FIELD

The present invention relates to anticancer agents. In particular the invention relates to glycerophosphorylethanolamine (GPEA) for use in treatment or prevention of cancer, and compositions including GPEA especially for decreasing risk of cancer caused by consumption of dairy products.

BACKGROUND

It has been reported that consumption of milk and other dairy products is associated with increased risk of cancer incidence and mortality [Neal D. Barnard, Milk Consumption and Prostate Cancer, Physicians Committee for Responsible Medicine; Ji, et al. Br J Cancer. 2014 Oct 14. doi: 10.1038/bjc.2014.544.]. It has also been reported that breast cancer patients who eat cheese, yogurts or ice cream could decrease their chances of survival. It has been suggested that oestrogen found in milk and other dairy foods may encourage tumor growth. On the other hand, it has been reported that certain milk components may have anticarcinogenic effects [Br. J. Nutrition, 84, 2000, S161 -166].

Accordingly, the reported effects on milk and dairy products on human health are bilateral, and the consumption of milk and other dairy products appears to have its pros and cons. Thus, there is a need for a, preferably naturally occurring, component that could suppress the potentially harmful effects of consumption of milk and other dairy products. SUMMARY

The following presents a simplified summary in order to provide a basic understanding of some aspects of various embodiments of the invention. The summary is not an extensive overview of the invention. It is neither intended to identify key or critical elements of the invention nor to delineate the scope of the invention. The following summary presents key concepts of the invention in a simplified form as a prelude to a more detailed description of exemplifying embodiments of the invention.

In the present invention it was observed that glycerophosphorylethanolamine (GPEA) is able to reduce proliferation of cancer cells.

According to one aspect the present invention concern glycerophosphorylethanolamine for use in the treatment and/or prevention of cancer. According to another aspect the present invention concerns pharmaceutical composition including GPEA.

A number of exemplifying and non-limiting embodiments of the invention are described in accompanied dependent claims. The verbs "to comprise" and "to include" are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", i.e. a singular form, throughout this document does not exclude a plurality. BRIEF DESCRIPTION OF FIGURES

Figures 1 -4 show the proliferation of a hormone dependent breast cancer cell line (MCF-7) as the function of GPEA concentration. Estradiol (E2) concentration was 0, 1 .3 nM, 33.3 nM and 3333 nM, in Figs 1 -4, respectively. Incubation time was 24 h.

Figure 5 shows the effect of GPEA on E2-induced stimulation of Scr phosphorylation in MCF-7 breast cancer cells (10 sec incubation; MFI = mean fluorescence intensity).

Figure 6 shows the effect of GPEA on ERK phosphorylation in MCF-7 breast cancer cells (10 sec incubation. MFI = mean fluorescence intensity).

Figure 7 shows the proliferation of a hormone independent prostate cancer cell line (PC-3) as the function of GPEA concentration. Incubation time was 24 h. DETAILED DESCRIPTION OF THE INVENTION

According to one embodiment the present invention concern glycerophosphorylethanolamine (GPEA) for use in the treatment and/or prevention of cancer. The cancer is preferably breast, colon or prostate cancer, more preferably breast or prostate cancer, most preferably breast cancer. According to a preferable embodiment the treatment and/or prevention is inhibition of cancer cell proliferation. The cancer cells are preferably selected from breast cancer cells, colon cancer cells and prostate cancer cells, more preferably breast cancer cells and prostate cancer cells, most preferably breast cancer cells. According to one embodiment the breast cancer cells are hormone dependent breast cancer cells. An exemplary hormone is oestrogen, in particular estradiol (E2). Exemplary E2 dependent breast cancer cells are MCF-7 cells.

According to another embodiment the prostate cancer cells are hormone independent prostate cancer cells. According to a particular embodiment the prostate cancer cells are independent to testosterone. According to an exemplary embodiment the prostate cancer cells are PC-3 cells.

According to another embodiment the present invention concerns a pharmaceutical composition including GPEA. GPEA can be used as such or as its pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" is defined in the review article of Berge et al. [J. Pharm Sci, 66, 1977, 1 ]. Structure of GPEA is shown below.

For the considered therapeutic or preventive uses, GPEA may be administrated as such or in the form of suitable pharmaceutical composition, using conventional techniques and excipients. An exemplary administration route is the oral route. Examples of suitable pharmaceutical compositions include capsules, tablets, solutions, syrups, sachets and vials. The required amount of the active compound may depend on the particular condition to be treated. Any kind of pharmaceutically acceptable solid or liquid carrier or excipient known to those skilled in the medicinal and pharmaceutical arts, may be used in the pharmaceutical preparation.

Amounts and regimens for the administration of the pharmaceutical compositions can be determined readily by those with ordinary skill in the clinical art of treating symptoms and disorders. Generally, the dosage depends on considerations such as age, gender and general health of the patient to be treated; kind of concurrent treatment, if any; frequency of the treatment and nature of the effect desired; duration of the symptoms; and other variables. A desired dose may be administered in one or more applications to obtain the desired results. If desired, the pharmaceutical compositions according to the present embodiments may be provided as unit dosage forms. Means and methods for formulating the present pharmaceutical preparations are known to persons skilled in the art, and may be manufactured in a manner which is in itself known, for example, by means of conventional mixing, granulating, dissolving, lyophilizing or similar processes.

According to one embodiment the present invention concerns a composition, preferably an pharmaceutical composition including GPEA and one or more excipients, wherein the amount of GPEA in the composition is more than 3.5 mg/g, preferably at least 10 mg/g, most preferably at least 100 mg/g. Exemplary excipients are antiadherents (e.g. magnesium stearate), binders (e.g. saccharides such as sucrose, lactose, starches, cellulose, xylitol, sorbitol and maltitol, proteins such as gelatine, synthetic polymers such as polyvinylpyrrolidone and polyethylene glycol), coatings (e.g. hydroxypropyl methylcellulose), disintegrants (e.g. crosslinked polyvinylpyrrolidone, crosslinked sodium carboxymethyl cellulose and sodium starch glycolate), fillers (e.g. plant cellulose, mannitol, sorbitol, calcium carbonate, and magnesium stearate), flavours (e.g. mint, cherry, anise, peach, apricot, liquorice and vanilla), colours, lubricants (e.g. talk, silica, stearin, magnesium stearate and stearic acid), glidants, sorbents, preservatives (e.g. antioxidants, cysteine, methionine, citric acid, sodium citrate, parabens), and sweeteners (e.g. syrups and sugars).

According to one embodiment the composition includes freeze-dried colostrum, preferably freeze-dried bovine colostrum, added GPEA, and one or more excipient, wherein the composition includes more than 3.5 mg/g GPEA, preferably at least 10 mg/g GPEA, most preferably at least 100mg/g GPEA. According to an embodiment the composition includes freeze-dried bovine colostrum and 3-30% (by weight) added GPEA. According to an exemplary embodiment the composition is a 600 mg tablet that includes 200 mg freeze- dried bovine colostrum, and 20-200 mg added GPEA. According to another exemplary embodiment the composition is a 600 mg tablet that includes 200 mg freeze-dried bovine colostrum whey, and 20-200 mg added GPEA.

GPEA can be added to the composition during whey manufacturing process or afterwards. An exemplary process for manufacture of bovine colostrum whey has been disclosed in Fl 103089. GPEA is a physiological metabolite normally present in human body. It has been shown that as far as acute toxicology is concerned, oral route up to 3 g/kg does not cause either death of any symptomatology in mice, rats or rabbits [WO 88/07860]. It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described below but may vary within the scope of the claims.

Examples

Example 1

The effect of GPEA on the proliferation of MCF-7 cells

MCF-7 breast cancer cells (ATCC # HTB-22) were treated with various concentration of estradiol (0-3333 nM) and GPEA (concentrations from 0.027 to 270 μΜ) for 24 h (t = 37 °C; C0 ₂ = 5%) in 24-well plates (50 000 cells/well). WST-1 -cell proliferation reagent (Roche) was added and the colour chance was measured at 450 nm. Results are shown in Figures 1 -4.

Example 2 The effect of GPEA on the proliferation of PC-3 cells

The experiment was performed as above by using PC-3 prostate cancer cells (ATCC # CRL 1435, 35 000 cells/well) in the absence of hormones. Results are shown in Figure 7.

Example 3

The effect of GPEA on oestrogen-induced intracellular signalling in MCF-7 cells The MCF-7 breast cancer cells were treated with 10 nM E2 and 2 μΜ GPEA for three days in 24-well plates, 30 000 cells/well. The growth medium was changed to phenol red-free DMEM and the cells were incubated for another 24 h, rinsed with ice-cold PBS, solubilized in 200 μΙ_ lysis buffer (containing a protease inhibitor), and the plates were frozen at -70 °C for kinase assays. For total ERK, phosphorylated ERK and phosphorylated Src were quantified with commercial immunoassay kits (MILLIPLEX ^® MAP: Cell Signalling Buffer and Detection Kit, Phospho Src (Tyr419) Magnetic Bead MAPmateTM ja 2-Plex Total/Phospho ERK Magnetic Bead Kit) and the samples were analysed by Luminex® 200TM analyser. Results are shown in Figures 5 and 6.

Results Figures 2-4 show proliferation of breast cancer cells (MCF-7) in presence of various concentration of estradiol (E2) as the function of GPEA concentration. As a reference, the proliferation was also studied in the absence of E2 (Figure 1 ). In Figures 2-4, the experiments were performed in the presence of 1 .3 nM, 33.3 nM and 3333 nM estradiol, respectively. It is clearly seen that GPEA is able to inhibit proliferation of breast cancer cells promoted by E2. Proliferation of MCF-7 cells is known to be promoted by oestrogens. Accordingly, GPEA is able to inhibit proliferation of breast cancer cells in the present of stimulant hormone that is also present in milk and other dairy products.

To further verify the effect of GPEA on the inhibition of proliferation of breast cancer cells, additional experiments were performed, namely the ability of GPEA to prevent intracellular signalling by was investigated by means of Src or ERK phosphorylation.

Src is a proto-oncogene encoding a tyrosine kinase expressed in most human tissues, including breast tissue. Its function in the cell is to promote other signals. Src is normally inactive while its activation has been observed in several cancers, including breast and colon cancers. There is also a correlation between elevated Src activity and the severity of the disease.

ERKs (extracellular signal regulated kinase) are intracellular signalling molecules activated by phosphorylation. They belong to MAPK (mitogen activated protein kinase) family and they exist in different forms, e.g. ERK1 (MAPK3) and ERK2 (MAPK1 ). Figure 5 shows the effect of GPEA on E2-induced stimulation of Scr phosphorylation in MCF-7 breast cancer cells. As seen from Figure 5, oestrogen induces elevation of p-Src level (Figure 5, middle). The effect of E2 is inhibited by GPEA (Figure 5, right). Accordingly, GPEA prevents the E2-induced stimulation of Src activity in MCF-7 cells.

Figure 6 shows the effect on GPEA on ERK phosphorylation in MCF-7 breast cancer cells. According to this experiment, a clear inhibition of ERK phosphorylation is visible, indicating that GPEA prevents Src-activation and thereby, at least Src-mediated activation of ERK is prevented.

Figure 7 shows proliferation of prostate cancer cells (PC-3) as the function of GPEA concentration. As shown from the figure, GPEA is able to inhibit proliferation of prostate cancer cells. Further embodiments of the present invention are disclosed in the following numbered clauses

1 . A method of treating and/or preventing cancer in a human or animal subject in need thereof by administering an efficient amount a composition comprising GPEA to said subject.

2. The method according to clause 1 wherein said cancer is selected from breast cancer, colon cancer and prostate cancer.

3. The method according to clause 1 , wherein said cancer is selected from breast cancer, and prostate cancer.

4. The method according to clause 1 , wherein the cancer is breast cancer.

5. The method according to clause 1 , wherein the cancer is prostate cancer.

6. The method according to clause 1 , wherein the cancer is colon cancer.

7. The method according to any of clauses 1 to 6, wherein the composition comprises

GPEA or its pharmaceutically acceptable salt and one or more excipients.

The specific examples provided in the description given above should not be construed as limiting the scope and/or the applicability of the appended claims.