WO2012049647A1 - Phosphatidylcholine in the treatment ophosphatidylcholinef tumours - Google Patents

Phosphatidylcholine in the treatment ophosphatidylcholinef tumours Download PDF

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Publication number
WO2012049647A1
WO2012049647A1 PCT/IB2011/054524 IB2011054524W WO2012049647A1 WO 2012049647 A1 WO2012049647 A1 WO 2012049647A1 IB 2011054524 W IB2011054524 W IB 2011054524W WO 2012049647 A1 WO2012049647 A1 WO 2012049647A1
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phosphatidylcholine
cells
treatment
tumoral
ppc
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PCT/IB2011/054524
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French (fr)
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Vincenzo Cinosi
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Vincenzo Cinosi
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention regards the use of phosphatidylcholine in the treatment of tumoral pathologies .
  • composition of the lipid membranes in tumoral cells can be influenced by the fatty acids added to the culture medium or changing the type of fat nutrition to animals affected by tumours.
  • the biochemical modification of the membrane fatty acids turns into a modification of some physical properties, such as the membrane fluidity and the transport of active ingredients. These modifications have an impact on the dynamics of the membrane and they lead to an increase of the cellular susceptibility to antineoplastic drugs and lipid peroxidation.
  • Phosphatidylcholine is a diacyl glycerophospholipid characterised by a choline residue as a head group bonded to the phosphate group. It is one of the most important components of the biological membranes and in particular it is the most abundant phospholipid on the external layer of the plasma membrane.
  • Phosphatidylcholine is also the main component of lecithin, whose extract consists in a mixture of phosphatidylcholine, phosphoric acid, choline, fatty acids, glycerol, glycolipids, triglycerides and other phospolipids .
  • Phosphatidylcholine can be obtained from the egg-yolk or from soybean seeds, from which it can be extracted with hexane . It is also present in other food products, such as caviar, cauliflower, lentils, peas, rice, calf liver, milk. Depending on the origin, phosphatidylcholine may have substantial differences as a function of the residues of fatty acids in position 1 and 2 of glycerol and in particular the degree of unsaturation thereof.
  • Phosphatidylcholine is normally used as hypocholesterolemic agent and, more recently, in cosmetics.
  • the present invention is based on the surprising finding that administration of phosphatidylcholine to a mammal affected by a tumoral disease causes the selective degradation of the plasma membrane of the tumoral cells and thus induced - therein - an apoptotic process or similar thereto, leading to the death of the tumoral cell.
  • the healthy cells are not influenced by the treatment using phosphatidylcholine.
  • an object of the present invention is phosphatidylcholine for use in the treatment of a tumoral disease .
  • a further object of the invention is a formulation for injection or local release of phosphatidylcholine for use in the treatment of a tumoral disease.
  • Another object of the invention is a method for the treatment of a tumoral disease comprising the administration of a therapeutically effective amount of phosphatidylcholine to a patient affected by a tumoral disease .
  • the present invention has the object of providing phosphatidylcholine for use in the treatment of a tumoral disease in a mammal.
  • the mammal is preferable a human being, but veterinary use is included in the object of the invention.
  • tumour or cancer such as, by way of example, Fibrosarcoma, Liposarcoma, Chondrosarcoma, Osteosarcoma, Angiosarcoma, Myeloma, Hodgkin's disease, Non-Hodgkin lymphoma, Lymphatic leukaemia, Myeloid leukaemia, Papillary carcinoma, Spinocellular carcinoma, Squamocellular carcinoma, Basocellular carcinoma, Adenocarcinoma, Undifferentiated carcinoma, Glioblastoma, Neuroblastoma, Retinoblastoma, Melanoma, Liver cancer, Pancreas cancer, Rectal Cancer, Lung cancer, Gastric tumour, Ovarian tumour, Mammary tumour .
  • the tumoral disease treated according to the present invention is a hepatoma or a mammary tumour .
  • the phosphatidylcholine according to the invention can be any phosphatidylcholine.
  • egg or soybean seed phosphatidylcholine is used.
  • the phosphatidylcholine is polyunsaturated phosphatidylcholine .
  • the polyunsaturated phosphatidylcholine comprises functionalised phosphatidylcholine in position 1, 2 with linoleic acid. More preferably, such functionalised phosphatidylcholine in position 1 , 2 with linoleic acid constitutes at least 70% in weight or at least 80% in weight of the total polyunsaturated phosphatidylcholine.
  • Phosphatidylcholine is available in the market or it can be obtained by extraction from the egg-yolk, from soybean seeds or from other food products in which it is contained .
  • Extraction can be carried out according to conventional methods, using hexane as solvent.
  • the treatment method according to the invention comprises the administration - to a patient affected by a tumoral disease - of a therapeutically effective amount of phosphatidylcholine.
  • the dose of phosphatidylcholine proposed for the administration to a human ranges between 0.1 mg and 2 g and, preferably, between 1 mg and 300 mg of the active ingredient per dose unit.
  • the dose unit can be administered, for example, from 1 to 4 times a day.
  • the dose will depend on the method selected for the administration. It should be observed that it could be required to continuously vary the dose depending on the age and weight of the patient as well as the seriousness of the clinical condition to be treated.
  • the exact dose, the duration of the treatment and the method of administration will be at the discretion of the doctor or veterinarian.
  • a formulation for injection or for local release comprising a therapeutically effective amount of phosphatidylcholine together with pharmaceutically acceptable excipients and carriers constitutes a further object of the invention.
  • the phosphatidylcholine according to the present invention can be formulated for a parenteral administration by injection, in particular by intravenous injection.
  • the formulations for the injections can be presented in form of a single dose, for example in a vial, with an added preservative.
  • the compositions can be in such form as suspensions, solutions or emulsions in oily or aqueous carriers and they may contain formulary agents such as suspension, stabiliser and/or dispersion agents.
  • the active ingredient may be in form of powder to be reconstituted, before use, using a suitable carrier, for example using sterile water.
  • phosphatidylcholine can be formulated according to rectal compositions such as suppositories or retention enemas, for example containing the basic components of the common suppositories like cocoa butter or other glycerides.
  • the phosphatidylcholine may also be formulated as a deposit preparation.
  • Such long-lasting action formulations can be administered by implantation (for example subcutaneous, transcutaneous or intramuscular way) or through intramuscular injection.
  • the phosphatidylcholine can be formulated using appropriate polymer or hydrophobic material (for example in form of an emulsion in a suitable oil) or ion exchange resin.
  • compositions according to the invention can be prepared according to the conventional methods, such as those described for example in Remington's Pharmaceutical Sciences Handbook, Mack Pub. Co.,N.Y.,USA, 17th edition, 1985.
  • PPC Polyunsaturated phosphatidylcholine
  • PPC containing at least 80% in weight of PPC functionalized in position 1,2 with linoleic acid was used.
  • mice Male Wistar rats, weighing 180-200 g, were used as hosts for Yoshida AH-130 ascites hepatoma. This hepatoma was maintained for the transplant of tumoral cells in the rats using serial i.p. passages of 1 ml of suspension containing 2-3xl0 7 cells, conducted at 7-day intervals.
  • Two groups of 20 animals each, subjected to the i.p. injection of 2xl0 6 hepatoma cells were studied.
  • the animals were fed following a standard diet and water ad libitum.
  • One of the two groups was treated with the composition of the example indicated above, corresponding to 20 mg/kg/day of PPC.
  • the second group (CONTROL) was treated using the excipients of the aforementioned composition alone, at the same concentrations present in the dose administered to the other group. Administration started the day subsequent to the implantation of the tumour.
  • hepatoma cells were collected from the peritoneal cavity both of the treated rats and control rats, they were washed using a phosphate buffer solution (PBS) and thus analysed.
  • PBS phosphate buffer solution
  • the plasma membranes were isolated essentially according to the procedure outlined by Koizumi et al . (Koizumi, K., S. Shimizu, K. T. Koizumi, et al . 1981. Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse. Biochim Biophys Acta. 649:393-403) and it is interesting to observe that following these procedures allowed obtaining a complete removal of the contaminant erythrocytes from the collected tumoral cells. Furthermore, the plasma membrane fraction was obtained as a well defined band at the interface between the sucrose layers at 20% and 42%.
  • ATPase activity of the cellular membrane was defined according to the method of Nakao et al. (Nakao, K., S. Kurashina, and M. Nakao. 1967. Adenosinetriphosphatase activity of erythrocyte membrane in hereditary spherocytosis. Life Sci . 6:595-600). The activity of the Na+-K+-ATPase was obtained from the difference of the activity of the total ATPase and Mg2+-ATPase. The assay of 5 ' -nucleotidase activity was conducted according to the method of Gerlach and Hiby (Gerlach, U., and W. Hiby. 1974. Methods of Enzymatic Analysis. H. U. Bergmeyer, editor. Academic Press. New York. NY. 871-875).
  • the Yoshida cells collected from the control rats and from rats treated with PPC were immediately processed through a transmission electron microscopy (TEM) and a scanning electron microscopy (SEM) and thus examined using an Philips 208S transmission electron microscope (FEI Company, Eindhoven, Netherlands) and using a Cambridge Stereoscan 360 scanning electron microscope (Cambridge Instruments Ltd., Cambridge, UK) , respectively.
  • TEM transmission electron microscopy
  • SEM scanning electron microscopy
  • the collected cells were washed and thus fixed using 2.5% glutaraldehyde in 0.2 M caco-dilate buffer, 7.2 pH, for 1 hour, post-fixed using 1% Os04 in 0.2 M caco-dilate buffer, 7.2 pH, dried using incremental concentrations of ethanol and trapped in epoxy resin (Agar 100, AGAR Scientific, Stansed Essex, UK) .
  • Ultra-thin sections obtained using an LKB Ultratome Nova ultramicrotome (LKB, Bromma, Sweden) were stained using uranyl acetate and lead citrate.
  • FF freeze-fracture
  • the cells were left attaching for 15 minutes to glass plates pre-treated using 0.01% aqueous poly-L-lysine hydrobromide, then fixed using 2.5% glutaraldehyde in 0.1 M caco-dilate buffer, 7.2 pH, added with 2% sucrose for 20 minutes. After post-fixation using 1% Os04 in 0.2 M caco-dilate buffer, 7.2 pH, for 30 minutes, the cells were dried using ethanol incremental concentrations, dried to the critical point in in C02 (CPD 020 Balzers device) and spray-gold-coated (SCD 040 Balzers device) .
  • C02 C02
  • SCD 040 Balzers device spray-gold-coated
  • the protein content of the plasma membrane was determined according to the method of Lowry et al . (Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol-reagent. J Biol Chem. 193:265-275) using bovine serum albumin as standard.
  • the lipids were extracted from the plasma membrane purified with CHC13/MeOH 2:1 v/v. An aliquot of the lipid extract was used for determining cholesterols through gas-liquid chromatography as described in Vieu et al . (Vieu, C, B. Jaspard, R. Barbaras, et al . 1996. Identification and quantification of diacylglycerols in HDL on accessibility to lipase. J Lipid Res. 37:1153-1161). Additional amounts were used for measuring the phospholipids according to the phosphorous content thereof after lipid extraction.
  • the phospholipid classes were separated by means of two-dimensional TLC as previously described by Fourcade et al. (Fourcade, 0., M. F. Simon, C. Vlode, et al . 1995. Secretory pho spho 1 ipa s e A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells. Cell. 80:919-927) using - for the first dimension - a mixture of chloroform, methanol and ammonia (65/25/5) and - for the second dimension - a mixture of chloroform, methanol, acetic acid and water (45/20/671) as solvents.
  • the specific enzymatic activities of the purified plasma membranes were enriched 10, 8 and 6 times,, respectively, compared to the raw homogenate.
  • the electron microscopy of the plasma membrane fraction revealed a predominance of the membrane structure of various dimensions and confirmed the identification of the sub- cellular fraction.
  • the phosphatidylcholine in the outer layer of the membrane was considerably increased up to 47% (as shown in Table I); on the contrary, the level of sphingomyelin had reduced by about 17%.
  • the serine and inositol phosphoglycerides did not reveal considerable differences with respect to the control animals.
  • the SEM and TEM preparations revealed that the Yoshida cells of the control animals (treated as mentioned above) have the same morphological and ultra-structural characteristics of the cells as implanted.
  • the SEM observations revealed the typical morphology characterised by a spherical shape, with a diameter comprised between 10 and 15 microns and the surface covered with numerous and long randomly distributed microvilli.
  • the tumoral cells grown in animals treated with PPC and collected after the same time interval (7 days of treatment) revealed extremely clear morphological alterations. Most of these seemed swollen, the cell profile was no longer rounded and the microvilli in the surface had shortened and widened.
  • some cells revealed signs of serious cell damage, particularly consisting in the formation of numerous and large surface bubbles. In addition, many tumoral cells seemed mortally damaged, producing easily visible cellular debris.
  • the control animal cells revealed a well preserved morphology: the intracellular organelles seemed intact and numerous long microvilli were present in the cellular periphery, confirming the SEM observations.
  • the intramembrane particles were distributed randomly both in the exoplasmic and protoplasmic fracture face, while in the protoplasmic fracture face the number of cross-fractured microvilli was extremely lowered.
  • the distribution of the protein particles was modified with the presence of numerous rounded smooth areas .
  • these lipid domains could be due to the new phospholipid re ⁇ arrangement caused by the administration of PPC and it probably represents considerable modifications in the molecular composition and in the structural organisation of the plasmatic membranes of the tumoral cells.
  • various tissues collected from control and treated animals were examined using the TEM technology. Surprisingly, no considerable effect was observed in the analysed istotypes.
  • Lysophosphatidylcholine 1.4+0.4 1.6+0.2 The values were expressed as ⁇ SD mean determined by 5 separate membrane preparations.
  • mice Female C+ Mice, with spontaneous mammary carcinoma, were used for the experimentation through i.v. treatment of polyunsaturated phosphatidylcholine (PPC) - with pharmaceutical formulation of the type used for the hepatoma (Yoshida AH-130) carrier rats.
  • PPC polyunsaturated phosphatidylcholine
  • Two groups of 25 animals each, fed with standard diet and ad libitum water were studied. One of the two groups was treated using 20 mg/Kg/day of PPC.
  • the second group (control) was treated with the excipients of the aforementioned composition alone, at the same concentrations present in the dose administered to the other group.
  • the intravenous administration of PPC in one group started simultaneously with the administration of excipients of the composition (as indicated above) in the other group (control) .
  • the tumoral cells collected from the animals treated using PPC already after two weeks of treatment, revealed considerable variations in the composition of the plasmatic membrane thereof and considerable damage to the nucleus and cytoplasmatic corpuscles, with respect to the tumoral cells of the animals of the control group.
  • most tumoral cells revealed serious alterations of the structure thereof up to the breakage of the plasmatic membrane and the death of the cell.
  • the life of the animals averagely revealed the same duration (four weeks) as the untreated animals.
  • the tumoral masses became more and more invasive in the various locations, producing ulcerations with infiltrations of the tissues surrounding the mammary gland, as it normally occurs in the untreated animals.

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Abstract

The present invention regards the use of phosphatidylcholine in the treatment of tumoral pathologies. In particular, the phosphatidylcholine according to the present invention is polyunsaturated phosphatidylcholine. The phosphatidylcholine suitable for the invention can be obtained by extraction from egg-yolk or from soybean seeds. The tumoral pathologies that can be treated according to the invention are in particular hepatoma and mammary tumour.

Description

PHOSPHATIDYLCHOLINE IN THE TREATMENT OF TUMOURS
Fields of the invention
The present invention regards the use of phosphatidylcholine in the treatment of tumoral pathologies .
State of the art
It is known that the composition of the lipid membranes in tumoral cells can be influenced by the fatty acids added to the culture medium or changing the type of fat nutrition to animals affected by tumours. The biochemical modification of the membrane fatty acids turns into a modification of some physical properties, such as the membrane fluidity and the transport of active ingredients. These modifications have an impact on the dynamics of the membrane and they lead to an increase of the cellular susceptibility to antineoplastic drugs and lipid peroxidation.
However, what occurs to the cellular membranes, especially to the plasma membranes, as clearly proven, is a substitution of the acyl chains of phospholipid fatty acids without minimally disturbing the architecture of the two layers constituting the plasma membrane. The phospholipid or cholesterol content in the membrane does not vary significantly and the vitality of the tumoral cells is not influenced .
In the literature it was indicated that the modification of the membrane phospholipids for treatment in vivo is complex and difficult to implement.
Phosphatidylcholine (PPC) is a diacyl glycerophospholipid characterised by a choline residue as a head group bonded to the phosphate group. It is one of the most important components of the biological membranes and in particular it is the most abundant phospholipid on the external layer of the plasma membrane.
Phosphatidylcholine is also the main component of lecithin, whose extract consists in a mixture of phosphatidylcholine, phosphoric acid, choline, fatty acids, glycerol, glycolipids, triglycerides and other phospolipids .
Phosphatidylcholine can be obtained from the egg-yolk or from soybean seeds, from which it can be extracted with hexane . It is also present in other food products, such as caviar, cauliflower, lentils, peas, rice, calf liver, milk. Depending on the origin, phosphatidylcholine may have substantial differences as a function of the residues of fatty acids in position 1 and 2 of glycerol and in particular the degree of unsaturation thereof.
Phosphatidylcholine is normally used as hypocholesterolemic agent and, more recently, in cosmetics.
Summary of the invention
The present invention is based on the surprising finding that administration of phosphatidylcholine to a mammal affected by a tumoral disease causes the selective degradation of the plasma membrane of the tumoral cells and thus induced - therein - an apoptotic process or similar thereto, leading to the death of the tumoral cell. Vice versa, the healthy cells are not influenced by the treatment using phosphatidylcholine.
Thus, an object of the present invention is phosphatidylcholine for use in the treatment of a tumoral disease .
A further object of the invention is a formulation for injection or local release of phosphatidylcholine for use in the treatment of a tumoral disease.
Another object of the invention is a method for the treatment of a tumoral disease comprising the administration of a therapeutically effective amount of phosphatidylcholine to a patient affected by a tumoral disease .
Detailed description of the invention
The present invention has the object of providing phosphatidylcholine for use in the treatment of a tumoral disease in a mammal.
The mammal is preferable a human being, but veterinary use is included in the object of the invention.
The term "tumoral disease" is used to indicate a tumour or cancer such as, by way of example, Fibrosarcoma, Liposarcoma, Chondrosarcoma, Osteosarcoma, Angiosarcoma, Myeloma, Hodgkin's disease, Non-Hodgkin lymphoma, Lymphatic leukaemia, Myeloid leukaemia, Papillary carcinoma, Spinocellular carcinoma, Squamocellular carcinoma, Basocellular carcinoma, Adenocarcinoma, Undifferentiated carcinoma, Glioblastoma, Neuroblastoma, Retinoblastoma, Melanoma, Liver cancer, Pancreas cancer, Rectal Cancer, Lung cancer, Gastric tumour, Ovarian tumour, Mammary tumour .
According to an embodiment, the tumoral disease treated according to the present invention is a hepatoma or a mammary tumour . The phosphatidylcholine according to the invention can be any phosphatidylcholine.
In an embodiment egg or soybean seed phosphatidylcholine is used.
In an embodiment the phosphatidylcholine is polyunsaturated phosphatidylcholine .
In a particularly preferred embodiment, the polyunsaturated phosphatidylcholine comprises functionalised phosphatidylcholine in position 1, 2 with linoleic acid. More preferably, such functionalised phosphatidylcholine in position 1 , 2 with linoleic acid constitutes at least 70% in weight or at least 80% in weight of the total polyunsaturated phosphatidylcholine.
Phosphatidylcholine is available in the market or it can be obtained by extraction from the egg-yolk, from soybean seeds or from other food products in which it is contained .
Extraction can be carried out according to conventional methods, using hexane as solvent.
The treatment method according to the invention comprises the administration - to a patient affected by a tumoral disease - of a therapeutically effective amount of phosphatidylcholine. According to the present invention the dose of phosphatidylcholine proposed for the administration to a human (with body weight of about 70 Kg) ranges between 0.1 mg and 2 g and, preferably, between 1 mg and 300 mg of the active ingredient per dose unit. The dose unit can be administered, for example, from 1 to 4 times a day. The dose will depend on the method selected for the administration. It should be observed that it could be required to continuously vary the dose depending on the age and weight of the patient as well as the seriousness of the clinical condition to be treated. The exact dose, the duration of the treatment and the method of administration will be at the discretion of the doctor or veterinarian.
A formulation for injection or for local release comprising a therapeutically effective amount of phosphatidylcholine together with pharmaceutically acceptable excipients and carriers constitutes a further object of the invention.
The phosphatidylcholine according to the present invention can be formulated for a parenteral administration by injection, in particular by intravenous injection. The formulations for the injections can be presented in form of a single dose, for example in a vial, with an added preservative. The compositions can be in such form as suspensions, solutions or emulsions in oily or aqueous carriers and they may contain formulary agents such as suspension, stabiliser and/or dispersion agents. Alternatively, the active ingredient may be in form of powder to be reconstituted, before use, using a suitable carrier, for example using sterile water.
Ac co rdi ng t o the p re s en t invention, phosphatidylcholine can be formulated according to rectal compositions such as suppositories or retention enemas, for example containing the basic components of the common suppositories like cocoa butter or other glycerides.
Additionally to the compositions described previously, the phosphatidylcholine may also be formulated as a deposit preparation. Such long-lasting action formulations can be administered by implantation (for example subcutaneous, transcutaneous or intramuscular way) or through intramuscular injection. For example, the phosphatidylcholine can be formulated using appropriate polymer or hydrophobic material (for example in form of an emulsion in a suitable oil) or ion exchange resin.
In any case, the pharmaceutical compositions according to the invention can be prepared according to the conventional methods, such as those described for example in Remington's Pharmaceutical Sciences Handbook, Mack Pub. Co.,N.Y.,USA, 17th edition, 1985.
EXPERIMENTAL PART
EXAMPLE - Pharmaceutical formulation
Polyunsaturated phosphatidylcholine (PPC) was formulated as a composition for injection, mixing:
PPC 250 mg
Deoxycholic acid 122 mg
Benzyl alcohol 45 mg
Sodium chloride 17.5 mg
DL-alpha-tocoferol 0.5 mg
Water up to 5 ml
In particular, PPC containing at least 80% in weight of PPC functionalized in position 1,2 with linoleic acid was used.
Experiment on animals - Hepatoma
Male Wistar rats, weighing 180-200 g, were used as hosts for Yoshida AH-130 ascites hepatoma. This hepatoma was maintained for the transplant of tumoral cells in the rats using serial i.p. passages of 1 ml of suspension containing 2-3xl07 cells, conducted at 7-day intervals.
Two groups of 20 animals each, subjected to the i.p. injection of 2xl06 hepatoma cells were studied. The animals were fed following a standard diet and water ad libitum. One of the two groups was treated with the composition of the example indicated above, corresponding to 20 mg/kg/day of PPC. The second group (CONTROL) was treated using the excipients of the aforementioned composition alone, at the same concentrations present in the dose administered to the other group. Administration started the day subsequent to the implantation of the tumour.
On days 7 and 10 the hepatoma cells were collected from the peritoneal cavity both of the treated rats and control rats, they were washed using a phosphate buffer solution (PBS) and thus analysed.
Isolation of the plasma membrane
The plasma membranes were isolated essentially according to the procedure outlined by Koizumi et al . (Koizumi, K., S. Shimizu, K. T. Koizumi, et al . 1981. Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse. Biochim Biophys Acta. 649:393-403) and it is interesting to observe that following these procedures allowed obtaining a complete removal of the contaminant erythrocytes from the collected tumoral cells. Furthermore, the plasma membrane fraction was obtained as a well defined band at the interface between the sucrose layers at 20% and 42%.
In addition, mitochondria, cellular debris and whole cells were easily recovered, while the nuclei were sedimented at the bottom of the test tube.
All membrane isolation procedures were conducted at 4°C. ATPase activity of the cellular membrane was defined according to the method of Nakao et al. (Nakao, K., S. Kurashina, and M. Nakao. 1967. Adenosinetriphosphatase activity of erythrocyte membrane in hereditary spherocytosis. Life Sci . 6:595-600). The activity of the Na+-K+-ATPase was obtained from the difference of the activity of the total ATPase and Mg2+-ATPase. The assay of 5 ' -nucleotidase activity was conducted according to the method of Gerlach and Hiby (Gerlach, U., and W. Hiby. 1974. Methods of Enzymatic Analysis. H. U. Bergmeyer, editor. Academic Press. New York. NY. 871-875).
Electron microscopy
At the end of the treatment, the Yoshida cells collected from the control rats and from rats treated with PPC were immediately processed through a transmission electron microscopy (TEM) and a scanning electron microscopy (SEM) and thus examined using an Philips 208S transmission electron microscope (FEI Company, Eindhoven, Netherlands) and using a Cambridge Stereoscan 360 scanning electron microscope (Cambridge Instruments Ltd., Cambridge, UK) , respectively.
Regarding the TEM analysis, the collected cells were washed and thus fixed using 2.5% glutaraldehyde in 0.2 M caco-dilate buffer, 7.2 pH, for 1 hour, post-fixed using 1% Os04 in 0.2 M caco-dilate buffer, 7.2 pH, dried using incremental concentrations of ethanol and trapped in epoxy resin (Agar 100, AGAR Scientific, Stansed Essex, UK) . Ultra-thin sections obtained using an LKB Ultratome Nova ultramicrotome (LKB, Bromma, Sweden) were stained using uranyl acetate and lead citrate.
Regarding the "freeze-fracture (FF)" analysis, after fixing using 2.5% glutaraldehyde, the tumoral cells were incubated using glycerol 25% in PBS and maintained at ambient temperature for 30 minutes. The suspensions were then centrifuged at 1000 rpm for 10 minutes, placed on supports and quickly frozen in Freon 22 which was partly solidified by cooling using liquid nitrogen. The mounted supports were then transferred into a Bal-Tec BAF 060 freeze-etch (Bal-Tec Inc., Balzers, Liechtenstein) unit, fractured at -100°C at a pressure of 2-4xl0~7 mbars, shadowed with 2.5 nm of Pt-C (at an angle of 45°) and reproduced with 20 nm of C. The cells were digested overnight with Clorox and the reproductions were mounted on 300-mesh bare grids.
Regarding the SEM analysis, the cells were left attaching for 15 minutes to glass plates pre-treated using 0.01% aqueous poly-L-lysine hydrobromide, then fixed using 2.5% glutaraldehyde in 0.1 M caco-dilate buffer, 7.2 pH, added with 2% sucrose for 20 minutes. After post-fixation using 1% Os04 in 0.2 M caco-dilate buffer, 7.2 pH, for 30 minutes, the cells were dried using ethanol incremental concentrations, dried to the critical point in in C02 (CPD 020 Balzers device) and spray-gold-coated (SCD 040 Balzers device) .
Chemical and statistical analysis
The protein content of the plasma membrane was determined according to the method of Lowry et al . (Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol-reagent. J Biol Chem. 193:265-275) using bovine serum albumin as standard. The lipids were extracted from the plasma membrane purified with CHC13/MeOH 2:1 v/v. An aliquot of the lipid extract was used for determining cholesterols through gas-liquid chromatography as described in Vieu et al . (Vieu, C, B. Jaspard, R. Barbaras, et al . 1996. Identification and quantification of diacylglycerols in HDL on accessibility to lipase. J Lipid Res. 37:1153-1161). Additional amounts were used for measuring the phospholipids according to the phosphorous content thereof after lipid extraction.
The phospholipid classes were separated by means of two-dimensional TLC as previously described by Fourcade et al. (Fourcade, 0., M. F. Simon, C. Vlode, et al . 1995. Secretory pho spho 1 ipa s e A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells. Cell. 80:919-927) using - for the first dimension - a mixture of chloroform, methanol and ammonia (65/25/5) and - for the second dimension - a mixture of chloroform, methanol, acetic acid and water (45/20/671) as solvents.
The results were expressed as ±S D mean. The statistical comparisons were conducted using the Student t- test for impaired samples.
The experiments thus performed revealed that the Yoshida tumoral cells grown in rats treated with PPC were subjected to significant modifications in the composition of the plasma membrane thereof.
The specific enzymatic activities of the purified plasma membranes (Na+-K+-ATPasi , Mg2+-ATPasi and 5'- nucleotidase) were enriched 10, 8 and 6 times,, respectively, compared to the raw homogenate. The electron microscopy of the plasma membrane fraction revealed a predominance of the membrane structure of various dimensions and confirmed the identification of the sub- cellular fraction.
The plasma membranes of the treated animal cells, collected after 7 days of administration of PPC, revealed a considerable difference in the individual distribution of the phospholipids when compared to that of the control cells collected after the same period of time. The latter revealed the same individual distribution of phospholipids of the cells as implanted. The two membrane preparations, from treated and control rats, did not reveal any statistically relevant difference in the amount of cholesterol and phospholipids, with respect to the protein content. Also the molar ratio between cholesterol and phospholipids was similar. Albeit the fact that the total phospholipid content in the plasma membranes of the cells of the treated rats did not reveal any variations, the distribution of the different classes of phospholipids had varied markedly. The phosphatidylcholine in the outer layer of the membrane was considerably increased up to 47% (as shown in Table I); on the contrary, the level of sphingomyelin had reduced by about 17%. The behaviour of phosphatidylethanolamine revealed to be particularly interesting: this phospholipid, which reveals a considerable increase in many tumours, in the hepatoma cells of rats treated using PPC revealed a surprising reduction by about 37% (Table I) . The serine and inositol phosphoglycerides did not reveal considerable differences with respect to the control animals. In addition, there was no difference in the percentage composition of lysophosphatidylcholine in the membranes of both preparations .
The SEM and TEM preparations revealed that the Yoshida cells of the control animals (treated as mentioned above) have the same morphological and ultra-structural characteristics of the cells as implanted.
The SEM observations revealed the typical morphology characterised by a spherical shape, with a diameter comprised between 10 and 15 microns and the surface covered with numerous and long randomly distributed microvilli. On the contrary, the tumoral cells grown in animals treated with PPC and collected after the same time interval (7 days of treatment) revealed extremely clear morphological alterations. Most of these seemed swollen, the cell profile was no longer rounded and the microvilli in the surface had shortened and widened. Furthermore, some cells revealed signs of serious cell damage, particularly consisting in the formation of numerous and large surface bubbles. In addition, many tumoral cells seemed mortally damaged, producing easily visible cellular debris.
The TEM observations confirmed a considerable effect of the treatment using PPC on the ultra-structural characteristics of the Yoshida cells. The control animal cells revealed a well preserved morphology: the intracellular organelles seemed intact and numerous long microvilli were present in the cellular periphery, confirming the SEM observations.
In the rats treated for 10 days using PPC the ascitic hepatoma cells were subjected to considerable morphological and ultra-structural variations, which consisted in an altered form, loss of microvilli, intense cytoplasmic vacuolization and alteration of the cytoplasmic organelles. In particular, in numerous cells, most of the mitochondria revealed a condensed matrix with dilated and ruined crests.
Furthermore, the observations of the cells of control animals and animals treated with PPC, processed by the TEM according to the "freeze-fracture" method, allowed us to analyse the effect of PPC on the molecular organisation of the plasmatic membrane.
In the cells of control animals, the intramembrane particles were distributed randomly both in the exoplasmic and protoplasmic fracture face, while in the protoplasmic fracture face the number of cross-fractured microvilli was extremely lowered. In addition, in the interior monolayer of the plasmatic membrane the distribution of the protein particles was modified with the presence of numerous rounded smooth areas .
Without particular reference to any theory, these lipid domains could be due to the new phospholipid re¬ arrangement caused by the administration of PPC and it probably represents considerable modifications in the molecular composition and in the structural organisation of the plasmatic membranes of the tumoral cells. Lastly, in order to verify whether the administration of PPC to hepatoma carrier rats can also alter healthy cells, various tissues collected from control and treated animals were examined using the TEM technology. Surprisingly, no considerable effect was observed in the analysed istotypes.
The hepatoma carrier rats, treated with PPC according to the previously described protocol, also revealed a considerably longer survival than the control rats, with a life extension of a few weeks in some cases.
Table I
Lipid Control animal Cells of animals cells
treated with PPC
Lipid composition
Cholesterol :protein 137.3±14.1 134.2+13.4 (mcg/mg)
Cholesterol : phospholipid 0.3910.01 0.40+0.03
(mol/mol)
Phospholipid : protein 692.5161.3 657.8+23.1
(mcg/mg)
Main phospholipids (%)
Phosphatidylcholine 32.813.8 48.3+5.7
Phosphatidylethanolamine 27.312.0 17.2+2.4
Sphingomyelin 22.1+1.5 18.4+2.1
Serine and inositol 15.2+2.2 14.1+3.0 phosphoglycerides
Lysophosphatidylcholine 1.4+0.4 1.6+0.2 The values were expressed as ±SD mean determined by 5 separate membrane preparations.
Experiments on animals - mammary tumour
Female C+ Mice, with spontaneous mammary carcinoma, were used for the experimentation through i.v. treatment of polyunsaturated phosphatidylcholine (PPC) - with pharmaceutical formulation of the type used for the hepatoma (Yoshida AH-130) carrier rats. Two groups of 25 animals each, fed with standard diet and ad libitum water were studied. One of the two groups was treated using 20 mg/Kg/day of PPC. The second group (control) was treated with the excipients of the aforementioned composition alone, at the same concentrations present in the dose administered to the other group. Given that the animals revealed manifest signs of mammary tumour at around one year of life, the two groups of animals studied had the same number of months of life (about 12) and a similar body weight. The intravenous administration of PPC in one group started simultaneously with the administration of excipients of the composition (as indicated above) in the other group (control) . The tumoral cells collected from the animals treated using PPC, already after two weeks of treatment, revealed considerable variations in the composition of the plasmatic membrane thereof and considerable damage to the nucleus and cytoplasmatic corpuscles, with respect to the tumoral cells of the animals of the control group. During the third and fourth week of treatment using PPC, most tumoral cells revealed serious alterations of the structure thereof up to the breakage of the plasmatic membrane and the death of the cell. The analysis of the tumoral cells of the control animals, collected at the same time intervals as the treated ones, revealed that such cells still had the same morphological and ultra-structural characteristics of the tumoral cells examined before the beginning of each treatment. The average life of the animals that do not receive any treatment, after the occurrence of a tumour along the mammary line, reveals the duration of about four weeks. In the control groups subject of our study, the life of the animals averagely revealed the same duration (four weeks) as the untreated animals. Furthermore, the tumoral masses became more and more invasive in the various locations, producing ulcerations with infiltrations of the tissues surrounding the mammary gland, as it normally occurs in the untreated animals. Particularly interesting was the behaviour of the tumour in the animals treated using PPC: the tumoral masses did not increase like in the controls and there was neither extensive infiltration of the tissues surrounding the tumour nor evident occurrence of ulcerations. The animals revealed considerable improved general condition and the survival thereof could also reach three months after the beginning of the treatment. Furthermore, in some cases the life extension of the animals could also reach 6-7 months.

Claims

1. Phosphatidylcholine for use in the treatment of tumoral pathologies in a mammal.
2. Phosphatidylcholine according to claim 1, wherein said tumoral pathologies are hepatoma or mammary tumour.
3. Phosphatidylcholine according to claim 1 or 2, wherein said mammal is a human.
4. Phosphatidylcholine according to any one of claims 1 to 3, wherein said phosphatidylcholine is polyunsaturated phosphatidylcholine .
5. Phosphatidylcholine acording to claim 4, said polyunsaturated phosphatidylcholine comprising functionalised phosphatidylcholine in position 1, 2 with linoleic acid,
6. Phosphatidylcholine according to claim 5, wherein said functionalised phosphatidylcholine in position 1, 2 with linoleic acid constitutes at least 70% in weight or at least 80% in weight of the total polyunsaturated phosphatidylcholine .
7. Phosphatidylcholine according to any one of claims 1 to 6, wherein said phosphatidylcholine is egg or soy phosphatidylcholine ,
8. Phosphatidylcholine according to any one of claims 1 to 7, wherein said treatment is in parenteral form.
9. Phosphatidylcholine according to claim 8, wherein said treatment is by i.v. or with local release.
10. Phosphatidylcholine according to any one of claims 1 to 9, wherein a therapeutically effective amount of said phosphatidylcholine is contained in a pharmaceutical composition for injection or for local release together with pharmaceutically acceptable carriers and excipients.
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