ITRM20120573A1 - FUNGINO EXTRACT WITH ANTITUMOR ACTIVITY - Google Patents

Description

"Fungal extract with anticancer activity"

Field of Invention

The present invention relates to a fungal extract with antitumor activity. In particular, the extract consists of a purified polysaccharide fraction characterized by the filtrate of a culture of the basidiomycete Trametes versicolor. The invention also relates to the compositions that comprise it and its use as an antitumor, in particular its use as an antiproliferative and proapoptotic on cultured tumor lines and primary tumors, i.e. in the treatment of various types of neoplasms, including oncohematological ones.

Note Art

Numerous studies are known which have shown that a category of compounds of fungal origin, represented by the secondary metabolic products of fungi, is also characterized by interesting cytotoxic activities (antibacterial, antifungal, etc). In particular, the use of basidiomycetes, edible fungi that are consumed in the diet in the form of herbal teas and decoctions, has been studied. In fact, in Asian countries a variety of fungi such as Trametes versicolor, Lentinula edodes, Grifola frondosa, Ganoderma lucidum and Schizophillum commune are used as â € œmedicinal mushroomsâ € against various pathologies (Wasser and Weis, 1999). Some compounds such as thioproline, mannitol and β-glucans, present in mycelia, fruiting bodies and cultural filtrates of these basidiomycetes have antioxidant activity. Furthermore, the polysaccharide compounds and glycoproteins present in these mushrooms have shown biological anticancer, antibacterial, antiviral and liver protective activities (Kobayashi et al., 1993; Sun et al., 2004). However, the development of their application has not progressed very far due to the fact that, in the majority of cases, the fractions used were not purified. For this reason, many results appeared contradictory and it was not possible to accurately measure their possible toxicity and the repeatability of their effects.

In particular, the non-toxic basidiomycete Trametes versicolor, a fundamental component of oriental medicine, is an important stimulating agent for the immune system and also an antioxidant, antibacterial, and is the best-selling anticancer drug in Japan, also used in association with surgery, chemotherapy and radiotherapy.

Trametes versicolor contains two very important polysaccharide classes, protein bound K polysaccharides (PSK) and polysaccharopeptides (PSP) (Maeda et al., 1974; Chihara, 1990, 1992; Kim et al., 1999; Sia et al., 1999; Fisher and Yang, 2002), which have been tested in in vitro models and in animals and have shown a stimulation of the immune response through the activation of lymphocytes and the ability to control peroxidative processes (Yuan et al., 1996; Slamenova et al., 2003). PSP and PSK are extractable substances in decoction, strongly immunostimulating, antibacterial, antiviral, anticancer especially in immune-dependent tumors (mammary, prostatic), and viral etiology.

Trametes versicolor treats chronic conditions and degenerations, but above all it is indicated in CFS (Chronic Fatigue Syndrome) for its ability to significantly increase vital energy, moreover it is valid, with remarkable results, in all forms of expression of Sycosis: miasma that predisposes to pathologies of toxic impregnation expressed by the skin in the form of vegetations, warts, warts, papillomas and epithelial tumors.

It is also indicated for side effects found in chemotherapy and radiotherapy, such as those that produce Candidiasis, Viral diseases, Hepatitis C, HIV, Herpes, CFS, Tumor pathologies, Multiple Sclerosis, Anemia, Diabetes type II, AIDS, Autoimmune diseases, Fibromyalgia, Rheumatoid Arthritis, Toxoplasmosis, etc.

Patent application US 20060045887 describes compositions based on a crude extract obtained from a mycelium extracted from at least one basidiomycetus, a medicinal mushroom selected from the group consisting of Ganoderma adspersum, Hypsizygus ulmarium, Kuehneromyces mutabilis, Omphalotus olearius, Panus conchatus, Piptoporus betulinus, Pleurotus eryngii, and Trametes zonata. It is reported that these extracts have antiproliferative activity against human neoplastic cell lines, particularly those of chronic myeloid leukemia (K562) and prostate cancer (LNCaP).

Compared to US patent application 20060045887, our invention proposes the use of a polysaccharide fraction produced in the culture filtrate from a specific isolate of Trametes versicolor from which the biologically active fraction has been determined. US 20060045887 proposes the use of crude extracts obtained from different basidiomycetes, but it is not possible to compare crude extracts obtained from Trametes zonata mycelia with the purified extracts of Trametes versicolor. In the present invention a polysaccharide is reported, purified from an extract of a culture of the basidiomycete Trametes versicolor, subsequently characterized, usable in the pharmaceutical field, in particular as an antitumor, and which is particularly active against human leukemic cell lines and primary cells obtained from patients affected by various onco-haematological neoplasms. Summary of the invention

The object of the invention is the purified polysaccharide fraction, hereinafter also referred to simply as Trametan, obtained from the filtrate of cultures of basidiomycetes, in particular of the genus Trametes, more particularly Trametes versicolor.

Other objects of the invention are the following:

-) the purification method of this polysaccharide fraction; -) the compositions and formulations comprising such polysaccharide fractions which;

-) the uses of these polysaccharide fractions and in the medical field for prevention and therapy, in particular as antiproliferative and proapoptotic agents in the treatment of various types of neoplasms, in particular onco-haematological ones.

Particularly preferred mushrooms according to the invention are those deposited at the CABI Bioscience collection center, UK Center (IMI), United Kingdom, according to the Budapest Treaty.

Further objects will become apparent from the detailed description of the invention.

Brief description of the figures

Figure 1: Chromatogram of the TV117 sample obtained by separation on a Sephacryl S-300 column;

Figure 2: <1> H-NMR spectrum of the high molecular mass fraction; Figure 3: <1> H-NMR spectrum of the low molecular mass fraction;

Figure 4: Gas chromatogram of the highest molecular mass fraction. Glc = glucose; Gal = galactose; Man = mannose. The positions of the glycosidic bonds are indicated: t- indicates non-reducing terminal; the number preceding the residue indicates the carbon atom engaged in the glycosidic bond. Inositol is used as an internal standard;

Figure 5: Gas chromatogram of the lowest weight fraction. Glc = glucose; Gal = galactose; Man = mannose; Fuc = fucose. The positions of the glycosidic bonds are indicated: t- indicates non-reducing terminal; the number preceding the residue indicates the carbon atom engaged in the glycosidic bond. Inositol is used as an internal standard;

Figure 6: Chromatogram of separate TV117 sample by volume exclusion chromatography on Sephacryl S-300 column. Figure 7: <1> H-NMR spectra recorded at 25 ° C of the B and C fractions obtained by separation of the TV117 extract on a Sephacryl S-300 column;

Figure 8: Separate C peak chromatogram on Bio Gel P10 column in water;

Figure 9: <1> H-NMR recorded at 50 ° C of fractions 3-7;

Figure 10: <1> H-NMR recorded at 50 ° C of fractions 8-9;

Figure 11: <1> H-NMR recorded at 50 ° C of fractions 10-13;

Figure 12: <1> H-NMR spectra recorded at 25 ° C of peak B and peak C obtained from the Sephacryl S-300 column and of the fraction 3-7 obtained from the separation on Bio Gel P10 of the sample contained in peak C;

Figure 13: <1> H-NMR recorded at 25 ° C of the deacetylated Peak C;

Figure 14: <1> H-NMR recorded at 50 ° C of the deacetylated Peak C;

Figure 15: signal of the resonance of the protons of the CH3 group of the fucose residue;

Figure 16: resonance signals of the anomeric protons of peak sample C;

Figure 17: COZY NMR spectrum of the product from peak C to 50 ° C; Figure 18: HSQC NMR spectrum of the product from peak C to 50 ° C; Figure 19: TOCSY NMR spectrum of peak C at 50 ° C;

Figure 20: Volume exclusion chromatography of the TV117 sample on a Sephacryl S-300 column;

Figure 21: HP-SEC of the TV117 sample;

Figure 22. Growth curve obtained in three independent experiments in the Jurkat cell line exposed to scalar concentrations of Trametan (Polisac);

Figure. 23. Cell count reduction induced by scalar Trametan concentrations in blasts of AML patients; Figure. 24. Increase of apoptosis levels evaluated on blasts of patients with AML after 72-120h (a) and after 144-168h (b) of exposure to scalar concentrations of Trametan;

Figure. 25. Analysis of apoptosis levels (% subG0 / 1) in non-stimulated lymphomonocytes (a) and in those stimulated with PHA and subsequently exposed to scalar concentrations of Trametan (Polisac).

Detailed description of the invention

Legend:

Trametes versicolor: TV C

Malt extract (MEX)

CZAPEK yeast extract (1% w / v) (CZL)

Potato Dextrose Broth (PDB)

Potato Dextrose Agar (PDA)

o / n: all night

FROM: Dalton

Glc: Glucose, Man: Mannoose, Fuc: Fucose, Gal: Galactose, NMR: Nuclear Magnetic Resonance, GC: Gas Chromatography, GC-MS: Gas Chromatography Coupled to Detection with Mass Spectrometry, HP-SEC: High Pressure Chromatography by exclusion of volume, COZY: Correlation Spectroscopy NMR, TOCSY: Total Correlation Spectroscopy NMR, HSQC: Heteronuclear Single Quantum Correlation NMR.

The present invention relates to extracts obtained from Basidiomycetes (medicinal mushrooms), in particular the species identified as Trametes versicolor.

Said extracts are obtained starting from cultural filtrates after the growth of single fungal isolates in different cultural media, such as for example: MEX; CZL; PDB. Cultivation can typically be carried out for 14 days at 25 ° C in the dark under agitation (150 rpm).

Particularly preferred is the PDB medium (eg from suppliers Sigma-Aldrich, USA and Difco, USA).

On the fungal culture filtrate obtained by separation by centrifugation (3000 rpm for 15 min at 4 ° C), a precipitation is carried out with a polar non-solvent, for example methanol, ethanol, isopropanol, acetone, alone or in a mixture with each other or also mixed with water. Preferred blends are water / ethanol.

To obtain the best production of polysaccharides, different fungal isolates of TV grown in PDB (potato dextrose broth) were tested after incubation at 25 ° C for 14 days. The fungal inoculum was 1 ml of PDB containing 1 gr. of mycelium of the different VTs tested obtained from 10-day PDA colonies stored at 25 ° C.

Table 1 shows the total quantities of polysaccharide produced (mg / L) by different TV isolates inoculated in PDB and incubated at 25 ° C for 14 days

Table 1

Quantification of the polysaccharides produced in various culture filtrates from T. versicolor inoculated in PDB and incubated under stirring conditions at 25 ° C for 14 days.

Isolate Quantity of extract (mg / l) Polysaccharides / reducing sugars (%)

TV A 279 ± 25 3.11 ± 0.5

TV B 347 ± 32 3.81 ± 0.4

TV C 392 ± 21 4.96 ± 0.2

TV D 350 ± 12 3.76 ± 0.5

TV E 381 ± 15 4.37 ± 0.1

TV F 370 ± 42 4.14 ± 0.6

The culture filtrate of TV C was found to be the one with the greatest amount of polysaccharide and therefore it was used in all subsequent experiments.

It is preferable to subject the culture filtrate to volume reduction to favor an efficient precipitation of the polysaccharide component and then to selective precipitation. The precipitate is separated from the solvent by filtration or centrifugation, preferably centrifugation, and dissolved in water, for example bi-distilled water. Then follows a conventional treatment of elimination of the protein part, for example with pronase (mixture of proteases aimed at the complete degradation up to low molecular weight compounds of the peptides present, including the self-degradation of the proteases). Subsequently the mixture is subjected to dialysis to eliminate the low molecular weight compounds including those of the degradation with pronase, for example with membrane with molecular weight cuts of 12000, 5000, or 1000, but preferably 12000. The dialysis product is separated by chromatography at volume exclusion on column, using resins with different separation ranges, for example a Sephadex S300 chromatographic resin (separation range for dextran 2x10 <3> - 4x10 <5> Da), to obtain a chromatographic separation, with cut of molecular weight at 2x10 <3> â € “4x10 <5> Da, of the polysaccharides present. For chromatographic separation, the polysaccharide is dissolved in a saline solution, preferably NaNO30.05 M using the same saline solution as eluent.

The separation allows to obtain two fractions, one with a higher molecular weight and one with a lower molecular weight.

The chromatographic trace of figure 1 shows the compounds present in the peak A and in the peak B, collected and used preliminarily for the bioassay, ie the cytotoxic activity on cell lines with different ontogenesis has been measured (see below). The component present in peak B, which, as described in the characterization part, will be a polysaccharide, with a molecular weight lower than 100,000 daltons, preferably between 35,000 and 15,000 daltons (see figures 20 and 21). This fraction was found to be the only one capable of exhibiting a significant (p <0.001) cytotoxic activity (> 90%), especially against myeloid-derived leukemic cell lines.

For this purpose, this compound was subsequently characterized from a chemical-structural point of view, extensively evaluating its cytotoxic activity on various oncohematological cell lines, on primary cells obtained from oncohematological neoplasms and on circulating mononuclear cells obtained from normal donors.

A characterization and definition of the structure of the compound was carried out which turned out to be a biologically active polysaccharide molecule of which the constituent monomers (glucose, mannose, galactose, fucose) and the relative percentages of presence in the molecule were defined as follows indicated in Table 2.

Table 2

Composition in monosaccharides and localization of glycosidic bonds

Molar ratio

Monosaccharides (related to fucose

total)

t-Fuc 0.20

3-Fuc 0.80

t-Man 1.51

2-Man 1.69

6-Gal 1.35

2,6-Gal 1.12

2,6-Hex possibly Glc 0.47

It has also been defined that the polysaccharide has short ramifications in its linear skeleton. This result is suggested by the characterization of the biologically active polysaccharide (also referred to in the following simply as Tramethane) carried out by chemical analysis and one- and two-dimensional NMR spectroscopy.

The purified extract obtained according to the present invention is therefore a polysaccharide fraction obtained from the filtrate of fungal cultures, in particular of Trametes versicolor. The fraction consists mainly of monomeric units of glucose, galactose, fucose and mannose to form a branched polymer with an average molecular weight of less than 100,000 daltons, preferably between 70,000 and 50,000, even more preferably around 30,000 ± 5,000 daltons.

The polysaccharide fraction can be used in the pharmaceutical field and can be advantageously formulated as an active ingredient in compositions containing vehicle excipients and / or adjuvants suitable for the preparation of pharmaceutical forms known to those skilled in the art for oral, parenteral, topical, spray administration. or intravenous. Particularly advantageous are the intravenous and especially oral administration, allowing the treatment even outside the hospital facilities.

Excipients, diluents, vehicles and / or adjuvants are defined as those substances which alone are unable to produce a pharmacological effect on the subject receiving said composition and which favor the stability, conservation, administration and absorption of the active ingredients. in pharmaceutical preparations.

All the excipients, diluents, vehicles and / or adjuvants necessary for the preparation of the compositions of the invention in solid or liquid form are those known to those skilled in the art and can be selected for example from substances such as water, physiological solution, glycerol, ethanol, polyethylene glycols, polyoxyethylenes, carbowax, glycols, PEGs, non-polar diluents (oils), buffers, emulsifying agents, diluents, preservatives, sweeteners, thickeners, stabilizers, flavorings and the like.

Dosages depend on the type of disease to be treated, the methods of administration and the response of the subject (man or animal) being treated and the doctor will be able to find the most appropriate routes and doses of administration.

The compositions can be obtained by mixing an adequate quantity of active principle together with the necessary excipients, diluents, vehicles and / or adjuvants, as known to the skilled in the art.

The polysaccharide extract in question is not described in the known art, in particular in US 2006/0045887, first of all because it is obtained from the culture of a single fungus, as opposed to the previous one which was obtained from a culture of seven different species of basidiomycetes. Furthermore, the effects reported in the patent application US20060045887 are of the antiproliferative type, as found both on neoplastic cells (two cell lines of oncohematological neoplasms, such as Chronic Myeloid Leukemia and Acute Lymphoid Leukemia, both Philadelphia Positive, a solid tumor cell line obtained from carcinoma prostatic) and normal haematopoietic cells with hemoglobin disorders (sickle cell anemia and B-thalassemia).

On the contrary, the extract according to the present invention is characterized not only by an antiproliferative (cytostatic) activity but also and unexpectedly of a pro-apoptotic (cytotoxic) type. It also carries out its action electively and preferentially on neoplastic cells, however represented above all by Acute Myeloid Leukemia not mentioned in the US patent, and not on normal haematopoietic cells (normal lympho-monocytes). Finally, the compound has been tested with positive results in its efficacy also on primary cells obtained from patients suffering from oncohematological neoplasms.

The following examples are to be considered illustrative and not limitative of the scope of the invention.

Extraction, purification and characterization of Trametano. T. versicolor TV C isolated and under registration at CABI Bioscience (UK) was grown for 14 days at 25 ° C in the dark under stirring (150 rpm) in liquid Potato Dextrose Broth (Sigma-Aldrich, USA and Difco, USA). The fungal culture was stored in Potato Dextrose Agar, PDA (Difco) at 30 ° C and renewed every 10 days.

Purification

The culture medium was lyophilized (lyophilization conditions: 48h at 0.03 mPa-freeze dryer Lio5P) of T. versicolor CF1 (1 g) was resuspended with 30 ml of ultra pure water and filtered to separate the insoluble portion from the soluble one . The soluble portion was placed at 4 ° C for about 2h. At the same time, an absolute Ethanol solution equal to four times the volume of the sample to be precipitated was cooled, again at 4 ° C. Once both solutions have cooled, the culture filtrate is added to the ethanol, immersed in an ice bath, stirring the mixture with a glass rod. The precipitate was obtained following a centrifugation of 20â € ™ at 4000 rpm at 4 ° C. The pellet was resuspended in 4 ml of ultra pure water and 4 ml of phosphate buffer 0.02M pH 7.5 in order to obtain the optimal conditions of both concentration and pH for the activity of pronase E (Sigma-Aldrich, USA). This cocktail of enzymes is able to digest all the proteins present in the solution and ultimately self-digest. The proteolytic activity occurs at a temperature of 37 ° C for one night. The elimination of salts and amino acids is carried out using dialysis membranes with a molecular cut of 12000Da against H2O, measuring the conductivity of the water every 3 hours. When the conductivity meter shows a value equal to that of ultra pure water (0.1 microSiemens / cm) it means that the sample is in osmotic equilibrium with water. The dialysates were lyophilized and used for subsequent analyzes.

Volume exclusion chromatography The T. versicolor CF1 extract (19 mg) was dissolved in 1.9 mL of NaNO30.05 M, centrifuged to remove any particulate matter, and subjected to separation by volume exclusion chromatography on a column containing Sephacryl S-300 resin (separation range for dextran 2x10 <3> - 4x10 <5> Da). The elution took place with a NaNO30.05 M solution, at a flow of 6 mL / hour and was monitored using a refractive index. The eluate was collected using a fraction collector. The elution profile obtained is shown in Figure 1: there are two main peaks at 10 (peak A) and 20 (peak B) elution hours, respectively. At about 27 hours there is the signal of the elution of the salt. Of the two signals of interest (10 and 20 hours) the first elutes at shorter times (indicated by arrow A) and therefore is characterized by a higher molecular mass, and the second (arrow B), more abundant, elutes later. and therefore it is characterized by a lower molecular mass. Preliminary biological activity tests performed on a large panel of human leukemic cell lines with different ontogenesis: myeloid (OCI-AML3, U937, NB4) and lymphoid (Jurkat), placed in liquid culture / - of the highest and lowest peak molecular mass at scalar concentrations (0.5-2mg / mL) have shown that the compound represented by the peak with the lowest molecular weight represents the biologically active fraction.

As is evident from Figures 2 and 3, the <1> H-NMR spectra of the highest and lowest molecular mass peak showed, on the one hand, that both samples contain carbohydrates as they give rise to typical spectra of these molecules, and on the other hand that they are structurally different compounds.

In order to confirm the different chemical nature of the two fractions, high and low weight, the composition in saccharides was determined by means of methylation analysis which, in addition to defining the identity of the saccharides, also provides information on the carbon atoms engaged in glycosidic bonds. The gas chromatograms obtained are shown in figures 4 and 5. The data obtained indicated that the high molecular mass fraction contains different monosaccharide residues compared to the fraction characterized by a lower molecular mass.

Volume exclusion chromatography data showed that the TV117 extract contains two fractions of different molecular weight. Structural characterization by NMR spectroscopy and methylation analysis showed that they are structurally different. Since the biologically active fraction is the low molecular mass fraction, the high mass fraction has not been further characterized.

Since the fraction of interest (called â € œfraction-activeâ €) has a shoulder at higher elution times, it has been further divided into two fractions (as shown in Figure 6 where they are indicated by B and C) which are were analyzed by NMR spectroscopy. The spectra of the two fractions were identical, demonstrating that the fractions have the same composition (Fig. 7).

In order to analyze in detail the fraction contained in the C peak, this has been divided into more homogeneous components in terms of molecular mass. For this reason, volume exclusion chromatography was carried out on a Bio Gel P10 column (with separation interval for the dextran standard between 1500 - 20000 Da), as shown in Figure 8. The elution was carried out with water at a flow of 6 mL / hour and was monitored using a refractive index. The eluate was collected using a fraction collector. The fraction eluted between 7 and 10 hours was divided into 3 parts (fractions 3-7, 8-9, 10-13) and the <1> H-NMR spectra were recorded.

As shown in Figures 9-11 the three <1> H-NMR spectra are all extremely similar to each other indicating that the three fractions are practically identical and that therefore the fraction contained in the C peak is homogeneous from a composition point of view. .

For comparison, Figure 12 shows the <1> H-NMR spectra of peak B, peak C obtained from the Sephacryl S-300 column and fraction 3-7 (obtained from the separation of peak C on Bio Gel P10). The comparison shows the undoubted equality of the three champions.

Having demonstrated the equality of the samples present in fractions B and C, the following structural analyzes were all performed on the sample present in peak B which was obtained in greater quantities. A methylation analysis was performed and the results are reported in Table 3.

Table 3

Methylation analysis by studying the partially acetylated methylated alditols of the sample present in fraction B.

Monosaccharides Relative molar ratio

t-Fuc 0.20

3-Fuc 0.80

t-Man 1.51

2-Man 1.69

6-Gal 1.35

2,6-Gal 1.12

2,6-Hex (possibly Glc) 0.47

t-Fuc: non-reducing terminal fucose, bonded only through the carbon atom n 1;

3-Fuc: fucose bound through carbon atoms n 1 and 3;

t-Man: non-reducing mannose, bonded only through the carbon atom n 1;

2-Man: mannose bound through carbon atoms n 1 and 2;

6-Gal: galactose bound through carbon atoms n 1 and 6;

2,6-Gal: galactose bound through carbon atoms n 1, 2 and 6; 2,6-Hex (possibly Glc): hexose, probably glucose bound through carbon atoms n 1,2 and 6.

The total quantity of fucose (t-Fuc 3 Fuc) set equal to 1 was taken as a reference value.

If we add up all the terminal monosaccharides (t-Fuc t-Man) we get 1.71 and if we add all the monosaccharides that have branches (2,6-Gal 2,6-Hex) we get 1.59 with a very good agreement. In fact, each branching must correspond to a terminal saccharide present in the short side chain. Furthermore, the terminal Fuc is in low quantity while the terminal Man is in high quantities and therefore, considering that the residue called Hex is also in low quantity, we could have another information: the branching on the saccharide residue Hex it ends with a Fucosio and the branching on the Gal ends with a Mannosio.

At the same time, the NMR spectroscopy investigation continued using two-dimensional methods. For this study, the sample of peak C was chosen, identical to that of peak B, because, in general, samples with a lower molecular mass are characterized by lower viscosity of their aqueous solutions, with a consequent increase in the resolution of the NMR spectra. . To further improve the resolution of the spectrum, the C peak was deacetylated by treatment with 0.01 M NaOH for 5 hours at room temperature and under nitrogen flow.

The one-dimensional spectra of the de-acetylated sample are shown in Figure 13 and 14 at 25 ° and 50 ° C, respectively. As expected, the spectrum obtained at 50 ° C has a better resolution than the one at 25 ° C since the higher temperature lowers the viscosity of the solution.

The spectrum analysis was therefore performed on the one obtained at 50 ° C.

The signal at 1.23 ppm is given by the methyl group of fucose, whose presence is therefore confirmed. The doubling that can be seen depends on the fact that there is a magnetic coupling between the proton of the methyl group in position 6 and the proton in position 5 of the saccharide ring (Figure 15).

The zone of anomeric protons is always the one from which we start for the definition of the structure and in Figure 16 an expansion of the zone of interest is shown.

The signals were named using the letters of the alphabet starting from the lowest field resonance. Once the name of the anomeric proton signals has been given, we move on to the study of the two-dimensional NMR spectrum COZY which allows us to trace the protons linked in position 2 starting from the anomeric ones, as shown in Figure 17.

The signals (cross-peaks) in the box are those of interest since, starting from the anomeric signals (H-1) of the one-dimensional spectrum, they indicate where the relative signals of the protons in position 2 (H-2) fall. For example, taking the triplet at 5.28 ppm in the one-dimensional spectrum, the dashed vertical line indicates the position of the NMR signal of the triplet on the diagonal. The dashed horizontal line indicates the position of the adjacent proton, in this case in position 2 (4.09 ppm), belonging to the same monosaccharide residue. From the analysis of the COZY spectrum, the assignment data of the signals for the anomeric protons and in position 2, which are reported in Table 4, are obtained.

Table 4:

NMR signal assignments (partial data)

Abnormal H pos. <13> C position (ppm) H 2 pos. H 3 pos. NMR signal

(ppm) (ppm) (ppm) A 5.34 101.44 4.10

B 5.32 101.44 4.09

C 5.28 101.44 4.09

D 5.12 103.08 4.07

E 5.11 102.20 3.92 3.97

F 5.08 102.26 3.79

G 5.06 102.99 4.06

H 5.05 98.83 3.83

I 5.00 98.92 3.85

L 4.97 Not detected 3.57

The table also includes the signals of the NMR spectrum of <13> C, obtainable from the HSQC spectrum (Figure 18), which allows to correlate the positions of anomeric protons (which are known, Table 4) with those of the relative carbon atoms to which they are related.

It should be noted that, contrary to proton signals, <13> C resonance signals can be grouped into only 3 or 4 groups based on their chemical displacements, marked with rectangles as shown in Figure 18.

The ppm scale of the proton spectrum is the horizontal one, while the ppm scale of the <13> C spectrum is the vertical one. Each cross peak carries two information: the chemical shift value of the atom <13> C and that of the H atom bound to it (eg the dashed lines). The signals in dark blue belong to the CH2e groups and therefore refer to the atoms in position 6 (except fucose which in position 6 has a - CH3 group which resonates at 1.23 ppm, as evidenced by the one-dimensional <1> H spectrum). The NMR technique allows them to be recognized and therefore to be marked in a different way.

The rectangles indicate the four groups of signals of the anomeric <13> Cs reported in Table 4. Considering that the signals of the <13> C are always less scattered than the signals of the protons, a reasonable hypothesis is that each of the four groups of signals correspond to a specific monosaccharide (of the four determined by methylation analysis). However, since the saccharide called Hex is in low quantity (Table 3) and considering the limits of the NMR technique, the signals of the HSQC spectrum could refer only to the three most abundant monosaccharides (Fuc, Man and Gal).

Another datum that can be obtained from the HSQC spectrum is the position of the carbon atoms involved in the glycosidic bonds, which are shifted to higher ppm values than the unbound atoms themselves. As can be seen from Table 3 there are bonds in position 2, 6 and 3, the latter for fucose alone. The spectrum area of interest (on the horizontal axis) is that which goes from 3.5 to 4.5 ppm for the <1> H spectrum, and from 60 to 80 ppm for the <13> C spectrum. Typical chemical shift values for CH2 groups not engaged in glycosidic bonds are at 60-62 ppm. It is possible to assign cross peaks at 62 ppm for <13> C and 3.88 / 3.75 for protons to t-Man and 2-Man.

Instead the signals at about 67 ppm belong to the saccharide residues bound in C-6 (6-Gal, 2,6-Gal, and 2,6-Hex). They can be assigned as follows: the two signals at 3.97 / 3.72 ppm, for protons, and 66.68, for <13> C, being of low intensity, are due to the 2,6-Hex residue and the others to 67.63 (doubled in two by the shift of the two protons at 3.90 and 3.72 ppm) and 67.95 ppm at the residues of 6-Gal and 2,6-Gal, which for now cannot be distinguished. The <13> C atoms involved in glycosidic bonds, but not in the C-6 position, resonate between 75 and 82 ppm. The methylation analysis indicates that there are three residues bound in position 2 and the fucose bound in position 3: The HSQC spectrum shows three intense signals in the area of interest. Since the position of the protons in 2 is known from the COZY spectrum (Table 4), from the HSQC spectrum it can be verified whether some of these are engaged in glycosidic bonds. The signal at 3.85 ppm is found to belong to residue I (Table 4) and that at 4.10 ppm to residue A (Table 4). The third bound signal in 2 that is expected belongs to 2,6-Hex, which is probably not visible due to its low concentration. The signal at 3.97 ppm does not correspond to any H-2 and therefore can be attributed to the H-3 proton of 3-Fuc. This assignment was confirmed by examining the TOCSY NMR spectrum, which is shown in Figure 19.

The dotted line indicates the TOCSY correlations, which are relative to the resonance band E and which are two: one at 3.92 ppm, attributed to H-2 by COZY, and the other at 3.97 ppm attributed to H- 3. Consequently, the signal named E in Table 4 is that of the anomeric proton of 3-Fuc.

With the available data it is possible to assign signal E to 3-Fuc, signal A to 2-Man and signal I to 2,6-Gal. The latter have also been assigned on the basis of literature data which report higher chemical shifts for proton 2 of mannose than for galactose. Similarly, the L signal can be attributed to glucose as proton 2 of this monosaccharide resonates around 3.4-3.6 ppm. This information led to the compilation of Table 5.

Table 5

resonance signals with an assignment attempt

Signal ppm <13> C Possible ppm H anom ppm H2 ppm H3

NMR anomaly assignment A 5.34 101.44 4.10 2-Man

B 5.32 101.44 4.09

C 5.28 101.44 4.09

D 5.12 103.08 4.07

E 5.11 102.20 3.92 3.97 3-Fuc

F 5.08 102.26 3.79

G 5.06 102.99 4.06

H 5.05 98.83 3.83

I 5.00 98.92 3.85 2,6-Gal L 4.97 Not detected 3.57 2,6-Glc In addition, the production of the biologically active component of the TV117 extract has been further improved, by cultivating the fungus on Potato Dextrose Broth obtained from Sigma- Aldrich, USA which turned out to be, after chromatographic analysis (see below) of the medium, purer than that supplied by the company DIFCO, USA. In fact, as seen in Figure 20, volume exclusion chromatography on a Sephacryl S-300 column showed the presence of only the peak at lower molecular masses, with total absence of the peak at higher masses.

This sample was also analyzed by high pressure volume exclusion chromatography (HP-SEC) using three columns in series (Tosoh Bioscience, TSKgel G3000PW, G5000PW and G6000PW, i.d. 7.5 mm, length 30 cm) maintained at 40 ° C . Calibration was obtained with pullulan standards (Polymer Laboratories, Germany and Sigma for pullulan with MM = 1.600.000). The elution was carried out with a 0.15 M NaCl solution at a flow of 0.4 mL / min and monitored with a refractive index (Knauer, Labservice Analytica). The obtained chromatogram is shown in Figure 21. The elution profile obtained confirms the absence of the peak at higher masses and has established a numerical mean molecular mass (Mn) value for fraction B equal to 26000 Da, a value weight average molecular mass (Mw) equal to 27500 Da and polydispersity index equal to about 1.

This sample was subjected to a colorimetric test to determine the uronic acids which were found to be 3.3% (w / w), which corresponds to one uronic acid residue for every 38 saccharide residues.

Cytotoxic and antineoplastic activity of Tramethane The polysaccharide molecule thus obtained was then evaluated for its cytotoxic activities against a panel of human leukemic cell lines and primary cells obtained from patients affected by various onco-haematological neoplasms, with the aim of evaluate its antiproliferative and / or pro-apoptotic activity, as reported below. Furthermore, the same evaluation was performed on the normal counterpart represented by circulating mononuclear cells obtained from normal donor and human hematopoietic progenitors purified from cord blood.

Human cell lines:

Cell lines originating from onco-haematological neoplasms with myeloid ontogenesis (OCI-AML3, U937, NB4) and lymphoid (Jurkat) were cultured at 37 ° C and at 5% CO2 by means of RPMI 1640 (Gibco) culture medium, added with Serum of fetal calf (FCS) at 10% (Hyclone), 1% Glutamine and 1% Penicillin / Streptomycin (P / S). The culture medium was renewed every three days reporting the appropriate cell concentration for each cell line.

Primary cells obtained from acute leukemia patients:

Cell samples obtained from scraps not usable for diagnostic procedures of bone marrow aspirate (BM) of 4 patients with acute myeloid leukemia (AML) at diagnosis, belonging to Hematology, Department of Cell Biotechnology and Hematology, Sapienza, University of Rome. Written informed consent for in vitro studies was obtained for each patient in accordance with the protocols established by the â € ̃Human Subjects Committee of Helsinkiâ € ™. Samples were selected on the basis of both the number of available cells and cell viability.

Circulating mononuclear cells obtained from normal donors:

Normal mononuclear cells were obtained from peripheral blood (PB) samples from volunteer donors.

Preparations of cell populations in liquid culture exposed to treatment with Trametan:

For experiments performed on cell lines, cells were harvested in the logarithmic phase of growth and cultured at the appropriate RPMI concentration at 10% FCS / - Tramethane at scalar concentrations (0.5-2mg / ml). Lymphocytes obtained from normal volunteer donor PB and blasts from BM from AML patients were separated by stratification on Ficoll-Hypaque density gradient (Lymphoprep; 1.007g / ml). Cells used for in vitro studies were resuspended at a concentration of 1.0 x 10 <6> / mL, in RPMI at 10% FCS / - Tramethane at scalar concentrations (0.5-2 mg / mL). In particular, circulating mononuclear cells obtained from healthy donors were placed in liquid culture / - scalar concentrations of Tramethane in the absence and in the presence of a proliferative stimulus (PHA).

Cell growth and viability curves:

cell count and viability were determined on cell lines and primary cells by evaluating trypan blue exclusion and using a hemocytometer.

Determination of the cell cycle using the Acridine Orange (AO) method:

The cell cycle analysis was performed, with flow cytometric method, by staining with AO. This method allows the simultaneous measurement of the total cellular content of DNA and RNA, and therefore the discrimination of the percentage of cells in G0 phase from those in G1 phase (higher RNA content compared to the reference population represented by normal quiescent peripheral lymphocytes), as well as © the evaluation of the percentage of cells in S and G2 / M phase. The cells permeabilized with a binder solution (TRITON buffer for 500ml of distilled water: 10 ml of HCl 2N, 4.4 g of NaCl, 0.5 ml Triton-X100) were subsequently stained with AO (for 1 l of distilled water: 7.77 g of Citric-acid-monohydrate, 17.84 g of Na2HPO4, 0.37 g of EDTA, 8.7 g of NaCl, 7.3 Î1⁄4g / ml of AO) which simultaneously intercalates the double stranded DNA (emitting green fluorescence) and the single RNA helix (emitting red fluorescence).

Apoptosis study and cell cycle analysis: Apoptosis levels were assessed on cell aliquots by staining with Annexin V which binds to membrane phosphatidylserine (PS) externalized on the cell membrane during the initial stages of the apoptotic process. The cells, after two washes in phosphate buffer saline (PBS), were suspended in a binding solution (10 mM Hepes / NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) with the addition of Annexin V conjugated with fluorescein isothiocyanate (FITC) at a concentration of 1 Î1⁄4g / ml and incubated for 15 minutes in the dark at RT before flow cytometric analysis. Membrane integrity was simultaneously assessed by excluding phosphatidylinositol (PI) (0.25 Î1⁄4g / ml).

Statistical evaluation of the results:

Statistical significant differences between the groups were analyzed with the two-tailed Student's t-test (paired t test). Results were expressed as mean / - standard deviation (SD). Values of P <0.05 were considered significant from a statistical point of view.

Effects of Trametan on cell lines:

In a first phase of the study, the effects induced by the molecule on a large panel of human leukemic cell lines with different ontogenesis were analyzed: myeloid (OCI-AML3, U937, NB4) and lymphoid (Jurkat), placed in liquid culture / - Tramethane in scalar concentrations (0.5-2mg / ml). Cell aliquots were collected at 24, 48 and 72 hours after exposure and analyzed for:

Cell count (Trypan blue);

· Cell cycle (flow cytometric analysis by AO); · Apoptosis (flow cytometric evaluation with AO, Annexin V).

The results obtained have documented that exposure to Tramethane induces dose and time dependent cell growth arrest in all the hematopoietic cell lines studied (Fig. 22-24). The effects are markedly evident at a dose of 1mg / ml already after 48 hours of culture, however, prolonged exposure times and higher concentrations of the molecule enhance its efficacy (Fig. 22). Parallel to the arrest of growth, in the same cell lines, the parallel evaluation of the cell cycle (simultaneous determination of the total DNA and RNA content by AO) allowed to estimate the variations induced by the molecule in each compartment of the cell cycle (G0 , G1, S, G2M). The data obtained showed that, in each of the lines analyzed, tramethane does not induce cell cycle modulation and / or induction of apoptosis. The different cytostatic and non-cytotoxic activity will be subsequently investigated on a large panel of cell lines.

Effects on primary cells from AML:

In a subsequent phase we examined the biological effects induced in vitro by Tramethane on primary cells from 4 patients with onset AML, with the aim of testing their clinical potential. Therefore, primary cells (peripheral blood and / or bone marrow aspirate) enriched by Ficoll in their leukemic component (> 80%), were placed in liquid culture / - Tramethane at scalar concentrations (0.5, 0.8, 1, 2mg / ml) and analyzed for the same parameters listed above for cell lines. The results obtained from the analysis of cellular samples from LAM indicate that Trametano exerts antiproliferative and cytotoxic activity in a dose and time dependent manner in all (4/4) samples studied. In fact, Trametan (after 72-120 hours) induces a significant reduction in cell count from 1272500 ± 235425, in the control condition, to 885000 ± 392386 (p = 0.06), 787500 ± 493381 (p = 0.04), 675000 ± 470213 (p = 0.02), 391750 ± 356435 (p = 0.01) in the presence of 0.5, 0.8, 1, 2mgr / ml of Tramethane (after 72-120 hours) (Fig. 23).

Interesting difference observed in primary AML samples, compared to cell lines, is the significant increase in apoptosis levels (subG0 / 1) from 35.7% ± 13.8, in the control condition, to 48.9% ± 23.5, 67.9% ± 25.9, 65.9% ± 34.6, 77.2% ± 28.3 (p = 0.04) in the presence of 0.5, 0.8, 10, 2 mgr / ml of Trametano (Fig.24a). This effect was found to be even more marked at longer exposure times (144-168 hours), reaching statistical significance even at the lowest dose: from 41.6% ± 23.5, in the control condition, to 77.5% ± 31.1 (p = 0.04), 88.6% ± 17.3 (p = 0.01), 86.5% ± 22.8 (p = 0.01), 94.5% ± 8.4 (p = 0.01) respectively 0.5, 0.8, 10, 2 mgr / ml of Trametano (Fig. 24b) .

Effects on normal cells:

In order to analyze the activity of the extract of the invention on normal hematopoietic cells, mononuclear cells obtained from peripheral blood of normal donors were studied. The lympho-monocytes were then exposed in liquid culture / - Tramethane at scalar concentrations (0.5, 0.8, 1, 2mgr / ml), in the absence and in the presence of a proliferative stimulus (PHA). The results obtained show that in normal circulating mononuclear cells Trametan is able to induce only a moderate pro-apoptotic activity, at 72 hours, regardless of the dose used (Fig. 25a). These effects do not change or increase after pre-treatment with PHA (Fig. 25b).

In conclusion, this study demonstrated that in leukemic cell lines with different ontogenesis, scalar doses of Trametan induce a reduction in cell growth, however, not associated with a cytotoxic effect. On the contrary, the preliminary data obtained on cellular samples from patients with AML demonstrate an effective in vitro cytotoxic activity of Trametan, as documented by an arrest of cell growth associated with an induction of time and dose-dependent apoptosis.

As regards the structural characterization of the Trametano biopolymer (name assigned by the authors of the patent and not existing in literature) with biological activity according to the present invention, the data presented here indicate that it is a polysaccharide consisting of a linear skeleton composed of residues of galactose with the addition of some glucose residues. The ratio of galactose to glucose is 4: 1. The polysaccharide also has at least three side chains, two of which consist of short sequences of mannose residues and one consisting of a short sequence of fucose residues. Structural data on oligomers obtained from the partial hydrolysis of the polysaccharide will allow to define the details of the chemical structure of its repeating unit.

Claims (10)

CLAIMS 1. Fungal extract obtained from a culture of cellular mycelium of basidiomycetes and characterized in that it contains a polysaccharide fraction with a molecular weight of less than 100,000 dalton consisting of linear polysaccharides bearing galactose and glucose residues in a ratio of 4: 1 with side chains consisting of both short sequences of mannose residues and short sequences consisting of fucose residues. 2. Fungal extract according to claim 1 wherein the basiodiomycete is Trametes, in particular Trametes versicolor. 3. Fungal extract according to any one of claims 1-2 wherein the polysaccharide fraction is a polysaccharide with a molecular weight comprised between 100000 and 15000 daltons. 4. Fungal extract according to any one of claims 1-3 wherein the polysaccharide fraction is obtained by elimination of the insoluble cultural part by centrifugation, precipitation with non-solvent, re-dissolution and separation by chromatographic methods. 5. Fungal extract according to any one of claims 1-4 for use in the pharmaceutical field. 6. Fungal extract according to claim 5 to be used for antiproliferative and proapoptotic purposes and for the prevention and treatment of various types of neoplasms, in particular onco-haematological pathologies. 7. Method for obtaining the extract according to claims 1-6 comprising the steps of: - cultivation in a culture medium chosen from PDB, MEX and CZL - separation from the culture medium by elimination of the insoluble culture part, precipitation with non-solvent, redissolution and separation by chromatographic methods. - elimination of the peptides present by their degradation with enzymatic methods followed by dialysis to eliminate the molecules produced with a molecular weight lower than 10000 Da. - separation and isolation of the molecular weight fraction lower than 100,000 daltons by chromatographic methods. 8. Pharmaceutical compositions comprising the fungal extract according to any one of claims 1-6 for use in the pharmaceutical field. 9. Pharmaceutical compositions according to claim 8 for antiproliferative and proapoptotic purposes and for the prevention and treatment of various types of neoplasms, in particular onco-haematological pathologies. 10. Use of the extracts according to claims 1-6 for the preparation of pharmaceutical compositions for antiproliferative and proapoptotic purposes and for the prevention and treatment of various types of neoplasms, in particular onco-haematological pathologies.