WO2006067498A1 - Eicosapentaenoic acid for the treatment of cancer - Google Patents

Abstract

Eicosapentaenoic acid (EPA) in free acid form having a purity of about 99 per cent was administered to patients with a history of colorectal polyps for three months. It was found that crypt cell proliferation was significantly reduced, and apoptosis significantly increased, in the colonic mucosa of the treatment group. No such significant changes were seen in the non-treatment group.

Description

EICOSAPENTAENOIC ACID FOR THE TREATMENT OF CANCER

The present invention relates to the use of omega-3 fatty acid compositions containing high levels of eicosapentaenoic acid (EPA).

Omega-3 fatty acids are long chain polyunsaturated fats containing a double bond at the n- 3 position. The main dietary forms of omega-3 are alpha-linolenic acid (ALA), EPA, and docosahexaenoic acid (DHA). ALA is found in certain plant and vegetable oils, for example flaxseed and canola oil, while EPA and DHA are found in fish oils, such as cod liver oil.

In their natural state in fish oils, EPA and DHA are esterified to glycerol and may occupy one or more of the hydroxyl groups on the glycerol molecule. The natural lipid is described as a "glycerol ester" or "triglyceride" and the EPA content in fish oils can range from a few percent up to a level of around 20%. The remaining fatty acids on the glycerol backbone include saturated fatty acids, monounsaturated fatty acids and other polyunsaturated fatty acids.

Omega-3 fatty acids are considered to have a wide range of health benefits. Figure 1 shows how fat consumption by humans has altered over time. It can be seen that in the last

100 years or so, total fat consumption has increased, and of this the proportions of omega-6 polyunsaturated fats and trans-fatty acids have increased, whereas the proportion of omega-3 polyunsaturated fats has decreased. There has therefore been much interest recently in dietary supplementation with omega-3 polyunsaturated fats.

Fish oils have been indicated as having a protective effect on the development of colorectal cancer in animal models of colorectal carcinogenesis. Furthermore, epidemiological studies have shown a reduced risk of colorectal cancer in populations consuming high amounts of fish/fish oil. The protective effect is thought to be due to the relatively high levels of omega-3 fatty acids in these oils. Patients with a history of colorectal polyps are at increased risk of developing colorectal cancer. Previous studies have shown these patients to have increased rates of crypt cell proliferation and reduced levels of mucosal apoptosis in biopsies of normal-appearing colonic mucosa taken from these patients.

Bartram et al. (Gastroenterology 1993; 105:1317-22) investigated the effect of fish oil and corn oil on rectal crypt cell proliferation in a double-blind crossover trial carried out over two 4-week periods. All participants consumed a controlled diet of identical nutrient composition, supplemented with either capsules providing 1 Ig of fish oil per day (containing 4.4g omega-3 fatty acids/day) or capsules providing 1 Ig of corn oil per day. The total labelling index from rectal biopsy specimens was significantly lower during the fish oil period when compared to the corn oil period, indicating a reduced level of rectal crypt cell proliferation. No analysis of the effects on apoptosis was carried out.

Anti et al. (Gastroenterology 1994; 107: 1709-18) studied the effect of differing levels of fish oil supplementation on rectal mucosal proliferation. Sixty patients with sporadic colorectal adenomas were randomised into 4 groups receiving 2.5, 5.1 or 7.7g of omega-3 fatty acids or placebo for 30 days. With respect to the placebo group, all 39 patients receiving fish oil showed reduced levels of proliferation, with significant decreases in the total labelling index, as well as significant changes in labelling indices in compartments 2, 3 and 4&5 (the upper regions of the crypt) combined, being exhibited. There were no significant differences in the changes in labelling indices between the 3 different fish oil dosage groups. Again, the effect of the fish oils on cell apoptosis was not studied.

Gee et al. (Journal of Nutrition 1999: 129: 1862-5) measured crypt cell proliferation in rectal biopsies taken pre-, during, and 8-12 weeks post-surgery in patients with left sided colorectal cancers receiving either fish oil (providing 1.4g EPA and 1.Og DHA per day) or safflower oil capsules (2g per day). The average duration of capsule consumption prior to surgery was just over 12 days. Although the proportion of EPA present in the mucosal fatty acids from biopsies taken at surgery was significantly higher in the fish oil versus placebo group, no significant changes in rectal proliferation were shown during the period of supplementation. Cheng et al. (2003; Cancer Lett. 193, 17-24) carried out a two year investigation into the effects of increased omega-3 fatty acid intake on colonic cell proliferation and apoptosis. The study group consisted of patients polypectomised for polyps/tumours of the colorectum. The experimental group decreased their intake of total fat, decreased their intake of omega-6 polyunsaturated fatty acids, and increased their intake of omega-3 polyunsaturated fatty acids for two years. To facilitate the increased intake of omega-3 polyunsaturated fatty acids the experimental group took eight capsules of fish oil (equivalent to 100 mg per day of EPA and 400 mg per day of DHA). The control group simply reduced its fat intake. These researchers found that after two years (but not after 12 months) there was an increase in apoptosis of cells of the sigmoid colon mucosa in the experimental group. However, the study showed no significant difference in cell proliferation between the experimental and comparison groups after 12 or 24 months.

Thus, while the clinical studies conducted to date support the view that fish oils have a protective role against cancer, the measured effects on crypt cell proliferation and apoptosis have been inconsistent.

Further studies have also shown a beneficial effect for other clinical indications. Belluzzi et al (1996 N. Eng. J. Med. 334, 1557-60) tested a marine lipid concentrate in free fatty acid form which comprised 40 per cent EPA, 20 per cent docosahexaenoic acid and 40 per cent omega-6, omega-7 and omega-9 fatty acids on patients with Crohn's disease. A significant decrease in markers of inflammation was found in the fish-oil group compared with the placebo group by the end of the study. However, side-effects were produced in some patients.

One of the problems with fish oil is that it can also contain high levels of toxins such as polychlorinated biphenyls and dioxins. Long-term use of such capsules can thus result in elevated levels of these toxins in the body. Therefore, despite the health benefits associated with the consumption of fish oils, recent advice is not to consume too much so as to avoid ingesting too much of these toxins. According to a first aspect of the present invention, the use of an omega-3 fatty acid composition, containing eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of omega-3 fatty acids present in said omega-3 composition, in the manufacture of a medicament for the treatment or prophylaxis of cancer is provided.

According to a second aspect of the present invention, a method for the treatment or prophylaxis of cancer is provided, comprising administering to a human or animal patient an effective amount of an omega-3 fatty acid composition containing eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of omega-3 fatty acids present in said omega-3 composition.

According to a third aspect of the present invention, a pharmaceutical composition is provided comprising an omega-3 fatty acid composition containing eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of omega-3 fatty acids present in said omega-3 composition.

The term "pharmaceutical composition" as used herein refers to a composition comprising an active ingredient, in the case of the present invention an omega-3 composition as defined above, and a vehicle for delivery of said active. The vehicle comprises one or more excipients and/or carriers. The pharmaceutical composition may be used for therapeutic purposes and/or as a dietary composition. In a particularly preferred embodiment of the present invention, the pharmaceutical composition is for use in the treatment or prophylaxis of cancer.

Fish oils in their natural state contain an EPA:DHA ratio (w/w) significantly lower than that used in the present invention. Methods of preparing high purity EPA in free acid form have been disclosed in the prior art, for example as taught in EP-A-0347509 (100% pure EPA), US 2004/0236128 (100% pure EPA), US 5840944 (at least 99% pure, although only purification of the ethyl ester is exemplified), and Wigmore et al., Nutr. Cancer 2000; 36(2): 177-84 (95% pure). However, high purity EPA is difficult and expensive to produce, as only a relatively small amount of high purity EPA can be obtained from a large amount of lower purity EPA.

Previous studies by Lawson et al. (Biochem Biophys Res Commun 1998;152:328-35 and 1988;156:960-3) have shown the free acid form of EPA to be better absorbed from the intestine. When consumed with a low fat meal (8g total fat), the free fatty acid form of EPA was completely absorbed, as compared with two-thirds of the EPA in triacylglycerol form and one-fifth of EPA in ethyl ester form. The absorption of EPA as triacylglycerols and ethyl esters increased to 90 and 65 per cent respectively, when given with a high fat meal (44g total fat)

The inventors have now discovered that an omega-3 composition containing EPA in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of omega-3 fatty acids present in said composition provides significant and surprising benefits in terms of efficacy in the treatment and prophylaxis of cancer, as compared to the omega-3 compositions used in the prior art containing lower EPA:DHA ratios.

The term "cancer" as used herein refers to both benign and malignant growths/tumours. For example, the omega-3 compositions identified above can be used for the treatment or prophylaxis of colorectal cancer, Barrett's oesophagus, breast cancer, pancreatic cancer, and familial adenomatuous polyposis.

In a preferred embodiment, the omega-3 composition contains eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of fatty acids present in said composition. More preferably, the omega-3 composition contains eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the composition. In such compositions the levels of any unwanted contaminants, such as omega-6, omega-7 and omega-9 fatty acids, monounsaturated and saturated fats, toxins such as polychlorinated biphenyls and dioxins, and any other impurities are minimised. The omega-3 composition can be obtained from fish oil as a fish oil isolate. Alternatively, other sources of EPA could be used. For example, EPA can also be obtained from other marine animals. In addition, other sources of EPA may, in the future, become available.

hi a preferred embodiment, the eicosapentaenoic acid is present in an amount of 90 or more per cent (w/w), more preferably in an amount of 95 or more per cent (w/w), most preferably in an amount of about 99 per cent (w/w), including 100 per cent (w/w).

The omega-3 composition can be present in the medicament / pharmaceutical composition / administered to the patient as the sole active ingredient.

The omega-3 composition is advantageously formulated as part of a composition for oral administration, and such a composition may combine the omega-3 composition with suitable excipients and/or carriers to provide a delayed release formulation allowing the site of release in the gastrointestinal tract of the composition to be controlled. For example the composition for oral administration may take the form of a capsule containing the omega-3 composition. The capsule may have a coating comprising hydroxypropylmethylcellulose phthalate. This preferably allows release of the EPA at a pH from 4.0 to 5.5, more preferably at a pH of from 5.0 to 5.2 and most preferably at a pH of about 5.0. This prevents release of the EPA in the stomach.

Preferably the patient is provided with about 0.5 to about 3 grams per day, more preferably about 2 grams per day of eicosapentaenoic acid. The EPA can conveniently be provided in unit dosages of from about 500mg to about 2g.

According to a fourth aspect of the present invention, the use of an omega-3 fatty acid composition, containing eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of omega-3 fatty acids present in said composition, in the manufacture of a medicament for the treatment or prophylaxis of a gastrointestinal, psychiatric or neurological disorder is provided. According to a fifth aspect of the present invention, a method for the treatment or prophylaxis of a gastrointestinal, psychiatric or neurological disorder is provided, comprising administering to a human or animal patient an effective amount of an omega-3 fatty acid composition containing eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of omega-3 fatty acids present in said composition.

According to a further embodiment of the third aspect of the present invention, a pharmaceutical composition for use in the treatment or prophylaxis of a gastrointestinal, psychiatric or neurological disorder is provided, comprising an omega-3 fatty acid composition containing eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of omega-3 fatty acids present in said omega-3 composition.

According to a sixth aspect of the present invention, the use of an omega-3 fatty acid composition, containing eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of omega-3 fatty acids present in said composition, to increase cell apoptosis and/or decrease cell proliferation is provided.

The inventors have discovered that the omega-3 compositions used according to the fourth through sixth aspects of the present invention are likewise of surprising benefit in treatment of the conditions indicated.

Preferred embodiments of the present invention are described below, by way of example only and with reference to the accompanying figures, in which:

Figure 1 illustrates the change in human fat consumption over time;

Figure 2 shows Ki67 mean total labelling index before and after treatment (cell proliferation assay); Figure 3 shows crypt total labelling indices after EPA treatment in individual patients;

Figure 4 shows crypt total labelling indices in individual patients of the control group; Figure 5 shows the labelling index in the upper 40 per cent of the crypt after EPA treatment in individual patients;

Figure 6 shows the labelling index in the upper 40 per cent of the crypt in individual patients of the control group; Figure 7 shows the frequency of Ki-67 labelling at each crypt cell position before and after

EPA therapy;

Figure 8 shows the change in labelling indices after EPA therapy;

Figure 9 shows the frequency of Ki-67 labelling at each crypt cell position before and after no treatment; Figure 10 shows the change in labelling after non treatment;

Figure 11 shows apoptotic cells per biopsy;

Figure 12 shows the data shown in Figure 11 on a different scale;

Figure 13 shows the percentage contribution of EPA to total mucosal fatty acids in individual patients given EPA capsules; Figure 14 shows the percentage contribution of EPA to mucosal total fatty acids in individual patients of the non-treatment group; and

Figure 15 shows the mean percentage contribution of EPA to mucosal total fatty acids before and after treatment.

The aim of this study was to investigate the effect of 99 per cent pure EPA in a free fatty acid form on cell proliferation, apoptosis and polyamine metabolism in normal-appearing colonic mucosa from patients with a history of colorectal polyps.

99 per cent pure EPA in a free fatty acid form was produced using supercritical fluid chromatography. 150 kg of unpure EPA can be converted into 50 kg of 99 per cent pure

EPA using this method.

In order to isolate EPA from the triglyceride it is necessary to free the fatty acids by hydrolysis or ester exchange in order that purification can be effected. Purification can be achieved by techniques such as fractional distillation, molecular distillation and chromatography. A particularly desirable chromatographic method employs super-critical fluids using, for example, carbon dioxide as the mobile phase, such as described in European Patent EP 0 712 651 B1, entitled "Process for recovering a substance or group of substances from a mixture". It has been found that using such techniques EPA may be purified to levels approaching 100 per cent. For practical reasons the EPA may be purified as its ethyl or methyl ester and hydrolysed back to the free fatty acid form. Purification enables a product to be prepared which is highly concentrated and free from other fatty acids that are less desirable in the finished product, hi addition other chemicals entities such as mono- and di-glycerides, hydrocarbons, pesticide residues and the like can be removed. The highly purified EPA is thus suitable for human ingestion as it contains substantially reduced levels of toxins, compounds contributing to unpalatability or undesirable fatty acids such as saturated fatty acids. The free fatty acid form of EPA can be absorbed in any part of the gut easily without need of enzymes.

EXAMPLE

The effect of 2g per day of 99 per cent pure EPA in a free fatty acid form for three months on colonic crypt cell proliferation, apoptosis and polyamine metabolism in normal- appearing colonic mucosa from patients with a history of colorectal polyps was investigated.

Thirty patients with a history of one or more colorectal polyps were recruited for the study, of which 28 completed the study. Fourteen patients were randomised to the EPA group, and 14 patients to the non- treatment group. Patients in the EPA group took four 500 mg capsules of 99 per cent pure EPA in a free fatty acid form per day. Patients usually took two capsules at breakfast, and two with their evening meal. The capsules were coated with a coating that allows release of the contents of the capsule at a pH of 5.5.

Patients had colonic biopsies taken at the initial colonoscopy, and then biopsies were repeated after an interval of approximately three months. Slides were coded to blind them, and scored for proliferation (MIB-I to Ki-67 antigen) and apoptosis (M30 antibody cytodeath). For the cell proliferation study, the labelling status at each cell position in the crypt was recorded. For the apoptosis study the cells were graded on a scale of 0 to 3 (0=no positive cells; l=single positive cells; 2=small groups of positive cells; 3=large areas of adjacent positive cells). In addition, biopsies were taken for measurement of ornithine decarboxylase and mucosal fatty acid content.

Figure 2 shows the results of the crypt cell proliferation assay. Crypt cell proliferation in the EPA treated group was significantly reduced from a mean total labelling index of 28.3 (SD 7.85) to 22.4 (SD 5.5), p=0.034 (Paired T test) (see Figure 3) compared to the non- treatment group (Figure 4). The number of labelled cells in the EPA treated group decreased significantly in the upper 40 per cent of the crypt from a mean of 7.39 (SD 5.48) to 3.63 (SD 3.04), p=0.031 (see Figure 5). Crypt cell proliferation in the non-treatment group showed a non-significant increase from a mean of 26.1 (SD 6.73) to 29.72 (SD

8.05), p=0.186 (see Figure 6). Proliferation index distribution curves showed a significant downwards shift in the combined frequency of labelling at each crypt cell position after EPA treatment (p<0.0001) (see Figures 7 and 8), whereas the frequency of labelling increased significantly in the non-treatment group (p<0.0001) (see Figures 9 and 10).

Figure 11 shows the results of the apoptosis assay. Apoptotic cells were almost exclusively located in the luminal epithelium between the crypts. Apoptosis in the EPA group increased significantly from a mean of 0.14 (SD 0.28) apoptotic cells per biopsy to 12.47 (SD 23.30), p=0.001 (Wilcoxon signed rank test). The apoptosis grade after EPA increased significantly from a mean of 0.36 (SD 0.5, range 0-1) to a mean of 1.50 (SD

1.02, range 0-3), p=0.003. Three of the patients had such a large increase in apoptosis that their results are not shown in Figure 11. Figure 12 presents the same "after treatment" data as shown in Figure 11, but with a different scale so that the outlying results can be also included. Apoptosis in the non-treatment group showed a small, non-significant increase from a mean of 0.68 (SD 0.94) apoptotic cells per biopsy to a mean of 1.62 (SD 1.85), p=0.213. Neither was a significant change in the grading of apoptotic cells in the non- treatment group.

The percentage of EPA in colonic mucosal total fatty acids increased significantly from 1.05% (SD 0.69) to 2.73% (SD 1.66), p=0.008 in the EPA treated group. No significant change was seen in the non-treatment group (see Figures 13, 14 and 15). These results show that crypt cell proliferation was significantly reduced, and apoptosis significantly increased, in the colonic mucosa of patients treated with 2 g per day of 99 per cent pure EPA in a free fatty acid form for three months. No such significant changes were seen in the non- treatment group.

The present inventors have thus shown that dietary supplementation with high purity EPA leads to significantly increased levels of this fatty acid in colonic mucosa, a significant reduction in crypt cell proliferation and a significant increase in apoptosis. It is therefore to be expected that dietary supplementation with high purity EPA in a free fatty acid form will be of significant utility, in particular for preventing polyps and in the chemoprevention of colorectal cancer.

Moreover, the combined levels of increase in apoptosis and reduction in cell proliferation are significantly and surprisingly higher than would have been expected from the prior art studies conducted using fish oils and EPAfDHA mixtures.

The use of high purity EPA also allows the amount of unwanted contaminants (such as omega-6 and omega-9 fatty acids), which could have pro-inflammatory properties or other undesirable side effects, to be minimised.

Although 99 per cent pure EPA was used in the Example, similar surprising therapeutic effects are to be expected for any omega-3 composition used according to the present invention.

Moreover, high purity EPA in a free fatty acid form will not only be useful in chemoprevention of colorectal cancer as demonstrated in the Example. The present inventors also envisage its use in therapy of other cancers, other gastrointestinal disorders, (such as inflammatory bowel disease), and in neurological or psychiatric disorders (such as autism, attention-deficit hyperactivity syndrome, memory loss, Parkinson's disease and Alzheimer's disease, encephalitis, multiple sclerosis, pain, substance abuse and drug addiction, schizophrenia and depression).

Claims

1. The use of an omega-3 fatty acid composition, containing eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of omega- 3 fatty acids present in said composition, in the manufacture of a medicament for the treatment or prophylaxis of cancer.

2. A use according to claim 1, wherein the composition contains eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of fatty acids present in said composition.

3. A use according to claim 1, wherein the composition contains eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of said composition.

4. A use according to any preceding claim, wherein the composition is a fish oil isolate.

5. A use according to any preceding claim, wherein the composition is used as the sole active ingredient.

6. A use according to any preceding claim, wherein the eicosapentaenoic acid is present in an amount of 90 or more per cent (w/w).

7. A use according to any preceding claim, wherein the eicosapentaenoic acid is present in an amount of 95 or more per cent (w/w).

8. A use according to any preceding claim, wherein the eicosapentaenoic acid is present in an amount of about 99 per cent (w/w).

9. A use according to any preceding claim, wherein the medicament is for oral administration.

10. A use according to claim 9, wherein the medicament is a delayed release formulation.

11. A use according to any preceding claim, wherein the composition contains from about 500mg to about 2g of eicosapentaenoic acid in the free acid form.

12. A method for the treatment or prophylaxis of cancer, comprising administering to a human or animal patient an effective amount of an omega-3 fatty acid composition containing eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of omega-3 fatty acids present in said composition.

13. A method according to claim 12, wherein the composition contains eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of fatty acids present in said composition.

14. A method according to claim 12, wherein the composition contains eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of said composition.

15. A method according to any one of claims 12 to 14, wherein the composition is a fish oil isolate.

16. A method according to any one of claims 12 to 15, wherein the composition is administered as the sole active ingredient.

17. A method according to any one of claims 12 to 16, wherein the eicosapentaenoic acid is present in an amount of 90 or more per cent (w/w).

18. A method according to any one of claims 12 to 17, wherein the eicosapentaenoic acid is present in an amount of 95 or more per cent (w/w).

19. A method according to any one of claims 12 to 18, wherein the eicosapentaenoic acid is present in an amount of about 99 per cent (w/w).

20. A method according to any one of claims 12 to 19, wherein the composition is orally administered.

21. A method according to claim 20, wherein the composition is administered as part of a delayed release formulation.

22. A method according to any one of claims 12 to 21, wherein about 0.5 to 3g of eicosapentaenoic acid in the free acid form is administered per day.

23. A pharmaceutical composition comprising an omega-3 fatty acid composition containing eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of omega-3 fatty acids present in said omega-3 composition.

24. A pharmaceutical composition according to claim 23, wherein the pharmaceutical composition is for use in the treatment or prophylaxis of cancer.

25. A pharmaceutical composition according to claim 23 or 24, wherein the omega-3 composition contains eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of the total amount of fatty acids present in said omega-3 composition.

26. A pharmaceutical composition according to claim 23 or 24, wherein the omega-3 composition contains eicosapentaenoic acid in the free acid form in an amount of 80 or more per cent (w/w) of said omega-3 composition.

27. A pharmaceutical composition according to any one of claims 23 to 26, wherein the omega-3 composition is a fish oil isolate.

28. A pharmaceutical composition according to any one of claims 23 to 27, wherein the omega-3 composition is the sole active ingredient.

29. A pharmaceutical composition according to any one of claims 23 to 28, wherein the eicosapentaenoic acid is present in an amount of 90 or more per cent (w/w).

30. A pharmaceutical composition according to any one of claims 23 to 29, wherein the eicosapentaenoic acid is present in an amount of 95 or more per cent (w/w).

31. A pharmaceutical composition according to any one of claims 23 to 30, wherein the eicosapentaenoic acid is present in an amount of about 99 per cent (w/w).

32. A pharmaceutical composition according to any one of claims 23 to 31, wherein the pharmaceutical composition is for oral administration.

33. A pharmaceutical composition according to claim 32, wherein the pharmaceutical composition is a delayed release formulation.

34. A pharmaceutical composition according to any one of claims 23 to 33, wherein the pharmaceutical composition contains a unit dosage of about 500mg to about 2g of eicosapentaenoic acid in the free acid form.