT colorimetric assay with PLTX in terms of in vitro growth inhibitory effects in human normal versus rodent and human cancer cell lines were highly reproducible. Figure 7 (A, B and C) illustrates a graph (percentage viable cells versus PLTX concentration) obtained for calculation of the IC50 for cancer and normal cells, after reading the 96-well plates including various types of cells treated for 72 h with PLTX (not shown).

Table 3: IC 50 in vitro growth inhibitory concentration by 50% after having cultured the cells with PLTX for 72 h. For calculation of the mean, the IC 50 value for HBL-100 cell line is included here. In rodents (rat and mouse, in grey), the NaK displays double mutation in the alpha- 1 subunit with 100-1000 times decrease in sensitivity to NaK inhibitors (Majatovic et al., 2007a and b; Lefranc et al., 2008). r, resistant to; s, sensitive to pro-apoptotic stimuli; pM, picomolar.

Pro-apoptotic

Cell line Normal / Cancer Origin IC50 (in stimuli PM)

MSC (fibroblast primoculture) - Normal Human >10 μΜ

NHDF (dermal fibroblast) - Normal Human >10 μΜ

Normal but transformed cells

HBL-100 (mammary epithelial) - Human 0.645 for immortalization

A549 (lung carcinoma) r Cancer Human 0.665

Hs683 (glioma) s Cancer Human 0.575

U373n (glioma) r Cancer Human 0.560

9L (gliosarcoma) - Cancer Rat 0.390

B16F10 (melanoma) s Cancer Mouse 0.440

Human and

Mean ± SEM 0.545 ± 0.05 murine

These morphological illustrations indicate that normal cells are less sensitive than cancer cells to the PLTX-mediated in vitro growth inhibitory effects, and that rodent cancer cells display similar sensitivity when compared to human cancer cells to these PLTX- mediated in vitro growth inhibitory effects. Various data from the literature also report that ouabain-related in vitro IC 50 growth inhibitory effects are in the 10-100 nM ranges of concentration (Mijatovic et al., 2007a and b), thus 10 4 to 10 5 higher than the < 10 "12 M (< 0.001 nM) IC 50 concentration evidenced by the Inventors for PLTX in various cancer cell lines.

Growth inhibitory effects on cancer cells by videomicroscopy:

Figure 8 illustrates the morphological pictures obtained with 0.01 and 1 nM PLTX on human Hs683 oligodendroglioma cells.

The concentration of 0.01 nM (i.e. 10 pM) represents -10 times the IC 50 in vitro growth inhibitory concentration associated with PLTX, while 1 nM represents -1000 times this IC 50 concentration (see Table 3). The IC 50 concentrations have been calculated after having cultured the various normal and cancer cells for 72 h with PLTX. Figure 8 shows that Hs683 cells began to die about 3 h after having been treated with 0.01 nM, while all the Hs683 cells already died at 1 h post-treatment with 1 nM PLTX. The morphological appearances of Hs683 cells treated with 1 nM PLTX for 1 h are typical of cell swelling and bubbling occurring after the impairment of various ion channels, not only NaK.

Figure 9 illustrates the morphological pictures obtained with 0.01 and 1 nM PLTX on human U373n glioblastoma cells using experimental conditions identical to those used for analyzing the behavior of Hs683 oligodendroglioma cells (Figure 8). As for Hs683 glioma cells, the concentrations used, 0.01 and 1 nM, are -10 and -1000 times the IC 50 in vitro growth inhibitory concentration associated with PLTX (Table 3). Figure 9 shows that U373n behaved as Hs683 when treated with PLTX, but with some delays. Indeed, U373n cells begin to die about 5 h (instead of 3 h for Hs683 cells; Figure 8) after having been treated with 0.01 nM PLTX, while all the U373n cells die 3 h (instead of 1 h for Hs683 cells; Figure 8) post- treatment with 1 nM PLTX.

The morphological appearances of U373n cells treated with 1 nM PLTX for 3 h are once more typical (as for Hs683 cells; Figure 8) of cell swelling and bubbling occurring after the impairment of various ion channels, not only NaK.

Growth inhibitory effects on leukaemia cell lines in vitro and IC n determination: PLTX was then further tested for the cell viability and proliferation on different human leukaemia cell lines corresponding to K562, JURKAT, U937 and MOLM14.

The results established that PLTX induces apoptosis in said leukaemia cell lines by down-regulating anti-apoptotic proteins, such as BID, MCL-1, BCL-2 and BCL-xL, via a proteasome-dependent degradation, and by inducing caspase activation (data not shown). The data further reveal that in spite of the teaching of QUINN et al. (1974), PLTX reveals a strong growth inhibitory effect after a short time of culture, and this regardless of the human leukaemia cells lines used (Table 4).

Table 4: IC50 in vitro growth inhibitory concentration by 50% after having cultured the cells with PLTX for different time (from 2 to 8 h). pM, picomolar.

Nd: not determined

Finally, the inventors thus established that the great potential of PLTX as an antitumor agent for the treatment of leukaemia. Simultaneously, the inventors confirms this potential by establishing a low PLTX toxicity on human PBMC with a maximum of 20% lethality at 100μΜ of PLTX (data not shown).

In vivo safety and pharmacokinetics study:

Despite its potential toxicity, the toxicological profile of the molecule is poorly understood, with the exception of the work disclosed in Del Favero et al. (2013) using oral administration.

For estimating the toxicity of PLTX by other administration routes, mice were administrated with different doses either subcutaneously (s.c.) intravenously (i.v.) and their survival was determine five days post-injection.

Table 5: mice survival after different PLTX doses (10 to 10,000 μg/kg) by s.c. or i.v.

administration.

PLTX (ng/kg) s.c. i.v.

10 Nd OK

30 OK Nd

100 OK Nd

300 OK Nd

1,000 Nd OK 2,000 OK Nd

10,000 Death Nd

Nd: not determined

The results show that no animal lethality was observed until a 10 μ /Ι^ administration, the animal surviving without visible adverse effects to a 2 μg/kg PLTX administration (Table 5). These results are in agreement with those presented in Del Favero et al. (2013) establishing an estimation of the NOAEL (no observed adverse effect level) to an oral administration of 3 μg/kg/day of PLTX. This result was confirmed by s.c. daily PLTX administration for 15 days to mice with an initial 2 μg kg administration (data not shown).

The PLTX pharmacokinetics was further determined by a s.c. PLTX administration of 2 μg/kg to two mice. Then, PLTX plasma concentration was determined at 30 min, lh and 3 hours following PLTX injection.

Table 6: plasma PLTX doses (10 to 10,000 μg/kg) by s.c. or i.v. administration.

The results shows a maximal PLTX plasmatic concentration at lh following the injection, which concentration rapidly decrease (table 6). This maximal plasmatic PLTX concentration corresponds to nearly 100 fold more than the efficient PLTX concentration on leukemic human cell lines.

Xenografts and in vivo drug treatment assays:

NOD/SCID (nonobese diabetic/severe combined immunodeficient)/yc null mice (NSG) are obtained from CHARLES RIVER Laboratory.

The acute myeloid leukaemia luciferase-expressing U937 (0.2M in Ι ΟΟμί of PBS) cells are transplanted into caudal vein of mice (6-8 week-old male).

Next, control medium or increased concentration of PLTX (10, 50, 100, 250 ng/kg and 1 μg/kg) are injected subcutaneously to said mice, said administration being repeated 5 days a week until day 14 post injection. Leukaemia expansion is followed in said mice by bioimaging using a Photon Imager (BIOSPACELAB).

Luciferin is injected i.p. (30mg/Kg in 100 μΐ PBS), followed by analysis 10 min after injection on anesthetized (3% isoflurane) animals. When the first leukaemia-related symptoms are observed (hunch-backed, a significant weight loss or ruffled coat), the mice are humanely euthanatized and survival rates is estimated by the Kaplan-Meier method. Plasmodium falciparum sensibility to PLTX:

The study is performed with on P. falciparum clones with distinct anti-malarial resistance patterns.

To evaluate rapidly Plasmodium falciparum growth in Vitro in the presence of PLTX, [3H]hypoxanthine is added to parasite microcultures and radioisotope incorporation is measured in the presence or absence of PLTX.

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