2. Description of the Relevant Art Arachidonic acid (AA) and oxygen-containing derivatives of AA, termed eicosanoids. play pivotal roles in controlling key cellular events that lead to acute and chronic inflammation. While it has been suggested for more than 50 years that dietary fat intake influences tumor development, only more recently has there been mechanistic evidence to link arachidonic acid metabolism with cell proliferation and apoptosis.

One area of research has been directe to understanding the association of total dietary fat or specific types of fat with cancer risk (Rose, In Diet and Breast Cancer. J Weiss, ed. Plenum Press, NY, 1994; Willett et al., JAMA 268: 2037,1992; Howe et al., J.

Natl. Cafzc. Inst. 83: 336.1991; Hunter et al., N. Engl. J. Med. 334: 356,1996). For example, countries such as Japan have historically had low rates of breast, colon and lung cancers. These rates are changing, however, and several investigators have suggested that this change is due in some part to a decrease in consumption of n-3 fatty acids as Japanese populations adapt Western diets, thus decreasing the n-3/n-6 fatty acid ratio (Akazaki and Stemmerman, J. Natl. Croc. Inst. 50: 1137,1973; Tominaga and Kuroishi, Cancer Lett.

90: 75,1995; Hirayama, Prev. Med. 7: 173,1978; Kato et al., Jpn J. Canc. Res. 78: 349, <BR> <BR> <BR> <BR> 1987; Landsetal., A) n J. Cli) ?. Nutr. 51: 991,1990; Goto, Nutr. Rev. 50: 398,1992; Shoda et<BR> <BR> <BR> <BR> <BR> <BR> <BR> al., Am J. Clirz. Nastr. 63: 741,1996; Kamano et al., Anticancer Res. 9: 1903,1989). This hypothesis is supported by several animal studies which reveal that n-3 fatty acids suppress both carcinogenesis and tumor progression (Telang et al., Anticancer Res. 8: 971,1988; Hillyard and Abraham, Cancer Res. 39: 4430,1979; Rose et al., J. Natl. Canc. Inst.

83: 1491, 1991; Rose, et al., Cancer Res. 53: 4686,1993; Rose et al, Cancer Res. 54: 6557, 1994; Rose and Connolly, J. Natl. Canc. Inst. 85: 1743,1993; Rose et al., J. Natl. Canc.

Inst. 87: 587,1995). Other in vivo studies suggest that high levels of n-6 fatty acids alone stimulate the development, growth and metastasis of several cancers (Welsch, Am. J. Clin.

Vtatr. 45: 192.1987; Rose, Am. J. Clin. Nutr. 66: 1513S, 1997). However, it is important to note that other studies have shown this pattern (n-6 fatty acids and n-3 fatty acids as pro- and anti-proliferative agents, respectively) is not observed in all animal models and n-6 fatty acids have, in fact, been shown to be protective in some in vivo models of cancer (Ramesh and Das, Cancer Lett. 123: 207,1998).

The in vitro influence of fatty acids (provided exogenously to the cell culture media) on cell proliferation and apoptosis has been examined in several cell types. Finstad and colleagues demonstrated that AA (n-6) and eicosapentaenoic acid (EPA, n-3) are potent inhibitors of proliferation in HL-60 cells and this inhibition is independent of further metabolism to eicosanoids (Finstad et al., Blood 84; 3799,1994). They further demonstrated that EPA and AA reduced the proliferation rate of HL-60 cells primarily by promoting apoptosis and cell differentiation. A survey of several papers reveal that docosahexaenoic acid, EPA and AA are inhibitors of cell proliferation in numerous cell types (Ramesh and Das, 1988; Finstad et al., 1994; Chow et al., Lipids 24: 700,1989; Soyland et al., Water. J. Clin. Invest. 23: 112,1993; Tessier et al., Biochem. Biophys. Res.

Comm. 207: 1015,1995).

When 20 carbon polyunsaturated fatty acids (PUFAs) such as AA enter a cell or are mobilized from intracellular phospholipids by phospholipases, there are selective pathways that traffic PUFAs back into phospholipids, thereby maintaining extremely low intracellular concentrations of AA. There are as many as 20 different arachidonate-containing phospholipid molecular species in any given inflammation or neoplastic cell. AA moves through these different molecular species in a linear fashion requiring several enzyme activities (Chilton et al., Biochim BiopAvs Acta 1299: 1,1996). Levels of free (unesterified) AA are tightly controlled within mammalian cells utilizing these remodeling pathways.

This is evidenced by the fact that measurable quantities of AA cannot be detected in most resting mammalian cells. Recent studies in the inventor's laboratory reveal that blocking

arachidonate-phospholipid remodeling leads to an accumulation of AA within neoplastic cells and a blockage of proliferation as well as an induction of apoptosis (Surette et al., Biochemist7n 35: 9187,1996; Winkler et al., J. Pharm. Exp. Ther. 279: 956,1996).

Several lines of evidence also suggest that certain phospholipase A, s (e. g. cytosolic PLA, [cPLA2]), which mobilize intracellular AA can play pivotal roles in regulating cell proliferation and inducing apoptosis. First, anti-FAS and TNF, both potent inducers of apoptosis, also induce cPLA2 activation and AA release in breast carcinoma cells.

Moreover, overexpression of bcl-2 in these cells blocks AA mobilization and renders cells resistant to apoptosis (Jaattela et al., Oncogene 10: 2297,1995). Secondly, TNF-resistant sublines of L929 express reduced levels of cPLA, and these cells regain their sensitive phenotype to TNF after expression of transfected cPLA2 (Hayakawa et al., J. Biol. Chem.

268: 11290,1993). Thirdly, there is a correlation between the activity of cPLA2 and susceptibility to TNF-induced apoptosis in melanocyte cell lines and melanoma tumor tissue (Voelkel-Johnson et al., J. Immtlnol. 156: 201,1996). Fourthly, cPLA2 is required for apoptosis in TNF-induced apoptosis, whereas it is dispensable for FAS-mediated apoptosis in L929 cells (Korystov et al., FEBS Lett. 388: 238,1996). Fifthly, cPLA, is a necessary component in the pathways leading to ceramide accumulation and associated apoptosis in L929 cells (Jayadev et al., J. Biol. Chem. 269: 5757,1994; Jayadev et al., J. Bio. Chem.

272: 17196,1997). Sixthly, disruption of cPLA, activation by 1,25-dihydroxyvitamin D3 protects human leukemia cells from TNF-induced apoptosis (Wu et al., Cancer Res.

58: 633,1998). Collectively, these studies are consistent with the idea that altering intracellular levels of certain PUFAs, in this case via PLA, activation, influences cell proliferation and apoptosis.

Once AA is mobilized by PLA, isotype (s), it can be metabolized by several enzymatic pathways including cyclooxygenase, lipoxygenase and p450. A large body of literature has accumulated connecting cell proliferation and apoptosis to the enzyme responsible for prostaglandin and thromboxane production, cyclooxygenase. A consistent finding in this area is that non-steroidal anti-inflammatory drugs ("NSAID") are effective in reducing colon tumors in both humans and rodents (Lupulescu Prostaglandins Leukotr.

Essen. Fatty Acids 54: 83,1996; Reddy et al., Cancer Res. 47: 5340,1987; Marnett, Cancer

Res. 52: 5575.1992; Rao et al., Cancer Res. 55: 1464,1995; Boolbol et al., Cancer Res.

56: 2556,1996; McCormick and Moon, Br. J. Cancer 48: 859,1983; McCormick et al., Cancer Res. 45: 1803,1985; Perkins and Shklar, Oral Surgery, Oral Medicine, Oral Pathologa53: 170,1982 ; Cornwall et al., J. Oral Maxillofacial Surg. 41: 795,1983).

NSAID have also consistently triggered apoptosis of cultured cells (Lu et al., Proc. Natl.

Acad. Sci., USA 92: 7961,1995; Piazza et al., Cancer Res. 55: 3110,1995; Shiff et al., J.

Clin. Illvest. 96: 491,1995). Since NSAID have long been recognized to block cyclooxygenase, most investigators have assumed that the anti-neoplastic effects of NSAID are due to decreased eicosanoid production. However, Chan and colleagues have recently suggested an alternative mechanism to account for NSAID effects in cancer. They found that NSAID treatment of colon tumor cells results in a dramatic increase in intracellular AA levels that, in turn, stimulates the conversion of sphingomyelin to ceramide, suggesting that an increase in free intracellular levels of AA (and not the decrease in prostaglandin production) mediates apoptosis after NSAID treatment of colon cancer cells.

Lipoxygenases may also play an important role in regulating cell proliferation and apoptosis. For example, 5-Lipoxygenase inhibitors induce apoptosis in human leukemia blast cells (Anderson et al., Scanning Microscopy 8: 675,1994; Anderson et al., Leuk. Res.

19: 789,1995; Anderson et al., Proc. Am. Assoc. Cancer Res. 36: A26,1995; Anderson et al., Anticaslcer Res. 16: 2589,1996). In addition, down-regulation of 12-lipoxygenase triggers apoptosis and this apoptosis can be partially blocked by 12 (S)-HETE [12 (S)- hydroxy-(SZ, 8Z. lOE, 14Z)-eicosatetraenoic acid](SZ, 8Z. lOE, 14Z)-eicosatetraenoic acid] and 15 (S)-HETE [15 (S)-hydroxy- (SZ, 8Z, llZ, 13E)-eicosatetraenoic acid], but not 5 (S)-HETE [5 (S)-hydroxy- (6E, 8Z, 1 lZ, 14Z)-eicosatetraenoic acid] (Tang et al., Proc. Natl. Acad. Sci. USA 93: 5241, 1996).

As mentioned above, dietary fatty acids have long been associated with the risk of developing cancer. However, the mechanism (s) that link changes in intracellular levels of PUFAs such as AA to cellular and biochemical events such as apoptosis and mitogenesis has not been clearly elucidated. Jayader and colleagues showed that AA stimulates sphingomyelin hydrolysis in HL-60 cells; ceramide has been shown to be a pivotal mediator of apoptosis in several normal and neoplastic cells (Jayadev et al., 1994).

Recently, Chen and colleagues have examined apoptosis produced by AA in cells overexpressing cytochrome p450, CYP2E41, and found that the capacity of AA to induce apoptosis is dependent on the expression of this p450 isozyme (Chen et al., J. Biol. Chem. <BR> <BR> <BR> <BR> <P>272: 14532,1997). These authors propose that the elevated production of reactive oxygen species by cells with this p450 causes lipid peroxidation and catabolism of polyunsaturated fatty acids such as AA, leading to the formation of toxic products such as malonodialdehyde. The authors proposed that these metabolic breakdown products were responsible for ethanol-induced hepatotoxicity. An alternative explanation of the increased cell death seen with enhanced p450 activity is that specific PUFA oxidation products, which have not been completely metabolized to malonodialdehyde, may specifically induce selective toxicity to cancer cells. The present disclosure includes such products.

SUMMARY OF THE INVENTION The present disclosure addresses the needs presented above by providing compositions including oxidized products of 20 and 22 carbon polyunsaturated fatty acids that are shown herein to have potent anti-proliferative activity when applied exogenously to tumor cells. These compositions and methods are based in part on the discovery that certain 20 to 22 carbon polyunsaturated fatty acids that have been oxidized have more potent anti- proliferative properties than the parent fatty acids. This observation led to the discovery that certain compositions that include oxidized products of these fatty acids have strong anti-proliferative activity. As an aspect of the present disclosure, isolated, or partially purified, oxidation products of 20 and 22 carbon polyunsaturated fatty acids are described and shown to exhibit anti-proliferative activity.

The present disclosure includes, therefore, compositions including oxidized 20 or 22 carbon polyunsaturated fatty acids, and compositions including active fractions of oxidized 20 or 22 carbon polyunsaturated fatty acids, wherein the compositions have anti- proliferative activity. Described herein are oxidized compounds derived from the oxidation of a substantially pure or homogenous single 20 or 22 carbon polyunsaturated fatty acid, as well as compositions that contain oxidation products of a mixture of two or more polyunsaturated fatty acids. For example, in order to obtain the anti-proliferative compositions described herein, one may begin with a composition containing substantially

a single polyunsaturated fatty acid and promote oxidation using a variety of methods to obtain oxidation products of that polyunsaturated fatty acid. Alternatively, one may begin with a plurality of polyunsaturated fatty acids and oxidize them separately or mix them together and then oxidize the mixture. Alternatively, one may oxidize one or more 20 or 22 carbon polyunsaturated fatty acids separately and fractionate the products of those oxidation reactions to obtain the active anti-proliferative fractions from each oxidized composition.

The individual active fractions may then be mixed to obtain an anti-proliferative composition.

Polyunsaturated fatty acids that are demonstrated herein to be useful in making the described compositions include arachidonic acid and docosahexaenoic acid. Based on the present disclosure, it is contemplated that other fatty acids, when oxidized, will yield equivalent anti-proliferative compositions, and that such compositions are included within the spirit and scope of the present invention.

The compositions described herein may be described in certain embodiments as oxidation products of, or products obtained by oxidation of one or more 20 or 22 carbon polyunsaturated fatty acids. Such products may be obtained by non-enzymatic means, for example, by exposure of one or more polyunsaturated fatty acids to molecular oxygen contained in ambient air, to partially purified molecular oxygen, to active oxygen species such as H, O,, or even to other oxidizing gases or compounds. The oxidation may also include superoxide species, free radicals, ozone, enzymatic oxidation or catalytic oxidation, for example. In certain embodiments, the oxidation products having anti-proliferative activity described herein may be produced by exposure of one or more 20 or 22 carbon polyunsaturated fatty acids to a stream of air at room temperature, or to peroxides in the absence or presence of transition metals at 37°C, or to superoxide using a xanthine/xanthine oxidase system for a time effective to at least partially oxidize the polyunsaturated fatty acids. Transition metals are known in the art to include elements from groups IB through VIIIB of the periodic table of the elements. A preferred transitional metal for use in oxidation of fatty acids as disclosed herein is a copper ion.

The compositions disclosed herein may also include a fraction partially purified from the total oxidation products. As is standard in the art, one may identify the fraction or fractions having the highest activity, in this case anti-proliferative activity or cytotoxicity, and isolate those fractions from the total oxidation products. Such isolated fractions may include all fractions that have any detectable activity, or they may include only the fractions with higher than average activity, or only those with the highest activity. Anti-proliferative activity or cytotoxicity, as used herein, describes the ability of certain compounds to inhibit the growth and/or development of tumor cells, or to cause tumor cell death. This anti- proliferative activity includes, but is not limited to, interrupting the cell cycle, inducing cell death by programmed cell death (apoptosis) or other mechanisms, attenuating, arresting, inhibiting, decreasing, stopping, or retarding the growth of a tumor cell, or a tumor mass, either in vivo or in vitro. Anti-proliferative activity may be measured by observing an arrest in cellular development or mitochondrial activity as described herein and as known in the art, or by any other means known in the art including measurement of tumor growth over time in an animal model.

A partially purified fraction of the oxidation products described herein may be obtained by reverse phase high pressure liquid chromatography (HPLC), for example, and especially reverse phase HPLC including a mobile phase of 100% methanol. The reverse phase HPLC may also include a C, 8 column. In certain embodiments a fraction may be partially purified by normal phase ion exchange chromatography, especially including a mobile phase of 50% ethyl ether and 50% hexane. In certain embodiments, the normal phase ion exchange chromatography will include a silica gel. It is also understood in the art of purification or separation of such compounds that sequential steps are used to obtain fractions with the highest activity. Therefore, any sequence of steps of purification known in the art may be used in any order as long as a fraction having anti-proliferative activity is obtained. Other purification techniques may include, but are not limited to, thin layer chromatography, gel filtration, electrophoresis, or capillary electrophoresis, for example.

Described herein are methods of making an anti-proliferative composition that includes exposing one or more 20 or 22 carbon polyunsaturated fatty acids to conditions effective to oxidize the 20 or 22 carbon polyunsaturated fatty acids to obtain oxidation

products and isolating a fraction of the oxidation products having anti-proliferative activity.

In certain embodiments the methods include oxidation of arachidonic acid, docosahexaenoic acid, or a mixture of the two. The method may include any means or mechanism of oxidizing the polyunsaturated fatty acids as described above, and in certain embodiments would include exposing a 20 or 22 carbon polyunsaturated fatty acid to a stream of air for a period of at least about 24 hours.

Disclosed herein are also methods of inhibiting growth of a tumor cell or a tumor, the methods including contacting the cell, cells or tumor with a composition including oxidation products from an oxidation of a 20 or 22 carbon polyunsaturated fatty acid, or a mixture of 20 or 22 carbon polyunsaturated fatty acids. The cells or tumor may be in an animal or human subject, and the method may include administering the composition to the subject. The tumor cells or tumor may be of any origin, including breast, brain, colon, lymph nodes, lung, bone, liver, skin, or from the hematopoetic system. Administration to a subject, according to the present method would include, but is not limited to oral, nasal, topical, rectal, vaginal, intra-arterial, intravenous, intramuscular, subcutaneous, or intramedullary administration.

Any of the compositions described herein as part of the present disclosure may be formulated as pharmaceutical compositions including an oxidation product of one or more oxidized 20 or 22 carbon polyunsaturated fatty acids, wherein the compositions have anti- proliferative activity. Such pharmaceutical compositions may be in the form of a topical cream or ointment, a liquid for injection, or an oral, nasal or suppository preparation.

Pharmaceutical or pharmacological compositions are known in the art to be suitable for administration to a human or animal subject and are preferably sterile, hypoallergenic and hypopyrogenic.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In certain instances the term"a"may mean one, or one or more.

Although any methods and materials similar or equivalent to those described herein can be

used in the practice or testing of the present invention, the preferred methods and materials are now described.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 depicts the effect of oleic acid, oxidized oleic acid, arachidonic acid, and oxidized arachidonic acid on proliferation of MDA-231 breast cancer cells.

FIG. 2 depicts the cell cycle inhibition of MDA-231 cells due to the presence of oleic acid, arachidonic acid, docosahexaenoic acid, and their oxidized forms.

FIG. 3 depicts the inhibition of MCF-7 breast cancer cell growth by arachidonic acid and oxidized arachidonic acid.

FIG. 4 depicts inhibition of cell cycle progression in MCF-7 breast cancer cells due to the presence of arachidonic acid and oxidized arachidonic acid.

FIG. 5 depicts inhibition of cell cycle progression of MDA breast cancer cells by various oxidized fatty acids.

FIG. 6 depicts fractionation of oxidized metabolites of arachidonic acid and the inhibition of cell cycle progression by the various fractions.

FIG. 7A depicts the results of fractionation of arachidonic acid (AA) and oxidized arachidonic acid (oxAA) by normal phase TLC in 90: 60: 6 hexane: ethyl ether: 90% formic acid. The ability of tumor cells to proliferate is shown under the graph for each fraction of the oxAA. The greatest antiproliferative activity is fraction 1 with a value of 2 relative to control (100).

FIG. 7B depicts the results of fractionation of arachidonic acid (AA) and oxidized arachidonic acid (oxAA) by reverse phase TLC in 65% methanol in water. The ability of tumor cells to proliferate is shown under the graph for each fraction of the oxAA. The greatest antiproliferative activity is in fraction 6 with a value of 40 relative to control (100).

FIG. 8 depicts the results of the primary C, 8 reverse phase HPLC of the TLC neutral origin fraction (fraction 1). The HPLC conditions are described herein below. The ability of tumor cells to proliferate is shown below each fraction. The compositions of fractions 4 and 6 prevented proliferation (numerical value for fractions 4 and 6 is zero relative to control <BR> <BR> <BR> <BR> (100)).<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <P>FIG. 9 depicts the results of secondary C, 8 reverse phase HPLC of fraction 4 of the primary HPLC shown in FIG. 8. The HPLC solvents are described herein below. Fraction 11 exhibits the highest antiproliferative activity, a value of 56 relative to control (100).

FIG. 10 depicts the results of secondary C, 8 reverse phase HPLC of fraction 6 of the primary HPLC shown in FIG. 8. The HPLC solvents are described herein below. Fractions 7,8 and 9 exhibit the highest antiproliferative activity, with values of 63,33 and 14, respectively, relative to control (100).

DETAILED DESCRIPTION An aspect of the present disclosure is that oxidation products of arachidonic acid, docosahexaenoic acid, and other 20 and 22 carbon polyunsaturated fatty acids can be exogenously supplied to cells to achieve an anti-proliferative effect. For example, certain newly synthesized 20 and 22 carbon polyunsaturated fatty acids, when exposed to a stream of air for 24 hours, strongly inhibit the cell cycle progression of MDA-231 cells. This endpoint may be quantitated by using flow cytometry to determine the number of cells that have entered the S phase of the cell cycle. Inhibiting cell cycle progression to S phase is an indicator of anti-proliferative activity. Exposure to air markedly shifts the dose response curve for arachidonic acid and docosahexaenoic acid in particular, such that the oxidized forms of these fatty acids are more potent anti-proliferative compounds than their precursor

fatty acids. In contrast, neither the 18 carbon fatty acid, oleic acid, nor its oxidized products affected the cell cycle progression of MDA-231 cells.

As described above, 20 and 22 carbon polyunsaturated fatty acids are oxidized by any of several means known in the field, including exposure to molecular oxygen or air, for example. A method of oxidation shown to be effective by the inventor is as follows: arachidonic acid in ethanol is placed in a glass tube and dried under nitrogen after marking the volume on the side of the tube. A moderate flow of air is blown over the dried arachidonic acid at room temperature. After 24 hours, the sample, now yellow in color, is brought to its initial volume with ethanol. The percent oxidation may be determined from a gas chromatography/mass spectrometry analysis of the oxidized sample and its original stock solution.

The total oxidation products of such exposure may be used in preparation of anti- proliferative formulations, or they may be divided into fractions and the fractions with the highest, or higher activity may be used. In addition, active fractions from separate oxidations of several different fatty acids may be pooled into a single composition in order to obtain an anti-proliferative composition.

Typically, oxidation products are fractionated by reverse phase HPLC. Oxidized arachidonic acid and docosahexaenoic acid are converted to several products that can be clearly separated by reverse phase HPLC, including separations utilizing two separate solvent systems : Oxidative products may be fractionated by reverse phase HPLC employing, for example, an ULTRASPHERE ODS column eluted with methanol/water/phosphoric acid (55: 45: 02), pH 5.7, at 1.0 ml/min. for 5 minutes followed by an increase in the methanol composition to 100% over 20 minutes. When it is necessary to obtain better resolution of these products, they may be further separated using an ULTRASPHERE ODS column eluted with acetonitrile (solvent B) and water/acetic acid (100: 01, viv, solvent A). The initial system may be 35% solvent B at a flow rate of 1.5 ml/min. The percentage of solvent B may be increased to 50% at 23 min. (over 5 min.), to 56% B at 30 min. (over 2 min.), and to 95% B at 75 min. (over 7 min.).

Fractions of the total oxidized products may be isolated by reverse phase HPLC as described above and fractions monitored at 234 nM [HETE-like compounds (12- hydroxyarachidonic acid)] or 270 nM [leukotriene-like compounds]. Isolated products may be converted to methoxime-pentafluorobenzyl-ester-trimethylsilyl ester derivatives.

Derivatized products as carboxylate anions may be analyzed by NICI GC/MS. An HPLC-electrospray mass spectrometry/mass spectrometry (HPLC-MS/MS) method may be used for characterizing the double bond positions and position of oxygen insertion of the oxidized fatty acid products.

In isolating the fractions having antiproliferative activity as described herein, aliquots may be collected as 1 minute fractions from the HPLC. Because there is a large number of products and only a few of these likely have antiproliferative properties, it may be necessary to utilize a colorimetric assay in multiwell plates to assay the effect of products in these fractions on cell growth and survival. For example, the MTT (3- (4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay to measure the proliferation of MDA-231 cells. In this assay the tetrazolium ring is cleaved in active mitochondria, and thus living cells, and this cleavage can be measured colorimetrically.

Fractions that contain antiproliferative activity may then be further fractionated by normal phase HPLC as described above. These fractions may then be assayed (MTT) for antiproliferative activity and the most active, or those with detectable activity may be pooled and used in the compositions described herein. The structure of compounds in these fractions that contain antiproliferative activity may be determined by NICI GC/MS or HPLC MS/MS.

Without limiting the present invention to any particular mechanism, the present disclosure is based in part on data that have indicated that inducing or blocking enzyme pathways, where the result is the accumulation of arachidonic acid, consistently blocks cancer cell proliferation. It is contemplated, therefore, that perturbations of key acylation or metabolic events that control intracellular levels of arachidonic acid can be utilized to influence the growth and death of cancer cells. As disclosed herein, oxidation of selected 20 and 22 carbon fatty acids results in products with extremely potent anti-proliferative

properties when applied to tumor cells exogenously. Two examples shown herein to be particularly effective are arachidonic acid and docosahexaenoic acid.

As shown in the data included herein, exposure to air markedly shifted the dose response curve for arachidonic acid (AA) and docosahexaenoic acid (DCHA) such that the oxidized forms of these fatty acids were much more potent anti-proliferative compounds than their precursor fatty acids. In contrast, several other 20 carbon fatty acids; dihomogammalinolenic acid [20: 3, n-6, DGLA], eicosatrienoic acid [20: 5, n-3, ESA], eicosapentaenoic acid [20: 5, n-3, EPA], their oxidized products; several 18 carbon fatty acids (oleic acid [18: 1, n-9, OA], linoleic acid [18: 2, n-6, LA] alpha linolenic acid [18: 3, n-3, LNA], gammalinolenic acid [18: 3, n-6, GLA]); and their oxidized products were shown to have little if any effect on the growth of cancer cells. Thus, these data reveal that oxidation of AA and DCHA results in potent anti-neoplastic compositions.

FIG. 1 shows the inhibition of cell growth of MDA-231 breast cancer cells measured at 72 hours relative to control cells. Oxidized arachidonic acid is shown to have a marked effect on the growth curve of the treated cells, whereas the other compositions had little if any effect. FIG. 2 graphically depicts data showing that MDA-231 breast cancer cells are inhibited from releasing into S phase after 18 hour incubations with oxidized and unoxidized fatty acids. Cells in this study were harvested and analyzed by flow cytometry.

The values shown in the graph are reported as % of the control values. The data acquired with MDA-231 breast cancer cells may be particularly important because these cells are estrogen receptor negative cancer cells and are thus very aggressive and non-responsive to hormone therapy. An effective composition for treating such tumors would be an important addition to available chemotherapeutic cancer therapies.

The inhibition of MCF-7 cell growth after 120 hour incubation with oxidized and unoxidized arachidonic acid is shown in FIG. 3. A comparison of inhibition of MCF-7 cell cycle progression into the S-phase after 18 hour incubation with arachidonic acid, oxidized arachidonic acid and oleic acid is shown in FIG. 4. Oxidized arachidonic acid is again shown to strongly inhibit tumor cell cycle progression, whereas oleic acid and arachidonic acid had little effect.

A number of polyunsaturated fatty acids were oxidized and the oxidized products were tested for their ability to inhibit proliferation of MDA-231 cells after an 18 hour incubation. The results are shown in FIG. 5. The oxidized products were derived from (from top to bottom in the graph) arachidonic acid (AA), docosahexaenoic acid (DCHA), dihomogammalinolenic acid (DGLNA), eicosatrienoic acid (ESA), eicosapentaenoic acid (EPA), oleic acid (OA), linoleic acid (LA), alphalinolenic acid (LNA), and gammalinolenic acid (GLA). These data also indicate a strong anti-proliferative effect of oxidized DCHA and AA on breast cancer cells.

Oxidized arachidonic acid has been fractionated to obtain active anti-proliferative fractions. As shown in FIG. 6 compositions including oxidized metabolites of arachidonic acid have been fractionated on a C, 8 reverse phase column (upper section) and a silica column (lower section). The strongest inhibition is seen in the C, 8 reverse phase fraction eluted with 100 % methanol. Other fractions exhibiting anti-proliferative activity include the C, 8 fraction eluted with 50 % methanol and 50 % water, and the silica column fractions eluted with 50 % ethyl ether and 50 % hexane, or 25 % ethyl ether and 75 % hexane.

In further studies, filtered house air was continuously passed over arachidonic acid for 7 days in a polypropylene mini-centrifuge tube (100 mg/tube). Oxidized arachidonic acid was initially fractionated by thin layer chromatography (TLC) utilizing a neutral lipid system 90: 60: 6 (hexane/ethyl ether/formic acid) or a polar lipid system 65: 35 (methanol/water). TLC fractions were purified and monitored for their capacity to block the capacity of breast cancer cells to move through cell cycle and divide (anti-tumor activity). In the neutral lipid system, the vast majority of the anti-tumor activity was located at the origin (fraction 1, see FIG. 7A). In the polar lipid system, the majority of the anti-tumor activity was localized in fraction 5 (FIG. 7B).

The origin fraction from the neutral lipid system was next further fractionated by high performance liquid chromatography (HPLC) on a C, 8 reverse phase column eluted with methanol and water as follows: 0-5 minutes-50% MeOH/H, O; 5-45 minutes-60% MeOH/H, O ; 45-60 minutes-75% MeOH/H, O ; 60-80 minutes-100% MeOH. Anti-tumor activity was found and isolated from fractions 4 and 6 (FIG. 8). These two fractions were

further separated by HPLC on a C, 8 reverse phase column eluted as follows: Fraction 4-0- 15 minutes-55% MeOH/H, O ; 15-30 minutes-60% MeOH/H20 ; 30-65 minutes-60-65% MeOH/H, O ; 65-70 minutes-65% MeOH/H, O ; 70-80 minutes-100% MeOH. Under these conditions, anti-tumor activity eluted at about 46 minutes in fraction 11 in the secondary HPLC system (FIG. 9). The activity found in fraction 6 was further purified as follows: 0- 10 minutes-55% MeOH/H20; 10-30 minutes-55-65% MeOH/H, O ; 30-35 minutes-65% MeOH/H. O; 35-73 minutes-65-75% MeOH/H2O ; 73-75 minutes-75% MeOH/H2O O ; 75- 90 minutes-100% MeOH. The anti-tumor activity from fraction 6 of the original HPLC eluted from 42 minutes to 70 minutes in fractions 7,8, and 9 (FIG. 10).

The fractions of oxidized polyunsaturated fatty acids as described may be formulated individually or pooled into one or formulations in order to obtain a composition having anti-proliferative activity as described herein. It is also understood that the fractions are further characterized by a technique such as mass spectrometry in order to further characterize these active compositions.

Pharmaceutical Compositions and Routes of Administration The therapeutic and anti-tumor compositions of the present disclosure will have an effective amount of the oxidation products or an active fraction of the oxidation products of one or more 20 and/or 22 carbon polyunsaturated fatty acids. Such compositions are generally dissolved or dispersed in an acceptable carrier or medium, preferably for oral, parenteral. or topical administration. In certain embodiments, the compositions may be formulated for intravenous, intraarterial, intramuscular, subcutaneous, intramedullary, nasal, vaginal, or anal administration.

The phrases"pharmaceutically or pharmacologically acceptable"refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or human, as appropriate. As used herein, "pharmaceutically acceptable carrier"includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well

known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in the therapeutic compositions is contemplated.

The compounds may be formulated for oral administration. Such pharmaceutically acceptable forms include, e. g., capsules, particularly gel capsules, or any other form of oral preparation, including liquids, syrups, suspensions or emulsions, inhalants and the like. A liquid formulation will generally consist of a dispersion of the fatty acid compositions in a suitable liquid carrier (s) for example, water and/or other solvents such as, for example, polyethylene glycols, oils, milk, phospholipids, with, in certain formulations, a suspending agent. emulsifier, preservative, anti-oxidant, flavoring, and/or coloring agents. Preferred ingredients may include any of the following: galactolipids, sphingolipids, lecithins, cellulose, malt or malt extract, gelatin, casein, cholesterol, egg yolk, sodium dodecyl sulfate, benzalkonium chloride, p-hydroxvbenzoic acid, vitamin C, vitamin E or alpha- tocopherol. A composition in the form of a dried powder may be prepared using any suitable pharmaceutical carrier (s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.

A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier (s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in liquid suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical formulations suitable for ingestion may include sesame oil, evening primrose oil, peanut oil, aqueous propylene glycol, and sterile powders. In all cases it is desirable to keep the formulation sterile and stable under the conditions of manufacture

and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

The active compounds may be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, lithium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and _latin.

The present disclosure includes compositions for parenteral administration such as injectable liquids, for example. Such compositions may include an aqueous component and an emulsifier. Compositions for injection may include up to about 1-6 % phospholipids, soy lecithins, soybean lecithins, or other known emulsifiers in an emulsion including oils, or unsaturated oils. Osmotic agents may also be included in such compositions including, but not limited to glycerin, glucose, sucrose, fructose, sorbitol, protein, and sodium acid phosphates. Compositions including oil in water emulsions for injection are described in US Patent No. No. 5,034,414, and 4,678,808 each incorporated herein in pertinent part by reference. Emulsions may be prepared by addition of an alkali and mixing,

possibly followed by a high pressure spray to prepare the liquid emulsion. As in known in the art, particles in such an emulsion are typically on the order of 1 micron or less in size.

Sterile compositions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered or steam sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient.

Upon formulation, the active ingredients will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.

The formulations are easily administered in a variety of dosage forms, such as tablets containing measured amounts of active ingredient, with even drug release capsules and the like being employable. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. However, it is understood that an effective amount of active ingredient would provide a concentration at the cell of from about 1 pM to about 100tM or the equivalent, or in certain embodiments as much as a 1M concentration. In terms of systemic administration, an amount of from about 0. lmg to about 100 mg, or even up to about 500 mg, or about 1 g per daily dose may be administered as appropriate to achieve anti-tumor activity.

While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.