Field of the Invention The present invention relates to the use of specific congeners of microbial sophorolipids to treat or prevent progression in cancer, including reduction of tumour growth in the gastrointestinal tract or integument, and reduction of gastrointestinal bleeding that can occur in a range of human pathologies. Background to the Invention

Compounds that are non-toxic, orally tolerated and specifically target epithelial neoplastic cells in the intestinal tract could have great potential in delaying progression of intestinal neoplasms that are associated progression to colorectal cancer such as Familial Adenomatous Polyposis (FAR) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC)/Lynch syndrome. Currently, the gold standard for treatment of these conditions is surgery followed by adjuvant chemotherapy, the latter of which does not discriminate between normal and transformed tissue leading to a variety of potentially serious complications, including leukopaenia, anaemia, nausea, vomiting and weight loss. FAR is caused by a germline mutation in the Adenomatous polyposis coli (ARC) gene located at 5q22.2, which increases an individual’s susceptibility to develop colorectal adenomas and carcinomas at a young age (1) and significant morbidity and mortality. The use of chemotherapy, radiotherapy and surgery has proven somewhat effective in the treatment of early diagnosed colorectal cancer; however these methods prove invasive and detrimental to the natural host environment (1). This highlights the need for a natural chemotherapeutic with the ability to differentiate and target only tumorigenic cells.

Sophorolipids (SL), first described in the 1960’s, by Gorin et al, (2) are extracellular glycolipids synthesized from the non-pathogenic yeast such as Candida bombicola. Naturally occurring SL mixtures contain a variety of amphiphilic species, composed of a hydrophobic fatty acid (C16-C18) tail and a hydrophilic carbohydrate sophorose head. SL mixtures are made up mostly of a lactonic (LSL), closed ring (Figure 1a) and an open acidic (ASL) structure (Figure 1b) (3).

Biosynthesis of SL occurs when fatty acids are converted to either a terminal or sub-terminal hydroxyl fatty acid via an enzymatic reaction using the mono-oxygenase enzyme - cytochrome P450. Next, glucose is coupled glycosidically to the C1 position of the hydroxyl group of the fatty acid via glycosyltransferase I. This reaction requires a nucleotide-activated glucose as a glucosyldonor.

A second glucose is then coupled to the C2 position of the first large glucose molecule via glycosyltransferase II. SL obtained at this point are classed as non-acetylated ASL molecules. A majority of the molecules formed continue on and are further modified via esterification (lactonization) or acetylation of the carbohydrate head forming LSL (4). The synthesis process produces mixtures of SL, differing in acetylation and saturation levels, which prove tedious to separate and purify.

Interest in SL has grown due to their low toxicity and biodegradability making them ideal to use in food (5), cosmetics (6) and pharmaceutical (7) industries. ASL and LSL possess their own unique biological properties, for example, ASL have superior solubility and foam-forming abilities while LSL tends to be the better anti-bacterial agent (4) (8). SL have also shown excellent anti-viral (9), antiinflammatory (10) and, most promisingly, anti-cancer (7) properties in vitro. However, the majority of studies published in vitro test the biological properties of natural LSL/ASL mixtures or derivatives (typically around 60-70% pure and containing both lactonic and acidic sophorolipids in addition to several other congeners such as free fatty acids) rather than a highly purified form of either.

The in vitro anti-cancer properties of SL have received a lot of attention in recent years, showing cytotoxic effects in human pancreatic (HP AC) (11), liver (H7402) (12), lung (A549) (12), brain (LN229, HNCG-2) (13), esophageal (KYSE109, KYSE450) (14), breast (15) and leukaemic (HL60.K562) (16) cell lines. SL mixtures have also been shown to induce cell death, inhibit migration as well as increase both free Ca ²⁺ levels and reactive oxygen species in the aforementioned cancer cell lines.

To date, few in vivo bioactivity studies have been reported; although SL have been observed to be well tolerated in mice, non-irritant when topically applied to the skin and eyes of rabbits and non-toxic when administered orally to either mice or rats (17). SL mixtures have also been observed to reduce inflammation and increased life-span in a model of severe abdominal sepsis in rats (18) as well as decreasing IgE levels in a murine asthma model (19). A previous study carried out by our group, orally administered a purified LSL to the pre-cancerous Apc ^min+/ colorectal model which resulted in an exacerbated growth of tumours along the intestinal tract. This was the first study to investigate the effects of SL in vivo.

The ability to produce a pharmacotherapeutic friendly compound depends on the purity of a product and their biological effects in vitro and in vivo. As noted above, we previously found that >90% purified LSL exacerbate tumour growth in the Apc ^{mil ~} model. However, in the study disclosed herein, we demonstrate the anti-cancer potential of a substantially pure preparation of ASL against colorectal cancer cells in culture and their ability to regulate tumour development in the Apc ^mm+/ mice. In addition, we demonstrate that substantially pure ASL reduces clotting time in plasma by promoting fibrin formation and has potential as a non-toxic, orally tolerated additive for reducing naturally occurring gastrointestinal bleeding or bleeding associated with gastrointestinal pathology, and thus confirms the therapeutic utility of substantially pure ASL. Summary of the Invention

Accordingly, in one aspect, the present invention provides acidic sophorolipids for use in therapy.

The acidic sophorolipids of the present invention are a substantially pure preparation of acidic sophorolipids. It will be understood that naturally occurring sophorolipid mixtures, which may be synthesized naturally by microorganisms such as the yeast Starmerella bombicola (syn. Candida bombicola), can contain a variety of amphiphilic species, composed of a hydrophobic fatty acid (C16- C18) tail and a hydrophilic carbohydrate sophorose head, and are made up mostly of acidic sophorolipids (ASL) or lactonic sophorolipids (LSL). The sophorolipid-producing microorganism can be genetic manipulated, according to standard techniques known in the art, to reduce or prevent the formation of the lactonic form of sophorolipid and thereby increasing the amount or proportion of the acidic form of sophorolipid. Thus, the acidic sophorolipids of the present invention can comprise, or consist of, a substantially pure preparation of acidic sophorolipids.

Optionally, the substantially pure preparation of acidic sophorolipids comprises at least about 80 v/v%, optionally at least about 85 v/v%, optionally at least about 90 v/v%, optionally at least about 91 v/v%, optionally at least about 92 v/v%, optionally at least about 93 v/v%, optionally at least about 94 v/v%, optionally about at least about 95 v/v%, optionally at least about 96 v/v%, optionally at least about 97 v/v%, optionally at least about 98 v/v%, optionally at least about 99 v/v%, acidic sophorolipids relative to the sophorolipid preparation in which the acidic sophorolipids are contained. Optionally, the substantially pure preparation of acidic sophorolipids comprises at least about 96 v/v% acidic sophorolipids relative to the sophorolipid preparation in which the acidic sophorolipids are contained.

Optionally, the substantially pure preparation of acidic sophorolipids comprises non-acetylated acidic sophorolipid molecules. Optionally, the substantially pure preparation of acidic sophorolipids comprises about 96% total sophorolipid with the remaining about 4% comprising principally short chain fatty acids derived from, for example, a food grade oil such as a food grade rapeseed oil used as a fermentation feedstock. In preferred embodiments, the sophorolipid fraction of the preparation may contain, for example, about 98.5% acidic, acetylated sophorolipid with a carbon chain length of C ₁₈ with the remaining about 1 .5% non-acetylated acidic sophorolipid.

Optionally, the substantially pure preparation of acidic sophorolipids is substantially free of lactonic sophorolipids. Optionally, the substantially pure preparation of acidic sophorolipids comprises less than about 20 v/v%, optionally at least about 15 v/v%, optionally at least about 10 v/v%, optionally at least about 9 v/v%, optionally at least about 8 v/v%, optionally at least about 7 v/v%, optionally at least about 6 v/v%, optionally about at least about 5 v/v%, optionally at least about 4 v/v%, optionally at least about 3 v/v%, optionally at least about 2 v/v%, optionally at least about 1 v/v%, lactonic sophorolipids relative to the sophorolipid preparation in which the acidic sophorolipids are contained. In preferred embodiments, the substantially pure preparation of acidic sophorolipids comprises less than about 1% v/v% lactonic sophorolipids relative to the sophorolipid preparation in which the acidic sophorolipids are contained.

Optionally, the substantially pure preparation of acidic sophorolipids is substantially free of impurities. Optionally, the substantially pure preparation of acidic sophorolipids is substantially free of impurities, such as residual quantities of short chain fatty acids derived from, for example, a food grade oil, such as a the rapeseed oil, fermentation feedstock. Optionally, the substantially pure preparation of acidic sophorolipids comprises less than about 5 v/v% impurities, optionally less than about 4 v/v%, optionally less than about 3 v/v%, optionally less than about 2 v/v%, optionally less than about 1 v/v%, in the sophorolipid preparation in which the acidic sophorolipids are contained. It will be understood that impurities include, but are not limited to, lactonic sophorolipids, and also include impurities present in the substantially pure preparation of acidic sophorolipids as a result of the sophorolipid synthesis and/or isolation processes.

Optionally, the invention provides acidic sophorolipids for use in the treatment or prevention of cancer. Optionally, the acidic sophorolipids are for use in the treatment or prevention of cancer, wherein said use comprises administration of a therapeutically effective amount of the acidic sophorolipids to a patient in need thereof. Optionally, the acidic sophorolipids are for use in the treatment or prevention of cancer, wherein the acidic sophorolipids reduce cancer cell viability without affecting the viability of non-cancerous cells. Optionally, the acidic sophorolipids are for use in the treatment or prevention of cancer, wherein the acidic sophorolipids inhibit, and/or prevent, anchorage-independent cancer cell growth. Optionally, the acidic sophorolipids are for use in the treatment or prevention of cancer, wherein the acidic sophorolipids reduce cancer cell migration. In other words, the acidic sophorolipids are for use in the treatment or prevention of cancer metastasis. Optionally, the acidic sophorolipids are for use in the treatment or prevention of cancer, wherein the acidic sophorolipids reduce cell-cell and/or cell-extracellular matrix adhesion. Optionally, the acidic sophorolipids are for use in the treatment or prevention of cancer, wherein the acidic sophorolipids induce apoptosis and/or necrosis in the cancer cells. Optionally, the cancer is selected from colorectal cancer. As will be understood by the skilled reader, the acidic sophorolipids described herein for use in the treatment or prevention of cancer are suitable for direct application to the site of a tumour due to their high tolerance and ability to induce necrosis and apoptosis. Thus, the acidic sophorolipids, formulated, for example, as appropriate gel forms, may be used in the treatment or prevention of any type of cancer such as oral cancer, oesophageal cancer, anal cancer and melanomas, as well as for non-malignant cancers such as basal cell carcinomas. In particular, the acidic sophorolipids may be used with conventional treatment options such as chemotherapy or radiotherapy, or prior to use of such conventional treatment options.

Optionally, the invention provides a method of therapy, wherein said method comprises administration of a therapeutically effective amount of acidic sophorolipids to a patient in need thereof. Optionally, the invention provides a method of treating or preventing cancer, wherein said method comprises administration of a therapeutically effective amount of acidic sophorolipids to a patient in need thereof. Optionally, the method of treating or preventing cancer comprises administration of a therapeutically effective amount of acidic sophorolipids to a patient in need thereof, wherein the acidic sophorolipids reduce cancer cell viability without affecting the viability of non-cancerous cells. Optionally, the cancer is selected from colorectal cancer, colorectal cancer, oral cancer, oesophageal cancer, anal cancer, and melanoma, and non-malignant cancer, optionally basal cell carcinoma.

Optionally, the invention provides use of acidic sophorolipids for the manufacture of a medicament. Optionally, the invention provides use of acidic sophorolipids for the manufacture of a medicament for the treatment or prevention of cancer. Optionally, the invention provides use of acidic sophorolipids for the manufacture of a medicament, wherein the acidic sophorolipids in said medicament reduce cancer cell viability without affecting the viability of non-cancerous cells. Optionally, the cancer is selected from colorectal cancer, colorectal cancer, oral cancer, oesophageal cancer, anal cancer, and melanoma, and non-malignant cancer, optionally basal cell carcinoma.

Optionally, the invention provides acidic sophorolipids for use in therapy in a patient by promoting blood coagulation in said patient. Optionally, the acidic sophorolipids are for use in therapy in a patient by promoting blood coagulation in said patient, wherein said therapy comprises administration of a therapeutically effective amount of acidic sophorolipids to a patient in need thereof.

Optionally, the invention provides acidic sophorolipids for use in reducing or preventing bleeding, optionally gastrointestinal bleeding, in a patient. Optionally, the acidic sophorolipids are for use in reducing or preventing gastrointestinal bleeding from intestinal polyps, or bleeding associated with, or caused by, intestinal polyps. Optionally, the acidic sophorolipids are for use in reducing or preventing gastrointestinal bleeding associated with, or caused by, cancer. Optionally, the acidic sophorolipids are for use in reducing or preventing gastrointestinal bleeding associated with, or caused by, colorectal cancer. Optionally, the invention provides acidic sophorolipids for use in reducing or preventing gastrointestinal bleeding associated with, or caused by, gastric ulcers. Optionally, the acidic sophorolipids are for use in reducing or preventing gastrointestinal bleeding associated with, or caused by, ulcerative colitis. Optionally, the acidic sophorolipids are for use in reducing or preventing gastrointestinal bleeding associated with, or caused by, irritable bowel syndrome. Optionally, the acidic sophorolipids are for use in reducing or preventing gastrointestinal bleeding associated with, or caused by, medication-induced bleeding, optionally aspirin-induced bleeding.

Optionally, the invention provides acidic sophorolipids for use in delaying or preventing progression of intestinal neoplasms that are associated with progression to cancer, optionally colorectal cancer. In other words, the invention provides acidic sophorolipids for use in delaying or preventing progression of intestinal neoplasms to cancer, optionally colorectal cancer. Optionally, the acidic sophorolipids are for use in delaying or preventing progression of intestinal neoplasms that are associated with progression to colorectal cancer such as Familial Adenomatous Polyposis (FAR) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC)/Lynch syndrome.

Optionally, the invention provides acidic sophorolipids for use in increasing, or restoring, haematocrit in a patient. Optionally, the acidic sophorolipids are for use in increasing, or restoring, haematocrit in a patient suffering from cancer. Optionally, the acidic sophorolipids are for use in increasing, or restoring, haematocrit in a patient suffering from colorectal cancer. It will be understood that increasing haematocrit refers to increasing haematocrit relative to level of haematocrit prior, optionally immediately prior, to treatment with acidic sophorolipid. Restoring haematocrit refers to restoring haematocrit to a level which is the same, or about the same, as that prior to a reduction in haematocrit experienced as a result of a disease or condition described herein.

Thus, it will be understood that the present invention provides acidic sophorolipids for use as a chemotherapeutic agent in the treatment or prevention of cancer. Optionally, the present invention provides acidic sophorolipids for use as a chemotherapeutic agent, in combination with one or more other chemotherapeutic agents, in the treatment or prevention of cancer.

Optionally, the present invention provides a pharmaceutical composition comprising the acidic sophorolipids described herein. Optionally, the pharmaceutical composition is for use in therapy described herein. Thus, it will be understood, that the above-described acidic sophorolipids may be comprised in a pharmaceutical composition and said pharmaceutical composition may be for use in the therapy of the diseases and conditions described herein.

Optionally, the present invention provides acidic sophorolipids for use in the treatment or prevention of bleedings caused, or associated with, one or more active agents. It will be understood that active agents comprise, or consist of, a pharmaceutical drug, medicine or agent. Optionally, the present invention provides a pharmaceutical composition comprising the acidic sophorolipids of the present invention and one or more further active agents. Optionally, the one or more active agents comprise a drug with a side-effect profile which includes bleeding, optionally increased bleeding, in a patient to which the drug is administered. Optionally, the one or more active agents comprise one or more drugs selected from nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, celecoxib, and naproxen; anticoagulants, such as heparin, lepirudin and warfarin; antiplatelets, such as prasugrel; novel oral anticoagulants (NOAs), such as apixaban and rivaroxaban; serotonin and norepinephrine re uptake inhibitors (SNRIs), such as duloxetine and venlafaxine; selective serotonin re uptake inhibitors (SSRIs), such as citalopram, fluvoxamine and sertraline. Optionally, said bleeding, or increased bleeding, comprises one or more of excessive bruising, nosebleeds, heavy menstrual bleeding, gastrointestinal bleeding, and rectal bleeding. Thus, it will be understood that inclusion of acidic sophorolipid as adjuvants for active pharmacological agents can prevent or reduce bleeding associated with administration of said active agents. In particular, inclusion of acidic sophorolipid as adjuvants for active pharmacological agents transiting the stomach and upper digestive tract can prevent or reduce gastrointestinal bleeding associated with oral delivery of said active agents.

Optionally, the acidic sophorolipids of the present invention are administered orally to a patient in need thereof. Due to the water solubility and surfactant activity of acidic sophorolipids, a wide range of different formulations of the acidic sophorolipids are easily possible, in particular in products such as mouthwash, toothpaste, skin creams, oral gels, suppositories, yogurts and other dairy products etc.

Optionally, the acidic sophorolipids of the present invention are anticipated to be administered, in humans patients, in amounts around 200-400 mg/day. However, the skilled physician will be able to determine the correct dose of the acidic sophorolipids depending on the site and severity of the condition, for example, cancer to be treated or prevented, by routine means known in the art.

Optionally, the present invention provides a foodstuff, or foodstuff ingredient, comprising the acidic sophorolipid of the present invention. It will be understood that the foodstuff, or foodstuff ingredient, comprising the acidic sophorolipid of the present invention can be used to conveniently treat patients suffering from conditions or diseases described herein, wherein consumption of the foodstuff or foodstuff ingredient allows administration of the acidic sophorolipid for treatment of said conditions or diseases. The foodstuff, or foodstuff ingredient, is not particularly limited and may comprise, for example, yogurt, probiotic drinks, ice-cream, mayonnaise, butter or margarine spreads, bread, etc. Optionally, the foodstuff, or foodstuff ingredient, can comprise a formulation having a water/oil interface, such as salad dressing.

“Acidic sophorolipids” and its acronym “ASL” are used interchangeably herein.

The term “patient” can include human and other mammalian subjects that receive the therapeutic treatment disclosed herein.

“Cancer” can include tumours, or neoplasms, which are benign, pre-malignant, or malignant, and each type of tissue cancer can include carcinomas, including, carcinoma in situ, invasive carcinoma, metastatic carcinoma and pre-malignant conditions. “Colorectal cancer” (CRC) includes bowel cancer and colon cancer.

The acidic sophorolipids of the present invention and/or the pharmaceutical compositions disclosed herein comprising the acidic sophorolipids may further comprise a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of, for example, the USA or EU. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Pharmaceutical compositions comprising the ASL compound can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition may be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin. The composition may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, or intramuscular administration to human beings. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. If a composition is to be administered by infusion, it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. If a composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

“Therapeutically effective amount” may be understood to mean a dose that produces the desired effect for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). Efficacy can be measured in conventional ways, depending on the condition to be treated. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP), or determining the response rates (RR). Therapeutically effective amount also refers to a target serum concentration, such as a trough serum concentration, that has been shown to be effective in suppressing disease symptoms when maintained for a period of time. Our in vivo data have demonstrated use of acidic sophorolipids in both normal (wildtype) and animal models of disease and indicates that the acidic sophorolipids are non-toxic and well tolerated at high doses (>50 mg/kg bodyweight, e.g. 100 mg/kg) which is consistent with oral delivery.

The phrase “treatment of cancer”, and the like, includes treatment of cancer to reduce tumour volume and/or reduce the rate of tumour growth. Tumour volume may be measured before or at the point of initial treatment and then again at any time after the treatment has begun, e.g. at day 28.

Actual dosage levels of the acidic sophorolipids of the invention or pharmaceutical composition comprising the acidic sophorolipids provided herein may be varied so as to obtain an amount which is effective to achieve the desired therapeutic response for a particular patient, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular therapeutic compound or composition employed, the route of administration, the time of administration, the rate of metabolism or excretion of the components of the therapeutic compound or composition being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound or composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the compound or composition required. For example, the physician or veterinarian could start doses of the ASL compound or composition at levels lower than that required to achieve the desired therapeutic effect and gradually increasing the dosage until the desired effect is achieved. In general, a suitable dose of compound or composition provided herein will be that amount of the compound or composition which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be oral; however, it is possible that suitably formulated ALS compound or composition can be administered intravenously, intramuscularly, intraperitoneally, or subcutaneously, and may administered proximal to a target site. If desired, the effective dose of a therapeutic compound or composition may be administered as two, three, four, five, six, seven, eight, nine, ten or more subdoses administered separately at appropriate intervals.

By “about”, as used herein, it is meant that the recited value may be precisely the recited value, optionally ± 10% of the recited value, optionally ± 20% of the recited value, optionally ± 30% of the recited value, optionally ± 40% of the recited value, further optionally ± 50% of the recited value.

As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. Similarly, a reference to “an active ingredient”, for example, includes one or more active ingredients.

Brief Description of the Drawings

The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which:

Figure 1 (a) depicts the structure of LSL, and (b) the structure of diacetylated C18:1 ASL;

Figure 2 depicts the effects of ASL on cell viability (a-b). Following 24h of treatment with ASL, there was no significant reduction in viability in the control cell lines CCD-841 -CoN and MRC5. We observed a significant reduction in O.D. 570 value at doses from 20-100 pg/mi in the HT29 and Caco2 cells (*p < 0.01) and at doses about 50 pg/ml in HT115, HCT116 and LS180 (**p < 0.001). Quantification of live, apoptotic and necrotic cells after treatment with vehicle or ASL (c-g). The vast majority of CCD-841 -CoN control cells show live viable cells at both concentrations with a 15% increase in apoptotic cells seen at 70 pg/ml ASL (c). HT29, HT115 and HCT116 cells treated with 20 pg/ml ASL show an increase in cell death inducing both apoptosis and necrosis equally (d, e, g) while Caco2 cells induced a higher number of apoptotic cells (f). At 70 pg/ml, the number of quantifiable cells was reduced with HT29 and Caco2 more susceptible. All colorectal cancer cell lines exposed to 70 pg/ml resulted in over <80% cell death. Representative photomicrographs of acridine orange / ethidium bromide staining in HT29 cell with 0 (left), 20 (middle) or 70 pg/ml (right) ASL (h). Condensed nuclei are characteristic of apoptosis while cells with red/orange nuclei clusters indicate necrosis. Graphs show a representative data set from three independent experimental replicates. Values indicate mean ± SEM (n=6). Statistical significance was assessed one-way ANOVA;

Figure 3 depicts migration of normal CCD-841 -CoN and colorectal cancer cells (a-b). (a): Photomicrographs of the scratch at Oh in CCD-841 -CoN (top) and HT29 (bottom), after 72h in vehicle-control and after 72h in 10 pg/ml ASL treated (b): Quantification of migration in CCD-841 - CoN, HT29, HT115, Caco2 and HCT116. A dose of 10 pg/ml ASL had no effect on the migration of CCD-841 -CoN after 72h. ASL treatments decreased the percentage of scratch migration in HT29 (17% ***), HT115 (22% **), Caco2 (15% ***) and HCT116 (25% **) compared to vehicle-control (c): Photomicrographs of HT29 colonies formed after vehicle-control or 30 pg/ml treatment (d): ASL treatments reduced the number of colonies formed by HT29 (3 vs 12 ***), HT115 (7 vs 18 **), Caco2 (7 vs 11 *) and HCT116 (4 vs 15 ***) compared to vehicle-control. Graphs show representative data from three independent experimental replicates. Values indicate mean ± SEM. Statistical significance was assessed using a student’s t-test. * p<0.01 , ** p<0.001 , *** p<0.0001 ;

Figure 4. Mice ( wt or Apc ^mm+/ ) were fed ASL or vehicle-only every other day for 70 days. There were no morphological changes to ileal segments from wt mice fed vehicle-control or 50 mg/kg ASL (a: top). Apc ^mm+/ ileum (a: bottom) treated with vehicle-only (bottom left) showed evidence of bleeding as well as numerous polyps with a diameter between 0.4mm-4mm (c). Ileal segments from Apc ^mm+/ - _mjce treated with ASL (a: bottom right) showed no evidence of intestinal bleeding and no significant change to polyp number (b: control:48 vs ASL:42) or diameter (c). Values represent mean ± SEM (n= 10/gr oup). Statistical significance was determined by one-way ANOVA or a student’s T-tests

Figure 5 depicts (a): Photographs of dissected spleen from a wt mouse and Apc ^mm+/~ mouse fed with vehicle-only or 50mg/kg ASL for 70 days (b): Graph indicating the difference in spleen weights. Ap _C ^min+/ - _mjce ti _{a(j a} significantly greater weight compared to wt mice (*** p<0.0001). ASL dosing resulted in a significant decrease in splenic weights compared to the vehicle control mice (** p < 0.001). Splenic histology from wt (6c left), Apc ^mm+/ mice fed vehicle control-only (6c middle) and Apc ^mm+/ fed ASL (6c right). WP =While Pulp, RP = Red Pulp wt spleens are characterised by the white pulp and the loose reticular structure of the red pulp. Apc ^mm+/~ mice demonstrated altered splenic pathology with increase red pulp regions (haematopoietic rich tissue). ASL fed mice showed an improvement in histopathology (c.f. 6c middle vs right) by decreasing the red pulp population (*p < 0.05). Haematocrit measured demonstrated a significant decrease in levels in wtvs Apc ^mm+/ (**p < 0.001). ASL resulted in a significant increase in haematocrit levels in the Apc ^mm+/~ compared to the vehicle control (*p<0.05). Graphs represented of mean ± SEM. Animals per group n= 10. Statistical significance was determined by students t-test.

Detailed Description

Materials & Methods

Sophorolipid production and purification.

The crude SL precursor was purchased from Soliance (France) as a typical lactone/acidic (~ 80/20) mixture (Sopholiance_S, Batch N°11103A). The C18:1 ASL was obtained by alkaline hydrolysis according to method 1 described by Baccile et al, (20). In brief, the acidic sophorolipid product may be prepared using the lactone esterase knock out strain of Starmerella bombicola described by Ciesielska et al. (2014) (38). The organism can be cultured in large scale aerobic stirred tank fermenters at 25°C using a fed batch system with continuous addition of food-grade rapeseed oil after the first 24 hours of culture. At the conclusion of the fermentation microbial cells are removed by ultrafiltration and hexane used to extract the sophorolipid product.

Cell culture

The colorectal cancer cell lines HT29 (ATCC® HTB-38), HT115 (phe-cultures 85061104), HCT116 (ATCC® CCL-247), LS180 (ATCC® CL-187), Caco-2 (ATCC® HTB-37), normal colonic epithelium CCD-841 -CoN (ATCC® CRL-1790) and lung fibroblasts MRC5 (ATCC® CCL-171) used in our studies were maintained in DMEM media supplemented with 10% foetal bovine serum (Invitrogen; Paisley, UK). All cultures were maintained at 37°C and at 5% C0 ₂ . The providence of all cell lines used in this study was confirmed by STR analysis (21) and morphological assessment of phenotype prior to use in bioassays. Cell viability assay

A total of 1x10 ⁴ cells per well were seeded (96 well plate: Nunc Thermos scientific, UK) and allowed to attach overnight before being serum starved for24h. Various concentrations of ASL (0.001 pg/ml - 100 pg/ml) were added and the cultures incubated for another 24h. Subsequently, 10 pi of a 25 mg/ml solution of MTT (3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide; (Sigma- Aldrich Company Ltd, Dorset, UK) was added to each well and the plate further incubated for 1 h at 37°C. The formazan crystals were solubilized with 100 pi of DMSO (Sigma-Aldrich Company Ltd, Dorset, UK) and the absorbance at 570nm was read on a spectrophotometer plate-reader (BMG- LABTECH, Omega, Aylesbury, Bucks UK). Each experiment was repeated three times with six internal repeats per group.

Acridine orange/ethidium bromide staining and quantitation To determine the number of necrotic or apoptotic cells induced by addition of ASL, cells were stained in situ with acridine orange (10mg/ml; (Sigma-Aldrich Company Ltd, Dorset, UK) and ethidium bromide (1 mg/ml (Sigma-Aldrich Company Ltd, Dorset, -Aid rich; UK)) and morphological changes were assessed by fluorescence microscopy (22). For assessment of apoptosis, a total of 3x10 ⁵ cells were seeded onto a 10mm coverslip (Agar Scientific; Stansted, Essex, UK) placed within a 6 well plate and the cells incubated overnight to form a confluent monolayer. Following serum starvation for24h, ASL (either 20 pg/ml or 70 pg/ml), vehicle-only control, or 5pM of etoposide (positive control) (Sigma-Aldrich Company Ltd, Dorset, UK) was added and the plate incubated for a further 24h. To determine the number of live cells remaining on the coverslip the samples were washed three times with ice-cold phosphate buffered saline (PBS; pH7.4, Oxoid; UK) 3 times, followed by incubation with a solution of 10 pi of 1 :1 acridine orange /ethidium bromide for 5 minutes and then the cells were washed 3x with ice-cold PBS and subsequently imaged with a Zeiss florescence microscope (Axio Scope 1 , Zeiss, Germany) at a range of objective magnifications. The operator was blinded to the experimental groups and random fields were selected (4 OX objective). A total of 300 attached cells/coverslip (or the maximum number of cells if this was <300) were identified morphologically and counted as necrotic (red/orange nuclei), apoptotic (green condensed or fragmented nuclei) or live (green non-condensed ovoid or rounded nuclei). Each experiment was repeated three times with six internal repeats per group. Data is presented as mean ± SEM of a single representative experiment.

Scratch assay

For wound healing scratch assay, 1.6x10 ⁶ cells were plated in each well of a 6 well plate (Nunc Thermos scientific, UK) and allowed to attach overnight. The “wound” was made by scratching a line in the centre of the confluent monolayer using a sterile toothpick. Cells were rinsed very gently with PBS three times and cultivated in serum free media supplemented with vehicle-control or 10 pg/ml ASL for 72 hours. Pictures were taken at 10x objective magnification at various time points using a Nikon microscope (Nikon ELWD Tl SCP, Japan). To quantify migration of cells into the scratch wound, the area of the gap was measured using ImageJ software (23). After 72h, the area of the remaining gap was measure and the difference between initial and final areas calculated. Experiments were plated in triplicate and repeated three times, with a single representative data set shown as mean ± SEM.

Soft agar colony formation assay

In order to determine if ASL disrupts tumour spheroid formation, a colony formation assay was performed in a 6 well plate. Briefly, a dilute suspension of HT29 cells was deposited between two layers of agarose. The bottom layer consisted of 0.5% agarose (Fisher Scientific, Loughborough,

UK Ltd) supplemented with DMEM containing 10% FBS, while the top layer contained an even dispersion of 1x10 ³ cells in DMEM containing 10% FBS and 0.35% agarose. Each individual agarose layer was supplemented with either vehicle-only control or 30pg/ml ASL. Plates were incubated for 14 days with media changes every third day. Colonies were stained with 0.001 % crystal violet (Sigma-Aldrich Company Ltd, Dorset, UK) and total colony numbers/plate were counted using a Stereo-microscope and a 4x objective lens (Leica CLS; Milton Keynes, UK). Experiments were plated in triplicate and repeated three times, with data shown as mean ± SEM of a single representative data set.

Animal model

All animal procedures were carried out in accordance with both local animal welfare committee (Ulster University) and national (UK Home Office) guidelines (24). For breeding purposes male Ap _C ^mm+/ - _{mjce W} ere housed together with female wild type (wt) mice and all the animals subjected to a 12/12 light cycle, with food and water being available ad libitum. Mice were genotyped as described previously, with both heterozygous Apc ^mm+/ and wt mice (male and female) used in experiments.

ASL dosing

At five weeks of age, both wt littermate and Apc ^min+/~ mice were treated orally (via a sterile p20 pipette tip) every other day with either vehicle-only or a solution containing 50 mg/kg (body weight) of ASL suspended in saline for 70 days. During treatments, body weights, general health and general behaviour were monitored bi-weekly. Food and water were weighed on a weekly basis to determine the effect of ASL treatment on eating/drinking habits.

Tissue collection and assessment

Mice were euthanized with an overdose of general anaesthetic (pentobarbitone), and cardiac puncture performed to collect blood prior that was stored in EDTA tubes (Aquilant Scientific, Down, Nl) prior to determining haematocrit (Cole-Parmer, Trickenham.UK). I nternal organs including the intestinal tract, colon, spleen, heart, liver, kidneys and lungs were carefully removed, weighed and immerse fixed in 10% buffered formal saline (pH7.4). The intestinal tracts were divided into 3 sections according to the description of Casteleyn et al., (2010) (25). After identification of the specific intestinal regions, samples were bisected longitudinally and the total number of polyps was recorded as well as their diameters measured with callipers. The specimens were then cut into ~2cm strips and placed in cassettes prior to standard wax embedding. To assess qualitative histopathological changes in the intestines and spleen, tissues were cut into 5pm sections using a microtome (Shandon; Cheshire, UK) placed on glass slides, cleared with xylene, dehydrated in descending grades of ethanol, stained with Mayer’s haematoxylin and eosin stain (Sigma-Aldrich, Dorset, UK) and examined with a Zeiss light microscope (Axio Scope 1 , Zeiss, Germany) at a range of objective magnifications.

Statistical analyses

All data in this study is presented as mean ± SEM of a single representative data set. Statistical analysis of in vitro data was determined using either one-way AN OVA or student’s t-test with the aid of GraphPad Prism (GraphPad software, San Diego, USA). All comparisons between in vivo groups were assessed using a students’ t-test. A value of p < 0.05 was considered statistically significant.

Results Production and purification ofASL

Acidic sophorolipid with a purity of 96% was produced through the use of a lactone esterase knockout mutant of the yeast Starmerella bombicola. The organism was grown on a fermentation medium containing food-grade rapeseed oil as the principal carbon substrate using a continuous fed batch system maintained at 25°C and pH 3.5. The culture can be adjusted to a suitable scale and will yield >50g/l of acidic sophorolipid product. A t the conclusion of the fermentation, the yeast cells were removed by ultrafiltration and the acidic sophorolipid extracted with hexane prior to removal of the solvent and drying. Composition and purity of the product was confirmed by HPLC/MS methods.

ASL has a selective effect on colorectal cancer cell viability

ASL concentrations ranging from 0.001-100pg/ml did not affect the viability of either non-transformed colonic epithelial (CCD-841 -CoN) or lung fibroblast (MRC5) cell lines (Figure 2a) after 24h in comparison to vehicle-only control treated cultures. However, ASL concentrations above 20 pg/ml resulted in significantly reduced viability of both HT29 and LS180 cell lines (**p <0.001), whereas concentrations over 40 pg/ml reduced cell viability of HT115, Caco2 and HCT116 tumour cell lines (*p <0.001) respectively.

ASL induces cell death and loss of adherence in vitro

ASL treatments of 20 pg/ml did not result in a significant increase in the number of apoptotic and necrotic CCD-841 -CoN cells (Figure 2c); however, a dose of 70pg/ml significantly increased the number of apoptotic cells to 15% of the total (* p <0.05). However, ASL treatment resulted in a dose-dependent increase in cell death and loss of adherence in all 4 human colorectal cancer cells we examined (Figures 2c-g). In comparison to vehicle-only treated controls (Figure 2c), exposure to a dose of 20 pg/ml ASL resulted in >50% total cell death in HT29 (62%, *p <0.01 ; Figure 2d), HT115 (30%; ** p<0.001 ; Figure 2e) and HCT116 (34%;** p<0.001 ; Figure 2g) with equivalent proportions of necrotic and apoptotic cells; whereas Caco2 cells showed a significantly higher proportion of cell death (59%; ** p <0.001 ; Figure 2g) as a result of apoptosis. The exposure of all colorectal tumour lines to 70 pg/ml ASL resulted in a marked reduction in the quantifiable cells present, such that fewer than 300 adherent cells in total were able to be counted. Exposure to 70 pg/ml ASL resulted in cell death in HT29 (>80%; **p <0.001), HT115 (* p <0.01), Caco2 (***p <0.0001) and HCT116 (* p <0.01) in the remaining adherent cells compared to the vehicle-only controls.

ASL reduces motility and anchorage-independent growth of tumour cells

To determine the effects of ASL on cell motility, a scratch wound healing assay was performed on normal colonic epithelial cells and 4 colorectal cancer lines (Figures 3a & b). A dose of 10pg/ml ASL had no significant effect on CCD-841 -CoN cell migration with 90% of the total scratch area being covered after 72h (Figure 3a; top panel). In contrast, 10 pg/ml ofASL resulted in a highly significant decrease (Figure 3b) in the proportion of the total scratch area covered after 72h in HT29 (17%; p <0.0001 ; Figure 3a lower panel), HT115 (22%; p <0.0001), Caco2 (15%; p<0.0001) and HCT116 (25%; p <0.0001). To determine if anchorage-independent growth and colony formation of colorectal cancer cells were affected by addition of ASL, colony growth was quantitated using the soft-agar colony formation assay (Figures 3c & d). After incubation of cells with 30 pg/ml ASL for 14 days a significant reduction in colony formation was observed in HT29 (p < 0.0001), HT115 (p < 0.001), Caco2 (p < 0.01) and HCT116 (p <0.0001) in comparison to vehicle-only controls.

ASL does not affect tumour number or size in Apc ^m,n+/~ mice

Apc ^mm+/ - _{or wt mjce were} randomly assigned to either vehicle-only or ASL dosing groups, irrespective of gender. The weights of wt and Apc ^mm+/ littermate mice fed with ASL solutions were not significantly different (endpoint of experiment: 25.2 ± 0.9g vs 22.9 ± 1 8g respectively; NS, p >0.05) and there were no differences in water or food consumption over the duration of the experiment (data not shown) gross morphological appearance of unfixed, flat-mounted ilea from wt mice (Figure 4a top) treated with either vehicle or 50 mg/kg ASL was characterised by a flattened, uniformly smooth mucous epithelium with prominent patent blood vessels. In vehicle-only treated Apc ^mm+/ - _mjce (Figure 4a; bottom left), there is clear evidence of polyp associated bleeding within the ileal segment. Following treatment with 50 mg/kg ASL for 70 days, there was no grossly discernible evidence of bleeding from intestinal polyps (Figure 4a; bottom right). The number of intestinal polyps in Apc ^min+/~ mice was not significantly different after the treatment of 50 mg/kg ASL for 70 days (vehicle = 48 ± 2 vs ASL = 45 ± 4 ; Figure 4b; p < 0.1). ASL treatments also had no effect on the modal size distribution of the polyps in comparison to vehicle-only (vehicle- 4mm vs ASL 4mm; Figure 4c; NS, p > 0.05).

ASL specifically reduce spleen weight and the proportion of red pulp in Apc ^m,n+/~ mice The weights and gross morphological appearances of heart, lungs, kidneys and liver were not significantly different in wt or Apc ^min+/ mice or in those fed either vehicle or 50mg/kg ASL (data not shown).

The spleen from vehicle-control fed Apc ^mm+/ mice were significantly heavier than their wt littermates (0.58 ± 0.2g vs 0.15g ± 0.5g respectively; Figures 5a & b; p < 0.0001). Administration of 50 mg/kg ASL for 70 days to Apc ^mm+/~ mice resulted in a decrease in splenic weight (Apc ^mm+/ = 0.55 ± 0.2 g vs ASL Apc ^mm+/~ 0.35 ± 0.4 g: Figure 5b; p < 0.001). Histological examination of sections from wt mouse spleen (Figure 5c; left panel) revealed conspicuous intensely basophilic areas of white pulp; these are separated by less dense regions of red pulp, areas responsible for removal of old or damaged erythrocytes. In vehicle treated Apc ^mm+/~ mice there is conspicuous clumping of red pulp and the proportion of this tissue is significantly increased when compared to wt (Figure 5c middle vs left panel; Figure 5d). Following treatment with 50 mg/kg ASL there was a significant reduction in red pulp size as compared with vehicle-only controls (Figure 5c right vs middle panel; Figure 5d). Haematocrit values were significantly higher in wt than Apc ^mm+/~ mice (48% vs 35%; p <0.001) and feeding Apc ^mm+/ mice with 50 mg/kg ASL resulted in a significant increase in haematocrit compared to the vehicle-only control (vehicle = 35% vs ASL = 39%; p < 0.05). Discussion

The ability of a chemotherapeutic agent to distinguish between normal and cancerous cells proves a highly desirable trait. Here we investigated whether a 96% pure preparation of ASL had this ability by assessing their differential effect in two normal cells (CCD-841 and MRC5) and five colorectal cancer lines (HT29, HT115, Caco2, HCT116 and LS180) in vitro. ASL showed no toxicity against the normal colonic epithelial (CCD-841 -CoN) and lung fibroblasts (MRC5) resulting in no change to viability at any dose between 0.001 pg/ml - 100pg/ml. However, doses of 20 pg/ml - 50 pg/ml ASL inflicted a potent response in the colorectal cancer cells and reduced their viability. A similar toxic effect of SL against pancreatic (11), liver (16), lung (12) and oesophageal (14) cancer cells in vitro has been observed at doses ranging from 40 pg/ml - 2 mg/ml and demonstrate specificity over cancer cell lines, resulting in no toxicity to normal non-transformed cell lines such as circulating blood monocytes. However, these studies have been carried out using impure and poorly characterised mixtures of SL applied to either a non-adherent or uncharacterised normal cell lines. The comparison of a SL, of uncertain purity, between an adherent and non-adhered cell line from different anatomical locations is questionable, especially when previous studies have hypothesised that SL have the ability of intercalate into the cytoskeleton of cells and inflict a response via deregulation of cellular adhesion (18). It has been shown that biosurfactants doses induce tension at the interfacial region of the bilayer resulting in phospholipid dehydration which affects lipid stability, ultimately resulting in cell death (26, 27). To assess cell death mechanisms caused by ASL, ethidium bromide/acridine orange staining was applied allowing allows the quantification of dose-dependent cell death. ASL treatments resulted in over 50% death in HT29 and Caco2 cells, both adenocarcinoma cancer lines, from as little as 20 pg/ml. The remaining carcinoma cell lines - HT115 and HCT 116 accumulated over 50% death only after 70pg/ml of ASL. All cell lines induced apoptosis and necrosis equally. Studies to date conflict which method of cell death SL induce. A previous study from our group demonstrated that a pure form of LSL induced predominantly necrosis in colorectal cancer (Callaghan et al. , 2016; PLoS One 11 (6): e0156845) (39). Other studies show that SL mixtures induced necrosis in the H7402 hepatoma cancer cell line (16), while apoptosis was the main mechanism of cell death induced in the HP AC pancreatic carcinoma cell line (11). Therefore, it is hypothesised that mechanism of cell death depend on both the structure and purity of the SL congeners as well as the characteristics of the tumour cell line used. The hypothesis that the amphiphilic properties of SL permit their incorporation in the cell membrane (28) may also prove beneficial against other well-documented characteristics of neoplastic growth and metastasis. The ability of ASL to inhibit the migration of cancer cells and obstruct anchorage- independent growth was assessed in the colorectal cancer cells. At a low dose of 10 pg/ml, ASL had no effect on the normal colonic cells (CCD-841 -CoN) allowing total scratch coverage after 72hrs. However, at the same dose, ASL reduced the total percentage coverage reduced the coverage area to only 17-25%. This effect has only been documented in one other study (15) where 5 mg f ¹ LSL inhibited the migration of cancer cells; however, the effects of this high dose on normal cells has not be recorded. Similar effects have been demonstrated with several other amphiphilic non-ionic surfactants such as Triton x100 (29).

The inhibition of anchorage-independent growth of cancer cells had previously been demonstrated with surfactants against breast cancer (30); however, this effect has not been tested with SL in vitro. ASL reduced the number of colonies formed after 14 days at 10 pg/ml compared to the vehicle control. It is hypothesised that the inhibition of anchorage-independent growth is regulated, in part, by adhesion proteins such as E-cadherin (31). This strengthens the hypothesis that SL induces a response via disruption of cell-cell or cell-matrix adhesion.

Investigations into the anti-cancer properties of SL in vitro currently populate the literature; however, there are no studies examining their therapeutic potential in vivo, with the exception of a paper published by our group looking at the effects of purified LSL in a pre-cancerous colon murine model. The Apc ^mm+/ mouse (32) is widely used in examining the relationship between food (33), genetics (34) and chemotherapeutics (35) in the development of intestinal adenomatous neoplasms (polyps) and these animals commonly present with an enlarged spleen and reduced haematocrit by 4 months of age (36). This is an acute model with a life span of <150 days, where the primary cause of death is not directly attributable to the development of numerous polyps but a result of extensive intestinal bleeding and anaemia (37).

Our previous studies demonstrated that the oral administration of purified LSL resulted in the exacerbated growth of adenomatous polyps along the intestinal tract therefore we hypothesised a similar reaction with the administration of a purified ASL.

The oral feeding of ASL to Apc ^mm+/ resulted in no significant change to either polyp numbers or their diameter along the intestinal tract when compared to the vehicle control. However, ASL administration demonstrated a systemic effect by reducing the spleen size in Apc ^mm+/~ mice compared to those dosed with the vehicle control. ASL also resulted in a 20% increase in the haematocrit of Apc ^min+/ mice. Changes to splenic size and haematocrit levels can be explained by a decrease in intestinal bleeding thus resulting in a reduction in anaemia. As the Apc ^mm+/~ mice are known to die as the result of the secondary effects, it can be hypothesized that the improvement of these effects may improve the life-span of these mice. The results in this study are a vast difference to those seen in the Apc ^min+/~ mice orally administered purified LSL.

In conclusion, ASL have the ability to discriminate between normal and colorectal cancer cell in vitro resulting in a decrease in the viability, migration and anchorage-independent growth in cancer cells only. ASL also slows the progression of the pre-cancerous Apc ^mm+/~ mouse model therefore highlighting the potential of ASL as a chemotherapeutic agent.

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