FRACTIONS OF SNAKE BLOOD, THEIR PREPARATION AND USE AS THERAPEUTIC AGENTS

Abstract: Fractions of snakes blood having therapeutic properties in vitro are described. The preparation of the above said blood fractions and their therapeutic properties are also described.

Description: Fractions of snake blood, their preparation and use as therapeutic agents Field of the invention The present invention refers to fractions of snake blood and to their preparation and use as therapeutic agents. The active principles present in snake blood show therapeutic activity on cell lines "in vitro". State of the art Chemotherapeutics are usually classified according to their chemical structure or their biological activity (see "The Pharmacological Basis of Therapeutics" by A. Goodman and M. Gilman, Pergamon Press, New York, 1990). In most cases anti tumour drugs act on the cell life cycle blocking the cell growth. The chemotherapeutics classification includes also natural compounds or derivatives thereof (as taxol or vincristine) which are capable to bind to tubulin and therefore inhibits cell mitosis. In view of the extreme importance of chemotherapeutic treatment of tumours it is self evident the importance of making available new drugs having anti tumour activity. Summary of the invention The invention refers to fractions of snake blood obtained by centrifugation of snake blood and their therapeutic use. Description of the drawings Fig. 1 : is a diagram showing the optical density of various cell lines and controls after incubation with P Serum 1:100 for 1 h. Fig.: 2A: reports the optical density of A431 cells cultures treated with various dilution of P Serum incubated at 56°C for 10' or 1 h at 37°C. Fig. 2B: reports the optical density of A431 cells cultures treated with various dilution of P Serum incubated at 56°C for 10' or 3 h at 37°C. Fig. 3: shows the optical density of various cell lines grown in the presence of P Serum at various dilution and FCS (10%). Fig. 4A: shows the number of viable cells per well of A431 cells treated with P Serum in the presence or absence of FCS. Fig. 4B: shows the number of viable cells per well of U373-MG cells treated with P Serum in the presence or absence of FCS. Fig.: 4C: shows the number of viable cells per well of HF cells treated with P Serum in the presence or absence of FCS. Fig. 4D: shows the number of viable cells per well of CVEC cells treated with P Serum in the presence or absence of FCS (10%). Fig. 5: is the picture of the electrophoresis on SDS-PAGE 12% of the supernatant obtained in Example 1. Detailed description of the invention The present invention makes available new compounds having anti tumour activity consisting of fractions of snake blood. According to the invention "fractions" means the supernatant obtained by centrifugation of the snake blood (hereinafter P Serum) or the precipitate (hereinafter pellet) obtained by centrifuging solution obtained treating the above said P Serum with a ethanol/water solution (such treatment being possibly repeated one or more times). Preferably, according to the invention, the snake blood used as starting product for the centrifugation is the blood of snakes of genus pythons. For preparing the fractions according to the invention, aliquots of snake blood are collected by intracardiopuncture and are centrifuged, the supernatant (P Serum) is collected and frozen. Alternatively the P Serum obtained as above described is treated with an ethanol/water solution and the so obtained solution is centrifuged, the supernatant is now eliminated collecting the precipitate (the operation can be repeated more then once) the precipitate remaining after the last centrifugation is collected and tested. The centrifugation of the snake blood is preferably performed at 3000 rpm for 5 minutes while the centrifugation of the ethanol/water solution is preferably performed at 15000 rpm for 30 minutes. The composition of the ethanol/water solution is preferably comprised between 80/20 - 50/50 (v/v) respectively. The invention will be better understood in the light of the examples hereinafter reported. Example 1 Blood aliquots are collected from a Seba python (python of the rocks). After centrifugation at 3000 rpm for 5' the supernatant (P Serum) is collected and subdivided in 0.5 ml aliquots which are stored at - 20°C. Example 2 A cold solution of ethanol in water (80%/20% respectively) is added to the P Serum (2 ml) obtained in Example 1 and left under stirring at 4°C for 30 minutes. The solution is centrifuged (15000 rpm, 30 minutes) the supernatant is eliminated and the precipitate (pellet) is collected. The pellet was resuspended in a solution of ethanol and water (80%/20% respectively) and centrifuged, the procedure was repeated three times. The last obtained pellet was resuspended in sterile PBS (1 ml). Example 3 A cold solution of ethanol and water (50%/50%) was added to the P Serum (1 ml) obtained in Example 1 and stirred at 4°C for 30 minutes. The solution is centrifuged at 15000 rpm for 30 minutes; the supernatant is eliminated and the pellet is collected. The pellet was resuspended in a solution of ethanol and water (50%/50%) and the procedure was repeated three folds. The last obtained pellets was resuspended in sterile PBS (0.5 ml). In this case the quantity of precipitate is lower then in the previous example. On the pellet obtained in example 3 an aminoacid analysis was performed, such analysis showed i.a. the presence of a lysine-squalene derivative which could be one of the main responsible of the activity of the examined component. The whole procedure as described above was repeated in FCS as control of non cytotoxic serum. Effect of P Serum on tumour cells viability The specific anti tumour effect of Serum P (as obtained in example 1) was determined performing tests on various cell lines, in particular: - human squamous carcinoma (A431) - human breast carcinoma (MCF7) - murine melanoma (B16) - human glioblastoma (U373-MG) As control the effects of P Serum on non tumour cell lines, as: - bovine endothelial microcirculus (CVEC) - human fibroblasts (HF) were tested. Experimental protocol 1 The cell viability was evaluated with the sulphurodamine B test according to Skehan P. et al., JNCI, 82: 1107-1112, 1990. 2000 cells per well (growth surface equivalent to 0.28 cm 2 ) are placed on a 96 wells microtiter plate in a medium containing foetal calf serum (FCS) (10%). After 24 hours the medium was washed and thereafter Serum P at different concentration (1:25, 1:50; 1:100, 1:200) is poured in the wells in the absence or in the presence of FCS, the incubation time of the tested samples was 1h and 18 h respectively. Finally the cell were washed with PBS and fixed with trichloroacetic acid (TCA 10%). The microtiter plate was kept at 4°C for 1.5 h and then washed with water (6 folds). Sulphurodamine B (0.4% in acetic acid 1%) was added in the wells and left for 30 minutes in order to allow its binding to cell macromolecules. After 30' the Sulphurodamine B is removed, the wells are washed (6 folds) with acetic acid (1 %) and the wells are dried. 150μl of Tris 10 mM (pH 10.5) are added to extract the colour from the cells, readings of the optical density are performed at 564 nm in a spectrophotometer. Results P Serum is very toxic on all the tested cell lines (tumour and non tumour) at dilution 1:25 and 1:50. This toxicity is evident already after 1 h incubation (see Table 1) and is still present after 18 h (see Table 2). Table 1 : Effect of P Serum on the viability of tumor and non-tumor cell lines (1 h stimulation) P Serum, 1 :25 P Serum, 1 :50 % dead cells % dead cells MCF-7 ND 70% A431 47% 77% B B1166 4 499%% 85% U373-MG 66% 68% HF 53% 60% CVEC 58% 61% Table 2: Effect of P serum on the viability of tumor and non-tumor cell lines (18 h stimulation) P Serum, 1:25 P Serum, 1:50 % dead cells % dead cells A431 74% 68% B B1166 4 477%% 71 % U373-MG 73% 65% HF 66% 50% CVEC 58% 58% Dilution of P Serum up to 1 :100 show a different effect on tumour cells in respect of non tumour cells. At a dilution 1:100 the P Serum shows low or absent toxicity on endothelial cells, a relatively high toxicity on B16 cells and a very high toxicity on other tumour cells (A431, MCF-7 and U373) (see Fig. 1). The effects of P Serum on cell viability are absent when the P Serum is incubated at 56°C for 10 minutes (see Fig. 2 A and B): in the figures are reported the effects on cell line A431 , and similar effects are observed in respect of other cell lines tested (data not shown). The effects of P Serum on the cell viability are maintained when this is incubated at 37°C for 1 - 3 h before the addition to the cell culture (Fig. 2 for A431 , similar effects were observed with others cell lines (data non reported)). 10% FCS (which is the optimal concentration in the cell medium for allowing cell growth) does not protect the tested cell lines from the toxic effects of P Serum (Fig. 3). In this test the cells are grown in the presence of P Serum (1:50 or 1:100) and FCS (10%) and the incubation is prolonged for 18 h. Experimental protocol 2 The experimental protocol 1 shows the absence of cytotoxic effects of highly dilute P Serum (1 :200 or higher) incubated for 1 h with tumour cell lines. In order to better verify whether this high P Serum dilution could show anti tumour activity for longer incubation times in subconfluental cell cultures the behaviour of P Serum dilutions 1:100, 1:200 and 1 :300 incubated for 48 h or 72 h on the following cell lines: - human squamous carcinoma (A431) - human glioblastoma (U373-MG) was tested. Moreover the effects of the same dilutions on non tumour cell lines like: - bovine endothelial microcirculus (CVEC) - human fibroblasts (HF) was tested. In these tests the cell viability was examined by analysing the morphology of the tumour cells fixed using Diff Quick coloration with optical microscopy. From the obtained results the following is observed. Even the highest dilutions of P Serum incubated for 2 - 3 days show cytotoxic activity in subconfluental cell cultures (tumour or non tumour). In particular the addition of FCS (10%) in the medium does not protect the tumour cells from the cytotoxic effect of P Serum (Fig. 4 A and B) while it protects the non tumour cells (Fig. 4 C and D). In conclusion even a dilution of P Serum 1 :200 is slightly toxic for non tumour cell lines (CVEC and HF) for long incubation times (48h or more) while this dilution does not show significant effects for short incubation times (1 - 2 h). Activity of the pellets The anti tumour activity of the pellets (as obtained in examples 2 and 3) was tested on A 431 (human squamous carcinoma cell line) and on the cell line of bovine endothelial microcirculus (CVEC) as non tumour control. The cell viability was measured as said above using the Sulphurodamine B test. The incubation time is 1 h and different dilution of pellets (1:25, 1:10 and 1:5) have been tested. The activity of the pellets was also compared with that of P Serum (dilution 1:25) and of the control (FCS). The results obtained with the pellets obtained according to example 2 show absence of cytotoxic effect on the non tumour cell line (CVEC) while only 65% of viable cells is measured in the tumour line A431 at the lowest dilution (1:5). The P Serum showed a toxic effect both on A 431 and CVEC as shown in Table 1. The anti tumour activity of the pellets obtained according to example 3 was tested on A431 and CVEC as control. The cell viability was tested using the sulphurodamine B test as above said. The incubation time is 1 h and various dilutions of pellets (1:25, 1:10, 1:5) were tested, moreover the activity of the pellets was compared to that of P Serum (1:25) and to that of the pellets obtained precipitating FCS (as control). The tests show absence of cytotoxic effect of the pellets on the non tumour cell line (CVEC) while only 60% of vital cells was observed in A431 cell line at all the tested dilutions (1 :20, 1:10, 1 :5). P serum showed a toxic effect for both A431 and CVEC as indicated in Table 1. The results of the tests support the idea that the anti tumour effect of Serum P is due to the presence of one or more proteins possibly having enzyme activity. Moreover, 2,5 μl of the supernatant obtained in Example 1 were submitted to electrophoresis on SDS-PAGE 12%. The results are reported in Figure 5 wherein column 1 refers to the sample loaded in non reducing conditions, column 2 is the Standars Pharmacia LMW and column 3 refers to the sample loaded in reducing conditions (0.1 M DTT). As it can be see from the figure (seen column 3) a band at around 30 kDa is present and it is considered that this band contains the protein which are responsible of the pharmacological activity. Other studies points towards the conclusion that the desired product is a protein of about 33 kDa having S-S bridges probably in the form of a dimer or oligomer. In addition to the above reported activity it was also demonstrated that P Serum, and the pellets, are capable of inhibiting the telomeric activity in cells as well as to show a pharmacological action in the treatment of non tumour pathologies as for example muscular dystrophy and hyttiosys.

Claims: Claims 1. Snake blood fractions obtained by centrifugation of snake blood. 2. Fraction according to Claim 1 wherein the fraction is the supernatant obtained by centrifuging the snake blood at 3000 rpm for 5 minutes. 3. Fractions according to Claim 1 wherein the fractions are obtained by the following process: a) snake blood centrifugation; b) treatment of the supernatant obtained in step (a) with a ethanol/water solution; c) centrifugation of the solution from step (b) and collection of the precipitate; d) possible repetition of steps (b) and (c). 4. Fractions according to claim 3 wherein the composition of the solution ethanol/water used in step (b) is comprised between 80/20 and 50/50 (v/v). 5. Fractions according to claim 3 wherein the steps (b) and (c) are repeated at least three times. 6. Fractions according to claims 1 - 3 wherein the snake blood is the blood of a snake of genus python. 7. Use of the fractions according to Claims 1 - 6 for the preparation of anti tumour pharmaceutical compositions. 8. Use of the fractions according to claims 1 - 6 for the preparation of pharmaceutical composition for the inhibition of telomeric activity or for the treatment of muscular dystrophy or hyttiosys. 9. Pharmaceutical compositions containing as active principle a fraction of the snake blood according to claims 1 - 6. 10. Use of a pharmaceutical composition according to claim 9 as anti tumour or for the inhibition of telomeric activity or for the treatment of muscular dystrophy or hyttiosys. 11. Basic protein containing S-S bridges and of about 33 kDa isolated by performing an electrophoresis on SDS-Page 12% of the fraction according to claims 2. 12 . Protein according to Claim 11 in the form of a dimer or an oligomer. Fractions of snake blood, their preparation and use as therapeutic agents. Abstract Fractions of snakes blood having therapeutic properties in vitro are described. The preparation of the above said blood fractions and their therapeutic properties are also described.

Inventors: DE FILIPPIS VINCENZO, GRANITO MARIO LEONARDO, MONTEMURNO CONCETTA ANTONIA, PALMIERI ANGELO MICHELE, PAZZAGLI MARIO, REFOSCO FIORENZO, SQUEO MARIA, STANISCI FRANCESCO, ZICHE MARINA, DE FILIPPIS, VINCENZO, GRANITO, MARIO, LEONARDO, MONTEMURNO, CONCETTA, ANTONIA, PALMIERI, ANGELO, MICHELE, PAZZAGLI, MARIO, REFOSCO, FIORENZO, SQUEO, MARIA, STANISCI, FRANCESCO, ZICHE, MARINA
Applicants: DE FILIPPIS VINCENZO, GRANITO MARIO LEONARDO, MONTEMURNO CONCETTA ANTONIA, PALMIERI ANGELO MICHELE, PAZZAGLI MARIO, REFOSCO FIORENZO, SQUEO MARIA, STANISCI FRANCESCO, ZICHE MARINA, DE FILIPPIS, VINCENZO, GRANITO, MARIO, LEONARDO, MONTEMURNO, CONCETTA, ANTONIA, PALMIERI, ANGELO, MICHELE, PAZZAGLI, MARIO, REFOSCO, FIORENZO, SQUEO, MARIA, STANISCI, FRANCESCO, ZICHE, MARINA
Application year: 2001
Main classification: A61K35
IPC codes: A61K35, A61P19, A61P21, A61P29, A61P35