WO2007085879A1

WO2007085879A1 - Compositions containing substances obtained from the blood and other biological fluid of snake : methods for their preparation and their use

Info

Publication number: WO2007085879A1
Application number: PCT/IB2006/000128
Authority: WO
Inventors: Marina Ziche; Mario Pazzagli; Angelo Michele Palmieri; Mario Leonardo Granito; Francesco Stanisci; Concetta Antonia Montemurno; Maria Squeo; Vincenzo De Filippis; Fiorenzo Refosco
Original assignee: Marina Ziche; Mario Pazzagli; Angelo Michele Palmieri; Mario Leonardo Granito; Francesco Stanisci; Concetta Antonia Montemurno; Maria Squeo; Vincenzo De Filippis; Fiorenzo Refosco
Priority date: 2006-01-26
Filing date: 2006-01-26
Publication date: 2007-08-02

Abstract

Compositions comprising fractions of blood and other biological fluid of snake (Pyton Sebae) having therapeutic activity in cell models in vitro are described, methods of preparations of the aforesaid fractions and pharmacological compositions contain them are also described.

Description

Compositions containing substances obtained from the blood and other biological fluid of snake: methods for their preparation and their use Field of the invention. The present invention refers to products having pharmacological properties in particular anti-tumour activity.

State of the art.

Chemotherapeutical drugs are classified according to their chemical structure or their biological activity (see "The Pharmacological Basis of Therapeutics" by To Goodman and M. Gilman, Pergamon Press, New York, 1990). In many cases the anti-tumour drugs act on the cell vital cycle blocking the growth of the cells.

The classification of the chemotherapeutical drugs includes also natural compounds or their derivatives (i.e. taxol or vincristine) able to bind to the tubulin and therefore to inhibit the mitosis.

In our previous patent PCT/EP01/14727 fractions obtained from the blood of snake through centrifugation and collection of the supernatants are described.

In view of the importance of chemotherapeutical drugs for treatment of the tumors, the interest in developing new drugs having antitumor activity is obvious.

Summary of the invention

The invention refers to compositions comprising compounds obtained from the blood and other biological fluid of snake.

Detailed description of the invention The present invention makes available new compositions having pharmacological activity comprising a component obtained from the blood and other biological fluids of snake.

According to the invention, as "a component obtained from snake blood"

(hereinafter SX) it is intended the supernatant fraction obtained by simple centrifugation of snake blood, whereas as "biological fluids" it is intended the liquor obtained from the third ventricle of the brain (hereinafter LQ). The composition according to the invention (hereinafter SX + LQ) shows to possess more specifically anti-tumour activity in respect to the sole SX. Preferably, according to the invention, the composition SX + LQ is obtained starting from the blood and from the liquor taken from snakes of python genus (Python Sebae)

The SX component of the mixture, according to the invention, is prepared as described in previous patent PCT/EPO 1/14727 to which reference is made for the complete description of the process. Briefly, aliquots of snake blood are collected by venipunture and centrifuged, at about 3000 rpm for 5 minutes, collecting the supernatant. Alternatively, as described in the aforesaid patent, the collected supernatant can be purified by means of alcoholic precipitation. Supernatant (SX) is then collected and possibly frozen.

The liquor (LQ) is found in the subarachnoid spaces of the snake head and it is a colourless and limpid liquid, secreted by special structures, the choroidal plexuses, that are scattered in the cerebral ventricles. LQ is collected, it also by venipuncture, from the third ventricle of the snake brain by a radiologically needle-guided procedure according to the following procedure. The snake is anesthetized with Ketamina 0,5 mg/kg and then maintained under gas anaesthesia (O2 and Isofluorane) administered by endotracheal intubation. After accurate disinfecting of all the cranial portion of the reptile head, about 2 millilitres of cerebrospinal fluid (LQ) are collected directly from the central cephalic cistern of the encephalic subarachnoid space using a syringe equipped with a needle 30G x 8mm that is inserted perpendicularly for about 1.5 millimetres through the medial cranial cleft under the third interparietal encephalic squama exactly at the centre of the skull at the junction between the parietal left, frontal and parietal right bones. The correct position of the needle in the cephalic cistern is radiologically confirmed. This procedure is harmless to the animal. LQ thus obtained is conserved to -2O⁰C in aliquots of 0.2 millilitres and used as such for producing the mixture SX+LQ as described hereinafter.

The composition of the mixture SX+LQ is preferably comprised between 95/5 and 70/30 (v/v). To the composition as above described chemical compounds such as

Interleukin 2 and compounds of the family of the Interferon such as Interferon alpha, Interferon beta, Interferon gamma and Interferon delta can be added.

According to a preferred embodiment, the composition SX + LQ according to the invention consists of 27 μl of SX, 2 μl of LQ, and 1 μl of each of the above said compounds at the concentration of 1 μg/ml.

The invention will be better understood in the light of the examples reported below.

Example 1 Aliquots of blood and liquor are obtained by venipunture from a Python Sebae

(or python of the rocks).

After centrifugation of the blood, supernatant (SX) is collected and aliquoted in 0.5 millilitres fractions which are stored to -20⁰C until the moment of the use. Liquor (LQ) is obtained by venipuncture from the cerebral third cerebral ventricle of the snake by means of the above described procedure and aliquoted in fractions of 0.2 millilitres that are conserved as such to -20⁰C until the moment of the use.

The definitive composition SX+LQ as above described is prepared then at the moment of the use.

Example 2

A cold solution of ethanol (80%) in water is added to Serum (2 millilitres) and left under gentle stirring at 4°C for 30 minutes.

The solution is centrifuged (15000 rpm, 30 minutes) and the supernatant and the precipitate are separated.

The precipitate (pellet) is resuspended in a solution of ethanol (80%) and water and the procedure is repeated three times. Finally the pellet is resuspended in sterile PBS (1 millilitre) giving SX.

This preparation is then added to the liquor (see example 1) at the moment of the use to constitute the wanted composition SX + LQ.

Example 3 Example 2 is repeated but the pellet is resuspended in a solution of ethanol (50%) and water and the procedure is repeated three times. Finally the pellet it is resuspended in PBS (0.5ml) sterile giving SX. With this procedure the amount of precipitated proteins is reduced in comparison to the previous example.

The cytotoxic effect of mixture SX + LQ according to the invention, has been estimated using three experimental models using two cell lines (A431 : human breast tumour cell line, and HF: human fibroblasts). These experimental models and the cell lines are analogous to those described previously in the Patent PCT/EPOl/14727. Description of the experimental models

Adherent cells are employed (5000 cells/well A431 in 10% serum for 24 hours and 1% serum for 18 hours; 3000 cells/well are employed HF in 10% serum for 24 hours and 1% serum for 18 hours). After this step the culture medium in which the cells had been grown was removed and was replaced by a new medium containing the products under study according to the protocols reported in Table 1 and described as follows.

It has been then estimated the effect of SX and LQ alone or in combination (mixture SX + LQ as above reported: final dilution 1:100) on cell lines of A431 and HF in presence of FCS (Fetal Calf serum) with a concentration of 0.1% or 10% respectively. Each experiment was replicate using direct microscopy observation of the cytotoxic effect or other quantitative biochemical techniques always in order to estimate the citotoxicity. The results are reported in detail in the following sections. 1° Experimental model

Cell morphology has been observed every 15 minutes for a overall experimental time of 1 hour. At the end of the incubation the cells have been fixed in methanol for 2 hours at 4°C and then stained with hematoxylin and eosin. Both during morphological examination and at the end of fixation and staining steps, the cells have been photographed by means of a digital camera directly connected to an inverted microscope. Results of the 1 experimental model: After 15 min (first observation), A431 and HF treated with SX show signs of suffering with increased refractiveness (as if cells begin to detach from the well walls), shape loss with formation of swells and cytoplasm protuberance (as if the cells take in liquids from the outside, swelling), or in some cases, formation of picnotic bodies. A431 cells treated with SX+LQ show a behaviour similar to the one observed in presence of the sole SX. However, the damage is lower. HF after 15 min treatment with SX+LQ do not show signs of suffering. At the end of the experiment (1 hour) the stimulation with SX + LQ induced in A431 cells 70-80% of mortality measured at the microscope as cell detachment from the plate, loss of their morphology, cells with picnotic nuclei, presence of vacuoles in the cytoplasm. The cytotoxic effect of SX + LQ on HF cells was much lower, even if present (20-30% of cell mortality). The addition of LQ, Interleukin-2 and Interferons, to SX, reduced its cytotoxic effect, in particular on HF, when compared to the effect of SX alone. 2° Experimental model

Use of trypan blue assay on cell in adhesion, stimulation for 1 hour according to the experimental protocol, removal of the culture medium, staining of died cells with trypan blue for 15 min, fixation in methanol for 2 hours at 4°C, counterstaining of alive cells with eosin. Before eosin counterstaining, died cells, incorporating Trypan blue, have been photographed as reported above. The experimental protocol is the same described in Table 1. Resultsof the 2 experimental model: By means of the Trypan blue test the cytotoxic effect of SX and the regulating effect of LQ have been confirmed. A431 and HF treated with SX are blue stained, indicating that the integrity of their plasma-membrane has been lost. The addition of LQ and of the other compounds to SX, significantly reduces the number of the cells died and improves the specificity of the cytotoxic effect on A431 when compared to HF. 3° Experimental model

Use of the so called MTT assay, cell stimulation for 1 hour following the experimental protocol, removal of the stimulus, adding of fresh medium containing 12mM of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)₅ incubation of the cells in presence of MTT for 4 hours at 37°C in 5%CO2, removal of the medium with residual MTT, adding of DMSO for dissolving the formazan salt that formed in mitochondria in alive cells deriving from the MTT substrate), spectrophotometric reading of the absorbance correlated to the presence of soluble formazan in the various wells. The absorbance due to the presence of the salt is directly proportional to the concentration of the formed salt and therefore to the number of alive cells, i.e. those capable of actively metabolize MTT, that are present in the various wells after treatment with the substances under study. The experimental protocol is described in Table 1. Resultsof 3 experimental model.

The modulating effect of LQ (in the presence of interleukin 2 and Interferons) on the cytotoxic activity of SX was confirmed again. SX alone, either in low serum concentration, 0.1% or in high concentration, 10%, inhibits the formazan production by metabolisation of MTT by about 60% and 70% in A431 and HF respectively. The mixture SX + LQ reduces such inhibition to about 25% and 70% in A431 and HF cells respectively. As demonstrated from the assays reported above the mixture SX+LQ remarkably improves the selective cytotoxic action of SX towards the tumor cell lines (see the results of experimental models 1-3).

In conclusion the cytotoxic activity of SX, that is mediated by one or more proteins possibly with enzymatic activity, is selectively addressed versus tumor cells by the addition of LQ (and interleukin 2, and the interferons alpha, beta and gamma) forming the mixture SX+LQ. Therefore, the described antitumor compound is represented by this mixture. TABLE 1.

Experimental Protocol Microtiter plate, 96 wells

1 2 3 10 11 12

A

B 0.1%CS 0.1%CS 0.1%CS 0.1%CS 0.1%CS 0.1%CS -

C SXl: 100 SXl: 100 SXl:100 SXl: 100 SXl: 100 SXl: 100 -

D +LQ1:100 +LQ1:100 +LQ1:100 +LQ1:100 +LQ1:100 +LQ1:100 -

E 10%CS 10%CS 10%CS 10%CS 10%CS 10%CS -

F SXl:100 SXl: 100 SXl:100 SXl:100 SXl: 100 SXl.100 -

G +LQl:100 +LQl:100 +LQl:100 +LQl:100 +LQl:100 +LQl:100 -

H

Wells B: 4-5-6; C: 4-5-6; D: 4-5-6; E: 4-5-6; F: 4-5-6; G: 4-5-6 A431 cells

Wells B:7-8-9; C: 7-8-9; D: 7-8-9; E: 7-8-9; F:7-8-9; G: 7-8-9 FU cells CS=calf serum

SX 1:100= SX+PBS (2:1) diluted 1:100 in the plate, in C (4-9) wells is in the presence of 0,1% CS, in F (4-9) wells is in the presence of 10% CS; +LQ= SX+LQ diluted 1 : 100 in the plate, in C (4-9) wells is in the presence of 0,1% CS, in F (4-9) wells is in the presence of 10% CS;

Molecular characterisation of the active principle in SX It is another object of the present invention the identification of the active principle that is present in SX. In order to identify the above said active principle, SX has been studied in particular by purifying and characterising its active protein as reported hereinafter.

Aliquots (200 μl) of SX, obtained as indicated above and more widely described in previous patent PCT/EPOl/14727, have been diluted 1:1 with 20 Tris-HCl mM, pH 7,5 and then centrifuged at 13000 rpm for 2 min and purified by means of ion exchange chromatography, using a Mono Q column (Amersham Pharmacia Biotech) (0,5 x 5 cm, granulometry 10 μm), connected to a system for liquid chromatography FPLC. The chromatographic column has been equilibrated with 20 mM Tris-HCl, pH 7,5 buffer, to a flow of 0.4 ml/min, and eluted with a gradient of NaCl in 20 mM Tris-HCl, pH 7,5, from 0 to 0.5 M in 30 min, to a flow of 0.4 ml/min. The absorbance of eluted material has been recorded at 280 nm. Reverse Phases Chromatography.

The eluted material at a salt concentration of about 0.4 M NaCl, corresponding to the fractions that show cytotoxic activity, has been collected in polypropylene tubes of 1.5 ml and lyophilised. An aliquot (about 50 μg) of such fraction has been subsequently solubilised in 0.5 ml of aqueous trifluoroacetic acid (0,1% TFA) and then purified by means of reverse phase chromatography (RP-HPLC), using an analytic column C4 Vydac (The Separation Group, Hesperia, CA, USA) (0,46 x 15 cm, granulometry 5μm). The column has been equilibrated with H2O-TFA 0,1% and eluted with a linear gradient of acetonitril-TFA 0,1% from the 30 to 60% in 35 min, with a flow of 0.8 ml/min. The absorbance of the eluted material has been recorded at 226 nm. The presence of two single chromatographic peaks at about 19 and 25 min have been observed. However, the material flowed in correspondence of P19 and P25 do not show cytotoxic activity. Determination of the Molecular Weight by SDS-PAGE and Mass Spectrometry. The corresponding fractions, called P19 and P25, have been collected in 1.5 ml polypropylene tubes and lyophilised. The electrophoresis analysis of Pl 9 and P25 on 12%polyacrylammide gel in the presence of SDS (SDS-PAGE)₃ followed by Coomassie Brilliant Blue R-250 staining, shows, in the limits of the experimental error, an identical electrophoresis mobility of the two fractions Pl 9 and P25, indicative of a similar value of molecular weight. Subsequently, the molecular weight of the material eluted in correspondence of the fractions Pl 9 and P25 has been determined by Mass spectrometry ESI- TOF. The fractions (10 μg) have been diluted in 20 μl H2O-acetonitril solution (1:1 v/v), containing 1% of formic acid, and loaded on the mass spectrometer Mariner (Perseptive Biosystems). How expected in view of the electrophoresis results, the obtained mass values of Pl 9 and for P25 are equal to 23132.6 ± 2 amu and 23151.7 ± 2 amu, respectively. The UV absorption spectra and the corresponding second order derivative spectra of Pl 9 and P25, recorded between 240 and 350 run, are very superimposable. This result indicates that P19 and P25 contain a very similar number of aromatic amino acids. The UV Spectra have been obtained in buffer Tris-HCl 20 mM, pH 7,5, using a double beam spectrophotometer, mod. Lambda-2 (Perkin-Elmer, Norwalk, CT, USA). Determination of the Number of Cys residues.

An aliquot (20 μg) of P25, purified by RP-HPLC, was submitted to a reaction of reduction and carboxymethylation of the possible Cystein (Cys) residues. The reaction of reduction is performed for 2 h at 37⁰C in 0,1 M Tris-HCl, pH 7,8, buffer, containing 1 mM EDTA and 0,125 M dithiothreitol (DTT). Subsequently, the reaction of carboxy-methylation of the Cys residues was carried out by adding iodoacetamide, up to a concentration of 0.25 M. The reaction has been carried out for 90 min at 37°C and the reaction mixture has been aliquoted by RP-HPLC. The reaction mixture (20 μg in 75 μl) has been loaded on a Vydac C4 column (4,6 x 150 mm, granulometry 5μm), equilibrated in H2O-0.1% TFA and eluted with a gradient of acetonitril-0.1% TFA, in the range from 30 to 60% in 35 min at a flow of 0.8 ml/min. The material eluted in correspondence of the only chromatographic peak has been harvested, lyophilised and analyzed by mass spectrometry, as reported previously, A value of mass for the reduced and carboxymethylated protein equal to 24080 ± 2 amu has been obtained. This value differs from that of non-modified P25 by about 929 amu and it well-matches with the presence of 16 Cys residues (16 x 58 amu = 929 amu).

Determination of the Molecular weight by analytic Gel-Filtration Chromatography. The apparent molecular weight of the active protein material eluted from the ion exchange column at about 0.4 M NaCl, has been analysed by analytic gel-filtration chromatography, using a column Superose- 12 (1 x 30 cm, Amersham Pharmacia Biotech) flowed at a flow of 0.3 ml/min in 20 HiM Tris-HCl, pH 7.5 buffer. The absorbance of the eluted material has been recorded at 280 nm. First, the column has been calibrated with a mixture of proteins having known molecular weight, in the range 14 - 120 kDa. The interstitial volume (Vi) and the empty volume (Vo) of the column have been calculated by loading the tri-peptide Gly-Tyr-Gly and the dextran blue 2x10⁶ Da. The value of the distribution constant (K_D) has been calculated according to the equation KD = (Ve - Vo)/(Vi - Ve), wherein Ve is the elution volume of the various proteins loaded in the column. The apparent molecular weight of the active protein material, purified by ion exchange chromatography, resulted 98 ± 10 kDa. This value indicates that, probably, the active protein, in the reported experimental conditions, exists in a non-covalent tetrameric form. This result well explains why the same fraction obtained by ion-exchange loses activity after inverse phase chromatography. Determination of the N-terminal Sequence. The fractions P19 and P25, purified by RP-HPLC, have been analyzed for their amino terminal sequence, using a protein automatic sequencer mod. 477- A (Applied Biosystems). In both cases the sequence His-Lys-Xxx-Glu-Ile- Xxx-His-Gly-Phe-Gly- Asp-Asp [SEQ. N° 1] was obtained, in which Xxx indicates the absence of a defined fenylthiohydantoin (PTH) in the chromatogram. Usually, that happens when in position Xxx a Cys residues involved in a disulphide bridge Cys-Cys is present. Therefore, the presence of the aforesaid segment results essential for the anti-tumour activity of the protein and proteins containing this sequence are to be considered useful for the wanted purposes.

An example of a protein according to the invention is a protein having the following amino acid sequence [SEQ. N° 2]: HKCEICHGFGDDCDGYQEECPSPEDRCGKILIDIALAPVSFRATHKNCF SSSICKLGRVDIHVWDGVYIRGRTNCCDNDQCEDQPLPGLPLSLQNGL YCPGAFGIFTEDSTEHEVKCRGTETMCLDLVGYRQESYAGNITYNIKG CVSSCPLVTLSERGHEGRKNDLKKVECREALKPASSD This protein, as it can be easily verified in literature (for example using the program BLAST), is extremely similar to the amino acid sequence of the Antitoxic Factor of Python reticulatus (Swiss Prot accession number: Q9I8P7), of which are generally described anti-poison properties when present in hexameric form.

Claims

1. Compositions comprising a component obtained from blood and other biological fluids of snake.

2. Compositions as claimed in Claim 1 in which said component is the supernatant fraction obtained by simple centrifugation of the snake blood

(hereinafter named SX) and said biological fluid is liquor (LQ) extracted from the third ventricle of said snake brain.

3. Compositions as claimed in Claims 1 and 2 containing also a product chosen in the group consisting of: Interleuchina 2, Interferons family including Interferon alpha, Interferon beta, Interferon gamma and Interferon delta or their mixtures.

4. Compositions as claimed in Claims 1 - 3 in which the fraction SX is obtained by centrifugation of aliquots of snake blood, collected by venipuncture, at about 3000 rpm for 5 min followed by the collection of the supernatant, possibly purified by alcoholic precipitation.

5. Compositions as claimed in Claims 1 - 4 in which said liquor (LQ) is collected by venipuncture from the third ventricle of the snake brain and used as such.

6. Compositions as claimed in Claims 1 - 5 in which the composition of the mixture SX+LQ is comprised between 80/20 and 60/40 (v/v)

7. Compositions as claimed in Claims 1 - 6 consisting of: 27 μl SX, 2 μl LQ, and 1 μl of Interleukin 2, Interferon alpha, Interferon beta and Interferon gamma 1 μg/ml.

8. Compositions as claimed in Claims 1 - 7 for pharmacological use.

9. Compositions as claimed in Claim 8 for anti-tumour use.

10. Pharmaceutical compositions containing as active molecule a composition as claimed in Claims 1 - 7.

11. Compositions as claimed in Claims 1 - 10 in which said fractions SX and LQ are obtained from the blood of a python.

12. Composition as claimed in Claim 11 in which the snake is a snake of the genus python (Python Sebae).

13. Method for withdrawal of liquor (LQ) from the third ventricle of snake brain wherein:

- the reptile is anesthetized with 0.5 mg/kg Ketamine and maintained under gas anesthesia with O₂ and lsofluorane administered by endotracheal intubation; - the entire cranial part of the snake head is disinfected;

- the cerebrospinal fluid (LQ) is withdrawn directly from the central cephalic cistern of the subarachnoid space of the brain using a 3OG x 8mm needle inserted at a 90 degree angle for about 1.5 mm across the medial cranial cleft under the third interparietal encephalic squama, exactly at the center of the skull where the left parietal, frontal and right parietal squama converge, under radiologic control of the position of the needle in the cephalic cistern.

14. Protein containing the amino acid sequence His-Lys-Xxx-Gm-Ile-Xxx-

His-Gly-Phe-Gly-Asp-Asp [ SEQ. N° 1 ]

15. Protein as claimed in Claim 14 having the following amino acid sequence:

[ SEQ. N° 2]:

HKCEICHGFGDDCDGYQEECPSPEDRCGKILIDIALAPVSFRATHKNCF

SSSICKLGRVDIHVWDGVYIRGRTNCCDNDQCEDQPLPGLPLSLQNGL

YCPGAFGIFTEDSTEHEVKCRGTETMCLDLVGYRQESYAGNITYNIKG CVSSCPLVTLSERGHEGRKNDLKKVECREALKP ASSD.

16. Use of a protein as claimed in Claims 14 and 15 for the preparation of pharmacological compositions having anti-tumour activities.