EP3856148A2

EP3856148A2 - Pharmaceutical formulation containing a selenitetriglyceride and a cytostatic for use in treatment of tumour

Info

Publication number: EP3856148A2
Application number: EP19865889.0A
Authority: EP
Inventors: Katarzyna WIKTORSKA; Lidia Mielczarek; Zdzis aw CHILMONCZYK; Piotr Suchocki; Pamela Krug; Maciej Mazur; Paulina G OWALA
Original assignee: Narodowy Instytut Lekow W Warszawie; Uniwersytet Warszawski; Warszawski Uniwersytet Medyczny
Current assignee: Narodowy Instytut Lekow W Warszawie; Uniwersytet Warszawski; Warszawski Uniwersytet Medyczny
Priority date: 2018-09-27
Filing date: 2019-09-26
Publication date: 2021-08-04
Also published as: PL241189B1; EP3856148A4; WO2020067910A3; WO2020067910A2; PL427216A1

Abstract

The invention relates to a pharmaceutical formulation having anti- tumour properties containing at least one cytostatic and selenitetriglyceride, preferably selol as a substance enhancing the effect of the cytostatic for use in the treatment of the tumour, wherein at least one cytostatic and selol are encapsulated in the lipid carrier.

Description

Pharmaceutical formulation containing a selenitetriglyceride and a cytostatic for use in treatment of tumour

DESCRIPTION

FIELD OF THE INVENTION

The invention relates to a mixture of selenitetriglyceride(s), preferably selol and a cytostatic, preferably a cytostatic selected from doxorubicin or 5-fluorouracil in a lipid carrier, which has improved anti-tumour properties compared with the properties of individual components of the composition, especially for breast tumour.

PRIOR ART

Cytostatics are commonly used in tumour therapy, but they exert a highly toxic systemic effect, which results in burdensome adverse effects in the patient. Doxorubicin (DOX) is an anthracycline family antibiotic, which is a widely used cytostatic in the treatment of various tumour types, such as breast, ovarian, prostate, gastric, thyroid tumour, small-cell lung cancer, liver tumour and many other tumours. Its effect consists in inhibiting the proliferation of tumour cells. One of the suggested mechanisms of action thereof involves intercalation to DNA and formation of free radicals, whereas another suggested mechanism is the proteolytic activation of the transcription factor CREB3L1. Due to high systemic cytotoxicity of doxorubicin, new forms of doxorubicin administration are sought for targeted therapy. A liposomal form of doxorubicin is used that has doxorubicin administration efficacy comparable to that of conventional doxorubicin administration with reduced cardiotoxicity [O'Brien M. E. et al.; Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HC1 (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer; Ann Oncol. 2004; 15(3):440- 449].

Another widely used cytostatic is 5-fluorouracil (5-FU), which is a pyrimidine analogue. It is one of the most effective chemotherapeutics used to treat colon, breast, head and neck, anus, gastric, oesophageal, skin cancers and other tumours. It's an anti-tumour drug from the anti-metabolite family. The key mechanism of its action involves inhibiting DNA synthesis by 5-fluoro-2’-deoxyuridine monophosphate metabolite (5-FdUMP) [Marlena Wawrocka- Pawlak; How 5-fluorouracil acts; Wspolcz. Onkol. 2005; 9(10): 414-423] However, 5- fluorouracil is also cytotoxic to normal cells, which implies adverse side effects such as haematological and gastrointestinal diseases [Nadler SH, Moore GE“A clinical study of fluorouracil” Surg Gynecol Obstet 127 (1968): 1210-4]

Combinations are known from prior art of compounds having anti-tumour properties with the so-called synergistic effect. The result of their joint action is improved anti-tumour effect than that of individual components. The paper [Li S.; Selenium sensitizes MCF-7 breast cancer cells to doxorubicin-induced apoptosis through modulation of phospho-Akt and its downstream substrates; Mol. Cancer Ther. 2007:6(3): 1031-1038] gives an example of the use of the combination of doxombicin and methylseleninic acid. This combination, when administered, increases the cytotoxicity of doxorubicin to MCF-7 cell line, which is resistant to doxorubicin for an incubation time of 24 hours. Selenium increases the susceptibility of MCF-7 cells to doxorubicin and abolishes their resistance to this cytostatic.

The anti-tumour properties are also found in selol, which is an original Polish discovery [Patent, Pol. PL 176530 (C1.A61K31/095)].

Selol is a mixture of selenitetriglycerides containing selenium with an oxidation state of +4 synthesized from sunflower oil in the Department of Bioanalysis and Drug Analysis at the

Medical University of Warsaw. The anti- tumour activity of selol is based on prooxidative properties of selenium +4. Selol is an organic selenium compound that has improved bioavailability as well as lower toxicity than inorganic selenium compounds. Studies on animals (mice and rats) have shown that selenium has a 56 and 30-fold, lower toxicity, respectively, than the inorganic compound, sodium selenite.

Prior art reports the use of liposomes as drug carriers, including anti- tumour drugs. Dmg carriers are intended to increase drug efficacy, i.e. increase its accumulation in tumour tissue, while reducing its toxicity to normal tissue. Publication US 4927571 A discloses a method for obtaining a liposomal formulation containing doxombicin for intravenous administration.

Publication WO 1992002208 A1 presents a formulation of doxombicin and lipids that is stable and protects the cytostatic from degradation during storage. Synergistic effect is known from prior art of selol and doxorubicin to murine breast cancer cells 4T1 [Ganassin R., et al; Nanocapsules for the co-delivery of selol and doxorubicin to breast adenocarcinoma 4T1 cells in vitro; Artif Cells Nanomed Biotechnol. 2017(27): 1-11]. However, the combination presented is highly toxic to normal NIH 3T3 murine cells. This means that the obtained combination in nanocapsules made of polyvinylmethyl copolymer and maleate is not selective to anti-tumour activity, which substantially restricts its usability. Moreover, the combination of selol and doxorubicin in nanocapsules as described in prior art is more toxic to normal cells than to tumour cells, which demonstrates its high toxicity and makes it difficult to actually use it in practice.

Prior art also includes information on the activity of selol as a modulator of gene expression, consequently enhancing the effect of doxorubicin on cervical cancer cells HeLa and KB-V1 [Dudkiewicz-Wilczyhska J., et al.; Comparison of selected gene expression profiles in sensitive and resistant cancer cells treated with doxorubicin and Selol; Contemp Oncol (Pozn). 2014 18(2): 90-94], however, there is no information on the synergistic effect of these compounds or on the toxicity to normal cells.

Prior art documents do not report examples of the use of a combination of selenitetriglyceride(s), preferably selol with cytostatic in the lipid carrier such as liposomes or micelles, in particular no combinations were known of selenitetriglyceride(s), preferably selol with doxorubicin or selenitetriglyceride(s), preferably selol with 5-fluorouracil in a lipid carrier to enhance the anti-tumour effect of the cytostatic e.g. doxorubicin or 5-fluorouracil, which, due to the form of administration in the lipid carrier would also reduce the toxicity of the combination of selenitetriglyceride(s), preferably selol and cytostatic to normal tissue. There are no reports in prior art on the use of lipid carriers, in particular liposomes as carriers of such combinations.

DISCLOSURE OF THE INVENTION

The aim of this invention is to provide an improved formulation having anti- tumour (anti-cancer) properties. It was found that lipid carriers in the form of liposomes containing the mixture of selenitetriglyceride(s), preferably selol and cytostatic, in particular doxorubicin and selol or 5-fluorouracil and selol, had increased anti-tumour activity, especially to breast tumour MCF-7 and MDA-MB-231 cells in in vitro studies. Importantly, the studied combinations have reduced toxicity to normal cells CRL-1790. Moreover, it was unexpectedly found that liposomes containing the mixture of doxorubicin and selol or 5-fluorouracil and selol are highly selective towards tumour cells. The studies were conducted using MTT and CVS cytotoxicity assays measuring the ability of compound to reduce cell viability and number, respectively. In both tests, combinations of compounds demonstrated synergism between doxorubicin and selol, 5-fluorouracil and selol. The inventors demonstrated that when administered jointly in the lipid carrier, selenitetriglyceride(s), preferably selol and the cytostatic, the selenitetriglyceride(s), preferably selol, increases the toxicity of cytostatics in tumour cells, in particular in breast tumour cells, while reducing toxicity to normal cells, so that they demonstrated that the combination of said compounds in a lipid carrier has a selective effect on tumour cells.

For example, for doxorubicin, the anti-tumour effect is substantially enhanced: up to 9- fold doxorubicin dose reduction is possible, while the combination of doxorubicin and selol known from prior art administered in nanocapsules [Ganassin R., et al, 2017] allows for up to 2-fold dose reduction while being more toxic to normal cells, which is not observed for the combination of the cytostatic and selol in the lipid carrier. The use of natural lipids to form liposomal formulation containing doxorubicin and selol completely changes the properties thereof.

The invention relates to a pharmaceutical formulation having anti-tumour properties containing at least one cytostatic and selenitetriglyceride(s), preferably selol as a substance enhancing the effect of the cytostatic for use in treatment of tumour, wherein at least one cytostatic and selenitetriglyceride(s), preferably selol, are encapsulated (enclosed, placed) in a lipid carrier.

Preferably, in the pharmaceutical formulation for use in the treatment of the tumour , the cytostatic is selected from the group comprising doxorubicin, 5-fluorouracil, methotrexate, cisplatin, pemetrexed, fludarabine, mercaptopurine, thioguanine, gemcitabine, cytarabine, capecitabine, epirubicin, idarubicin, daunorubicin, bleomycin, dactinomycin, mitomycin, mitoxantrone, etoposide, teniposid, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, topotecan, irinotecan, asparaginase, chlorambucil, cyclophosphamide, ifosfamide, estramustine, chlormethine, melphalan, carmustine, lomustine, streptozocin, cisplatin, carboplatin, oxaliplatin, busulfan, dacarbazine, procarbazine, temozolomide, and analogues thereof, or mixtures thereof.

The pharmaceutical formulation for use in the treatment of tumour is preferably used to treat tumour selected from breast tumour, ovarian tumour , prostate tumour, thyroid tumour, small-cell lung tumour, liver tumour, lymphomas, multiple myeloma, Hodgkin's disease, colon tumour, tumour of head and neck, squamous cell carcinoma of head and neck, anal tumour , gastric tumour, oesophageal tumour, skin tumour. Preferably, in the pharmaceutical formulation for use in the treatment of tumour, the cytostatic is doxorubicin, the selenitetriglyceride is selol, and the formulation is used in the treatment of tumour selected from breast tumour ovarian tumour, prostate tumour, gastric tumour, thyroid tumour, small-cell lung cancer, liver tumour, wherein more preferably the tumour is breast cancer, more preferably a breast cancer characterised by the absence of oestrogen, progesterone and HER-2/Neu receptors.

Preferably, in the pharmaceutical formulation for use in the treatment of tumour, the cytostatic is 5-fluorouracil, the selenitetriglyceride is selol, and the formulation is used in the treatment of tumour selected from breast tumour, ovarian tumour, colon tumour, tumour of head and neck, squamous cell carcinoma of head and neck, anal tumour, gastric tumour, oesophageal tumour, skin tumour, wherein more preferably the tumour is breast cancer, more preferably a breast cancer characterised by the absence of oestrogen, progesterone and HER-2/Neu receptors.

Preferably, in the pharmaceutical formulation for use in the treatment of tumour, the lipid carrier is liposomes or lipid micelles, preferably lipid liposomes, wherein more preferably liposomes and lecithin micelles constituting the carriers are produced from compounds or mixtures thereof selected from groups of natural lipids, including chicken egg yolk lecithin, soy lecithin and synthetic lipids, both non-deuterated and deuterated including phosphatidylcholines, preferably l,2-dimirystol-rac-glicero-3-phosphocholine, cholesterol, or distearoyl-phosphatidylethanolamino-poly( ethylene oxide) or a mixture thereof.

In the pharmaceutical formulation having anti- tumour properties for use in the treatment of tumour of the invention, containing a cytostatic, e.g. doxorubicin or 5-fluorouracil and the substance enhancing the effect thereof, acting in synergy with a cytostatic, constituted by selenitetriglyceride(s), preferably selol, anti- tumour substances are dispersed in the aqueous phase by being encapsulated in lipid carriers, preferably lipid liposomes.

Selenitetriglyceride(s) are intended to denote selenitetriglyceride(s) and/or a mixture of selenitetriglycerides, which is/are a combination of triglycerides and selenium at varying oxidation states, and the final product has varying contents of selenium, favourably contains 1 to 30% selenium. Preferably, the selenitetriglyceride is selol, which is a mixture of selenitetriglycerides containing selenium at +4 degree of oxidation.

Lipid carriers are intended to denote lipid association structures such as liposomes and lipid micelles. In order to obtain the formulation, in the first step an aqueous suspension containing a lipid, the selected cytostatic and selenitetriglyceride(s), preferably selol, is exposed to ultrasounds. The resulting solution is then alternately frozen using liquid nitrogen and thawed in 50°C in order to reduce the volume of the resulting liposomes. Finally, the solution is passed through a filter, preferably a syringe filter with the diameter of several dozen to several hundred nanometres. Once the sample is passed through the filter, preferably the syringe filter, the mixture is dialysed in order to remove the cytostatics not enclosed in the liposomes.

The invention also relates to the use of the formulation according to the invention in the treatment of tumour, including breast tumour. The problem of tumour therapy in Poland and in the world remains unresolved, and more effective therapy methods are still being sought. The presented invention: the combination of cytostatic with selenitetriglyceride(s), preferably selol in the lipid carrier, preferably doxorubicin and selol and 5-fluorouracil and selol in the lipid carrier, in in vitro studies showed improved cytotoxic properties to tumour cells, in particular for breast tumour than doxorubicin alone and 5-fluorouracil alone or selol alone, as well as combined administration of a cytostatic and selol not enclosed in a lipid carrier.

The pharmaceutical formulation comprising selol and the cytostatic in the lipid carrier according to the invention has reduced toxicity to normal cells, as confirmed by measurements of survival rates of normal cells CRL-1790 in in vitro studies. Consequently, the formulation containing selenitetriglyceride(s), preferably selol and the cytostatic in the lipid carrier is highly selective towards tumour cells.

The pharmaceutical formulation comprising selenitetriglyceride(s), preferably selol and a cytostatic in a lipid carrier is generally administered in the form of appropriate pharmaceutical forms, where the active substances enclosed in lipid carriers may be combined with the therapeutically acceptable carrier.

The choice of the therapeutically acceptable carrier will depend on the route of administration of the pharmaceutical formulation containing selenitetriglyceride(s), preferably selol and cytostatic in the lipid carrier, and on the need to protect it against inactivation or degradation, against penetrating into cells, tissues or body. Therapeutically acceptable carriers include solvents, dispersion media and auxiliaries (coating agents, surfactants, flavourings, anti-oxidants and the like).

The pharmaceutical formulation containing selenitetriglyceride(s), preferably selol and cytostatic in the lipid carrier according to the invention may be administered by various routes, including by injection, orally, topically and per rectum. The dose of the pharmaceutical formulation is determined taking into account the route of administration, the condition requiring treatment or prevention, and other specific circumstances.

BRIEF DESCRIPTION OF THE FIGURES OF THE DRAWING

To be better understood, the invention has been illustrated in embodiments and in the accompanying figures, where:

Fig. 1 shows TEM image (Transmission electron microscope) of liposomes loaded with selol and doxombicin (200 nm).

Fig. 2 presents the correlation between the Cl value (determining the type of the interaction observed) and the fraction of dead cells (determining the number of dead cells) for the combination of (A) doxombicin (DOX) and selol in liposomes in MCF-7 cell line; (B) doxombicin (DOX) and selol in liposomes in CRL-1790 cell line. The correlation between the Cl value based on experimental points and the dead cell fraction (solid line) was estimated.

Fig. 3 presents the correlation between the Cl value (determining the type of the interaction observed) and the fraction of dead cells (determining the number of dead cells) for the combination of (A) 5-fluorouracil (5-FU) and selol in liposomes in MCF-7 cell line; (B) 5- fluorouracil (5-FU) and selol in liposomes in CRL-1790 cell line. The correlation between the Cl value based on experimental points and the dead cell fraction (solid line) was estimated.

The invention is illustrated in greater detail in the below embodiments that do not limit the scope thereof.

EXAMPLES

In the below examples, unless indicated otherwise, standard materials and in vitro biochemical methods were employed or it was proceeded according to the manufacturer's recommendations for particular materials and methods.

Example 1. Obtaining DMPC liposomes containing selol and cytostatic (using the example of doxorubicin (DOX) and 5-fluorouracil (5-FU))

A. Liposomes were obtained by dissolving 2.4 mg DMPC (l,2-dimyristoyl-rac-glycero-3- phosphocholine) in a solution of 3 mg selol in 600 mΐ chloroform in a glass vial, after which chloroform was completely evaporated from the vial using argon from the tank. 1.5 mL solution of 1 mg of doxombicin in 10 mL was added to the vial and sonicated by ultrasound for 3 minutes. The solution was then alternately frozen using liquid nitrogen and thawed in 50°C in order to reduce the volume of the liposomes. The freezing and thawing procedure was repeated four times. Finally, the solution was passed through a 0.45 pm pore diameter syringe filter and the sample was dialysed to remove any doxorubicin not encapsulated in the liposomes.

B. Liposomes containing 5-fluorouracil and selol were obtained like above: a solution of 3 mg of selol in 600 pL chloroform was added to 2.4 mg DMPC, after which the chloroform was evaporated and 1.5 mL of 5-fluorouracil solution (4 mg/lO mL) was added, followed by the solution being sonicated, frozen and thawed four times, and at the end of the synthesis the solution was passed through a syringe filter, after which the sample was dialysed at 4 °C for 24h.

C. The following were obtained using the modified procedure for obtaining liposome carriers described in items A and B:

- liposomes containing only doxorubicin or only 5-fluorouracil (no selol was added during production);

- liposomes containing only selol (no cytostatic was added during production);

- empty liposome carriers (no selol or cytostatic were added during production).

Example 2. Determination of the sizes of liposomes obtained through microscopic measurements

In order to prepare the samples for the tests by transmission electron microscope (TEM), the sample of the liposomes obtained in Example 1 were diluted twenty-fold with PBS solution. Then one drop of the prepared sample was added onto the TEM grid and left to dry for 24h. Fig. 1 shows a representative microscopic image of liposomes containing selol and doxombicin. The liposomes obtained were approx. 400 nm in size.

Example 3. Determination of the encapsulation rate of doxorubicin and selol

In order to determine the efficiency of encapsulation of the active ingredients in the liposomes obtained in Example 1, the UV-V1S spectrum for the standard solution in THF (tetrahydrofuran) and the spectrum for sample of liposomes containing the active ingredients were recorded after evaporating water from the sample and dissolving in THF. The measurement of absorbance in order to determine the doxorubicin content was conducted at the 480 nm wavelength. For example, for liposomes containing doxorubicin and selol obtained according to Example 1, the encapsulation rate of doxorubicin was 15%. Due to the hydrophobic structure of selol, all of its content was enclosed in liposomes.

Example 4. Testing the interaction of doxorubicin with selol and 5-fluorouracil with selol

The cytotoxicity of the test cytostatic compounds administered in lipid carriers separately and in combination with selol after incubation time of 72 hours was studied.

Lipid carriers containing selol alone, doxorubicin alone and 5-fluorouracil alone as well as empty carriers were obtained as described in Example 1.

The tests were carried out on two cell lines of breast cancer: MCF-7 and MDA-MB-231. Normal CRL-1790 cells were used in testing as control. Toxicity of the compounds i.e. how they affect the number of viable cells was determined in two cytotoxicity assays (MTT and CVS).

Cell lines used:

MCF-7 cell line

The MCF-7 cell line is the breast cancer cell line derived from 69-year-old Caucasian woman. MCF-7 cells produce oestrogen and progesterone receptors. The MCF-7 cell line was purchased from the American Type Culture Collection (ATTC).

MDA-MB-231 cell line

The MDA-MB-231 cell line is the breast cancer cell line derived from 51 -year-old Caucasian woman. MDA-MB-231 cells do not express oestrogen and progesterone or HER-2/Neu receptors. The MDA-MB-231 cell line was purchased from the ATTC.

CRL-1790 cells

CRL-1790 cells are normal cells collected from the intestine of 21 -week-old female foetus. Morphologically, they resemble epithelial cells, but they do not contain keratin and there is no clear evidence to identify their origin. The CRL-1790 cell line was purchased from the ATTC.

Cell culture process

MCF-7, MDA-MB-231 and CRL-1790 cell lines were stored frozen in a liquid nitrogen tank at the temperature of -196°C. In order to start the culture, a vial containing the cells was heated with a stream of warm air from the dryer, after which the cell suspension was transferred to a culture bottle. The culture was carried out in the incubator in 5% atmosphere C0₂ and 37 °C. Cells were incubated to reach confluence of 80% and then passaged. Cells were detached using 0.25% trypsin solution in EDTA. For the CRL-1790 cell cultures, the cells were additionally scraped from the bottom of the bottle, since they did not completely detach from the medium during trypsinization. Tumour cells were diluted in fresh medium in the ratio of 1 :4, and normal cells in the ratio of 1 :2. For the experiment, a suspension of MCF-7 or MDA-MB-231 cells with a density of 40,000 thousand cells per 1 ml of the medium was plated into 96-well culture plates. For the CRL-1790 cell line, a cell suspension with a density of 80,000 cells per 1 mL was used. Carriers containing selol alone in varying concentrations, the cytostatic alone in varying concentrations and the mixture of these compounds inappropriate concentrations were then added to the wells. A control test was also performed where empty drug carriers were added.

CVS ( Crystal Violet Staining) test

After washing the cell-containing wells, 50 pL 0.5% crystal violet was added to each well and incubated for 10 minutes at room temperature. The violet solution was then pipetted off and 100 pL of 1% sodium lauryl sulfate (SDS) was added to each well. The absorbance of the solution was read at l=595 nm using the POWER WAVE XS spectrophotometer.

MTT (methylthiazol tetrazolium salt) assay

After washing, 50 pL MTT (250 pg/mL) was added to each cell-containing well, which were placed in the incubator for 3 hours. Isopropanol was then added (200 pL per well) to the plates. The absorbance of the solution was read at l=570 nm using the POWER WAVE XS spectrophotometer.

In order to determine the type of interaction between selol and cytostatics in the lipid carries in the form of liposomes, the Chou-Talalay method was used to determine the Cl (Combination Index) value, which indicates synergy if lower than 0.9, and additive effect, if between 0.9 and 1.1. For such interactions, the effect of the mixture is more potent than that of each substance alone. In addition, combined administration allows for using smaller doses of drugs compared to individual administration. Cl values above 1 indicate that there occurs antagonism, which means that the effect of the mixture is less potent than that of each substance separately.

Fig. 2 and Fig. 3 show representative examples of studies of the interaction of doxorubicin (DOX) and selol and 5-fluorouracil (5-FU) and selol in a lipid carrier in the form of liposomes in in vitro studies involving the breast cancer cell line MCF-7 and normal cell line CRL-1790 as control performed using the CVS assay. Liposomes containing a mixture of doxorubicin (DOX) and selol and 5-fluorouracil (5-FU) and selol in a lipid carrier in the form of liposomes have been found to have an anti- tumour effect superior to that of the cytostatic alone, as Cl values are below 0.9 for practically the whole range of dead cell fraction. In addition, combined administration allows for using smaller doses of drugs compared to individual administration. The dose reduction indices (DRI) determined indicate that it is possible to significantly reduce the doxorubicin dose (Table 1). In vitro studies on breast cancer cells MDA-MB-231 showed the synergy of action between doxorubicin and selol in the lipid carrier, as Cl values are below 0.9 - Table 2. The combination of 5-fluorouracil and selol in the lipid carrier is characterized by additivism and antagonism depending on the dead cell fraction.

Table 1. Cl and DRI values for the combination of doxorubicin and selol in lipid carrier, 5- fluorouracil and selol in lipid carrier— MCF-7 cell line

Table 2. Cl and DRI values for the combination of doxorubicin and selol in lipid carrier, 5- fluorouracil and selol in lipid carrier - MDA-MB-231 cell line

Table 3 presents Cl values for the tested combinations of doxorubicin and selol, 5- fluorouracil and selol in the lipid carrier in the form of liposomes in relation to normal CRL- 1790 cells.

Table 3. Cl values for the combination of doxorubicin and selol in lipid carrier, 5- fluorouracil and selol in lipid carrier - CRL-1790 cell line

Surprisingly, it was found that the pharmaceutical formulation containing a mixture of doxorubicin or 5 -fluorouracil (cytostatic) and selol in a lipid carrier had anti- tumour properties superior to those of the cytostatic alone, and its toxicity to normal cells is reduced, which was hitherto not reported or suggested. Combined administration of a cytostatic and selol in the lipid carrier is characterized by antagonism in normal cells (Table 3), which reduces cytotoxicity of the cytostatic in relation to normal tissue, while demonstrating synergy in tumour cells, which increases the cytotoxic effect of the cytostatic in tumour tissue (Tables 1 and 2). The demonstrated synergy of active substances is a highly beneficial factor, as it allows to reduce the effective concentration of the drug. Combined administration of selenitetriglyceride(s), preferably selol and cytostatic in the lipid carrier allows for reducing doxorubicin concentration for the largest dead cell fractions by almost 90%, and by almost 56 % in MCF-7 cells for 5- fluorouracil. The positive decrease in Cl value was also observed together with an increase in the dead cell fraction (Fig. 2A).

Example 5. Comparison of interaction of the formulation containing a cytostatic and selol jointly enclosed in liposomes and combined administration of a cytostatic and selol (using the example of 5-fluorouracil) In order to determine the merits of preparing the formulation, cytotoxicity of the tested compounds administered in lipid carriers was tested by administration in terms of both combined administration (administered jointly not in the lipid carrier) and in the form of a formulation containing selol and 5 -fluorouracil encapsulated j ointly in liposomes (administered jointly in the lipid carrier). Lipid carriers were obtained as described in Example 1. The study was performed using the MTT assay on MCF-7 breast tumour cells - Table 4.

Table 4. Cl values for 5-fluorouracil and selol for MCF-7 cells after a 72h incubation time for combined administration and for the formulation where the components are enclosed jointly in liposomes.

The studies showed that the formulation containing 5-fluorouracil and selol, which are jointly encapsulated in liposomes, has a sustainable synergistic effect on MCF-7 tumour cells for the whole range of dead cell fraction (Cl values: 0.76-0.92).

Combined administration of the active ingredients thereof not enclosed in liposomes for lower dead cell fractions is characterised by antagonism, and, conversely, by synergy for higher dead cell fractions. To sum up, it is more preferable to enclose cytostatic and selenitetriglyceride(s), preferably selol, jointly in the lipid carrier such as the liposome. Example 6. Selectivity of effect of doxorubicin with selol and 5-fluorouracil with selol in the lipid carrier in the form of liposomes

Based on a cytotoxicity study performed using the CVS assay, the selectivity of effect of the combination of doxorubicin and selol, 5-fluorouracil and selol in the lipid carrier in the form of liposomes obtained according to Example 1 in relation to tumour cells was studied. The IC25, IC50, IC75 parameters were determined using GraphPadPrism 5 software to determine the concentration of the test compound for which the survival rate of cells is 75%, 50% and 25%, respectively. Table 5 shows the data obtained. The concentrations shown are expressed as the concentration of doxorubicin and 5-fluorouracil [mM] This made it possible to determine the value of the selectivity coefficient, which is the ratio of the concentration of doxorubicin (or 5- fluorouracil) in normal cells to the concentration in tumour cells inducing the same cytotoxic effect.

Table 5. IC25, IC50, IC75 and IC75 and selectivity coefficient values for the combination of doxorubicin and selol, 5-fluorouracil and selol to breast tumour cells MCF-7 and normal cells CRL-1790

The obtained preparation of selol and cytostatic in the lipid carrier in the form of liposomes is selective in terms of cytotoxic effect: it has reduced systemic toxicity while also having the more potent anti-tumour effect, especially for breast tumour cells. The values of the selectivity coefficient show that for the combination of doxorubicin and selol in the lipid carrier in the form of liposomes, the toxicity to normal cells is approximately one and a half times lower, while being approximately twice as low for the combination of 5-fluorouracil and selol in the lipid carrier in the form of liposomes to normal cells. Anti-tumour drugs should have such properties, so that the pharmaceutical formulation containing selenitetriglyceride(s), preferably selol and the cytostatic in the lipid carrier according to the invention will be useful in the treatment of the tumour and has better properties than doxorubicin or 5-fluorouracil with selol in a polymer carrier. The combination of doxorubicin and selol in nanocapsules made of polymer known from prior art has no selectivity towards tumour cells, and, further, it is more toxic to normal cells than to tumour cells.

Claims

1. A pharmaceutical formulation having anti- tumour properties containing at least one cytostatic and selenitetriglyceride(s), preferably selol as the substance enhancing the effect of the cytostatic for use in the treatment of tumour , wherein at least one cytostatic and selenitetriglyceride(s), preferably selol, are encapsulated in a lipid carrier.

2. The pharmaceutical formulation for use according to the claim 1, characterised in that the cytostatic is selected from the group comprising doxorubicin, 5-fluorouracil, methotrexate, cisplatin, pemetrexed, fludarabine, mercaptopurine, thioguanine, gemcitabine, cytarabine, capecitabine, epirubicin, idarubicin, daunorubicin, bleomycin, dactinomycin, mitomycin, mitoxantrone, etoposide, teniposid, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, topotecan, irinotecan, asparaginase, chlorambucil, cyclophosphamide, ifosfamide, estramustine, chlormethine, melphalan, carmustine, lomustine, streptozocin, cisplatin, carboplatin, oxaliplatin, busulfan, dacarbazine, procarbazine, temozolomide, and analogues thereof, or mixtures thereof.

3 The pharmaceutical formulation for use according to the claim 1 to 2, characterised in that it is use in the treatment of tumour, preferably used to treat tumour selected from breast tumour, ovarian tumour, prostate tumour, thyroid tumour, small-cell lung cancer, liver tumour, lymphomas, multiple myeloma, Hodgkin's disease, colon tumour, tumour of head and neck, squamous cell carcinoma of head and neck, anal tumour, gastric tumour, oesophageal tumour, skin tumour.

4. The pharmaceutical formulation for use according to the claim 1 to 3, characterised in that the cytostatic is doxorubicin, the selenitetriglyceride(s) is selol, and in that the formulation is use in the treatment of tumour selected from breast tumour, ovarian tumour, prostate tumour, gastric tumour, thyroid tumour, small-cell lung cancer, liver tumour.

5. The pharmaceutical formulation for use according to the claim 4, characterised in that the tumour is breast cancer, more preferably breast cancer characterised by the absence of oestrogen, progesterone and HER-2/Neu receptors.

6. The pharmaceutical formulation for use according to the claim 1 to 3, characterised in that the cytostatic is 5-fluorouracil, the selenitetriglyceride(s) is selol, and in that the formulation is use in the treatment of tumour selected from breast tumour, colon tumour, tumour of head and neck, squamous cell carcinoma of head and neck, anal tumour, gastric tumour, oesophageal tumour, skin tumour.

7. The pharmaceutical formulation for use according to the claim 6, characterised in that the tumour is breast cancer, more preferably breast cancer characterised by the absence of oestrogen, progesterone and HER-2/Neu receptors.

8. The pharmaceutical formulation for use according to the claim 1 -7, characterised in that the lipid carrier are liposomes or lipid micelles, preferably lipid liposomes.

9. The pharmaceutical formulation for use according to the claim 8, characterised in that liposomes, lipid micelles, preferably lecithin micelles constituting the carriers are produced from compounds or mixtures thereof selected from groups of natural lipids, including chicken egg yolk lecithin, soy lecithin and synthetic lipids, both non-deuterated and deuterated including phosphatidylcholines, preferably l,2-dimirystol-rac-glicero-3-phosphocholine, cholesterol, or distearoyl-phosphatidylethanolamino-poly(ethylene oxide) or mixtures thereof.