Compositions for treating colonic neoplasia by the intraluminal application of antibiotics

Abstract

The present invention provides compositions comprising fosfomycin for reducing or eliminating bacterial promoters of colorectal cancer from colorectal adenomas, polyps and adenocarcinomas, as well as from the associated bacterial biofilm, by local 5 administration into the bowel lumen.

Description

Compositions for treating colonic neoplasia by the intraluminal application of antibiotics Field of invention The present invention provides compositions comprising fosfomycin and other antibiotics for local application to the colonic mucosa from the luminal side to eliminate or reduce the presence of bacterial promoters of the formation of colonic neoplasia of the adenoma-adenocarcinoma sequence, including fusobacteria, colibactin-producing (polyketide synthetase-positive) Escherichia coli (pks+ E. coli), enterotoxigenic Bacteroides fragilis (ETBF) and other microorganisms present in the bacterial biofilm associated with the initiation and progression of the carcinogenic sequence. The purpose of the invention is to reduce the pro-carcinogenic action ascribed to the biofilm and thus reduce the initiation and growth of colonic adenomas and their possible transformation into malignant adenocarcinomas. As such, it is relevant to the fields of oncology and gastroenterology as well as to other fields comprising the prevention and treatment of colorectal neoplasia. Background of invention Colorectal cancers (CRC) arise in stages, starting with mucosal dysplasia and aberrant crypt foci, which lead to the development of adenomatous polyps that are considered to be CRC precursors. Colonic polyps are usually classified according to their propensity to progress to malignancy (adenomatous as opposed to hyperplastic) as well as by their structure (sessile, pedunculated, or flat), shape (tubular, villous, serrated) and size (small 1-5 mm, medium 5-10 mm, and large ≥10 mm). Hyperplastic polyps are usually small, located in the rectum and sigmoid colon, and have little malignant potential except for some subsets of serrated hyperplastic polyps. Adenomatous polyps or adenomas account for approximately 70% of colon polyps and have the potential to progress to CRC over time if not screened and removed by colonoscopy or sigmoidoscopy (Dulal & Teku 2014). In the following, colonic or colorectal polyps, adenomas and adenocarcinomas are referred to collectively as colonic or colorectal tumors or neoplasia. The term “fusobacteria” refers to members of the genus Fusobacterium of Gram-negative anaerobic bacteria, species of which, including F.nucleatum, are found in the oral cavity, where they play a role in periodontal disease. Fusobacterium spp. are enriched in human colonic adenomas relative to surrounding tissues and in stool samples from colorectal adenoma and carcinoma patients compared to healthy subjects (Kostic et al 2013). Fusobacteria target colorectal adenocarcinoma cells by means of their Fap2 lectin, which specifically binds to the D-galactose-β(1-3)-N-acetyl-D-galactosamine (Gal-GalNAc) residues that are overexpressed in the surface carbohydrates of colorectal and certain other adenocarcinomas. Once in the tumor, fusobacteria can enhance cellular proliferation (Rubinstein et al 2013; Chen et al 2017; Yang et al 2017), create a tumor-favorable inflammatory environment (Kostic et al 2013) and protect the cancer cells against killing by natural killer cells and tumor-infiltrating T cells. F. nucleatum also promotes the resistance of CRC to chemotherapy (Yu et al 2017), and, because similar cellular mechanisms are involved, to radiotherapy. High fusobacterial abundance in CRC correlates with poor disease outcome (Flanagan et al 2014). At the same time, there has been a call for antibacterial agents targeted specifically to fusobacteria to avoid the side effects of systemically administered broad-spectrum antibiotics. Fusobacteria such as F. nucleatum are also present in bacterial biofilms associated with colonic tumors, particularly with tumors of the ascending colon. In these biofilms, bacteria of the Bacteroides and Prevotella genera often the most prominent, together with Enterobacteriaceae such as Escherichia coli, while Clostridium spp., actinobacteria and various bacilli, including the putatively protective lactobacilli, can also be found in addition to fusobacteria. The biofilm associated with colonic tumors is characteristically thick and continuous, invading the deeper mucous layer and lying in contact with colonic epithelial cells both over the tumor and in adjacent normal epithelium. Tissues underlying the biofilm show a decrease or alteration of the tumor suppressor E-cadherin, enhanced expression of the pro-inflammatory and angiogenic cytokine IL-6 and the proliferation-associated Ki-67 protein, as well as Stat3 activation. This points to a pro-carcinogenic effect of the biofilm (Dejea et al 2016). Among specific ways in which the bacteria present in the biofilm can initiate cancerous transformation is a direct mutagenic effect on DNA or interference with host DNA repair. This is the case for enterotoxigenic Bacteroides fragilis (ETBF), superoxide-producing Enterococcus faecalis, and colibactin-producing pks+ E. coli. Many of the biofilm bacteria can enhance Wnt-mediated signaling pathways or other specific pro-inflammatory pathways that are commonly mutated and / or overexpressed in CRC, as seen in the above-mentioned bacterial strains and F. nucleatum. There are also local immunological effects of the biofilm, as illustrated by the promotion of a tumorigenic Th17 response by ETBF that has been demonstrated in a mouse model (Chung et al 2018). These mechanisms promoting CRC initiation are also likely to play a similar role in CRC progression (Drewes et al 2016). Colonic biofilm and CRC tumors, especially tumors of the ascending colon, seem to enter into a symbiotic relationship, whereby each component can promote the formation and growth of the other in a vicious circle. A factor in this is an increased production of N1,N12-diacetylspermine, which originates from both the biofilm and the tumor (Johnson et al 2015). The inventors consider that the technical effect of the local administration of fosfomycin into the colonic lumen is due to the removal of pro-carcinogenic biofilm from the colonic mucosa, which will break the vicious circle, reduce colonic tumor size, and tend to normalize immune responses in and around the tumors, as well as reduce the initiation of recurrent tumors. Summary of the invention Accordingly, the present invention seeks to target the above-mentioned bacteria that promote the formation of colorectal tumors and eliminate tumor-associated bacterial biofilm by providing a pharmaceutical composition and a method for treating these conditions by the local administration of the compositions into the lumen of the bowel where the tumors and or biofilm are located, said invention comprising essentially the following items: A composition comprising the antibiotic fosfomycin as an active ingredient for use in the treatment of a colorectal tumor, wherein said use comprises reducing or eliminating fusobacteria and any biofilm in and around a colorectal tumor in a subject with said tumor, wherein the composition is to be administered locally into the bowel lumen and onto the site of said tumor and biofilm associated with it, and wherein the composition is to be administered at 1 to 30 days prior to removal of the colorectal tumor, and / or after removal of the colorectal tumor at one or more intervals of up to 3 months after its removal.The composition for use according to the previous item, which further comprises one or more additional antimicrobial or antibiotic agents as active ingredients. The composition for use according to the previous items, wherein the composition is to be administered at 1 to 30 days prior to removal of the colorectal tumor. The composition for use according to the previous items, wherein after removal of the colorectal tumor, the composition is to be administered at one or more intervals of up to 3 months after its removal. The composition for use according to the previous items, wherein the composition is to be administered at 1 to 30 days prior to removal of the colorectal tumor, and after removal of the colorectal tumor at one or more intervals of up to 3 months after its removal. The composition for use according to the previous items, wherein the one or more additional antimicrobial or antibiotic agents is / are chosen from a non-limiting list comprising metronidazole or a carbapenem such as ertapenem, or meropenem, or imipenem. The composition for use according to the previous items, wherein the additional antibiotic agent is metronidazole. The composition for use according to the previous items, wherein the active ingredients are formulated as a dry powder or granulate to be dissolved in an aqueous medium before delivery into the bowel lumen. The composition for according to any of the previous items, wherein said colorectal tumor is selected among an adenoma, a polyp and a cancer. The composition according to any of the previous items, wherein said composition is administered via a colonoscope. The colonoscope may be a two-channel colonoscope. The composition according to any of the previous items, wherein said composition is formulated to form a mucoadhesive gel on being delivered into the bowel lumen. The composition according to any of the previous items, wherein the concentration of fosfomycin delivered into the bowel lumen is in the range of 4 mg / g to 16 mg / g. The composition according to any of the previous items, wherein the concentration of metronidazole delivered into the bowel lumen is in the range of 1 mg / g to 4 mg / g. A kit of parts for treating a colorectal tumor, wherein said kit comprisesa. a first solution comprising the composition according to any of the previous items, andb. an aqueous second solution comprising a gelling agent. The kit according to the invention, wherein said kit comprisesc. instructions for administering the first and second solution in parallel via a two-channel colonoscope and mixing the first and second solution in the bowel lumen to form a mucoadhesive gel. The kit according to the invention, wherein said aqueous second solution comprises about 125 mg of low-acyl gellan gum. The kit according to the invention, wherein said first solution and said second solution each have a volume of about 50 mL. The kit according to the invention, wherein said mucoadhesive gel is about 100 mL containing about 8 mg / g fosfomycin and about 2 mg / g metronidazole. A method of treating a colorectal tumor in a subject by reducing or eliminating fusobacteria and any biofilm in and around said colorectal tumor and removal of the colorectal tumor, wherein said method comprises the steps of:a. Administering a composition comprising the antibiotic fosfomycin as an active ingredient to said subject having said colorectal tumor, wherein said composition is administered locally into the bowel lumen and onto the site of said colorectal tumor and biofilm associated with it,b removing said colorectal tumor,wherein said step a is performed 1 to 30 days prior to said step b, and / or at one or more intervals of up to 3 months after step b. In the following detailed description of the invention, details of the scope of the invention and its practical performance will be given.  Detailed description of the invention The purpose of the present invention is to provide a means of reducing or eliminating cancer-promoting fusobacteria in and around colorectal tumors as well as the bacterial biofilm associated with these tumors. This is achieved by the direct local administration of a solution of appropriate antibiotics into the bowel lumen at a level which ensures the application of the solution to the site of the tumor and / or biofilm. The colonization of the tumors by fusobacteria is associated with increased tumor growth and resistance to both endogenous antitumor immune mechanisms and exogenous antitumor therapies. Some of the mechanisms by which these effects are achieved have been elucidated. It is therefore proposed that the elimination or drastic reduction of fusobacteria colonization of these tumors will reverse these adverse effects and in the case of cancers increase the efficacy of other anticancer therapies. Eliminating the bacterial biofilm associated with these tumors (i.e. covering the tumor and adjacent areas of normal colonic epithelium) will also exert these effects, as well as removing the pro-carcinogenic action of the biofilm that may promote tumor recurrence and tumor growth. Local administration of the antibiotics via the bowel lumen, typically through application via a colonoscope, will result in a very high antibiotic concentration at the luminal surface of the tumor and the biofilm. The antibiotics will penetrate into the tumor cells where the fusobacteria are located and into the overlying and adjacent biofilm to achieve bactericidal concentrations, while at the same time being only poorly absorbed into the blood, so that any systemic side effects of the antibiotics are substantially reduced. This treatment can be applied both before and after removal of the tumor to inhibit the pro-carcinogenic action of any biofilm and hence inhibit tumor recurrence. Quantitative polymerase chain reaction analysis of F. nucleatum DNA in cancerous colorectal tumors demonstrates that the bacteria are concentrated in the superficial, luminal portion of the tumors (Yamamura et al 2017). Antibacterial treatment by applying high concentrations of antibiotic from the luminal side should be particularly effective in eliminating the bulk of the bacterial load at the same time minimizing side effects associated with conventional systemic administration of the broad-spectrum antibiotic. The same consideration applies to the elimination of the overlying and adjacent bacterial biofilm. Active ingredients of the compositions of the invention Compositions according to the present invention comprise essentially the antibiotic fosfomycin, either alone or in combination with one or more additional antibiotics, which may be chosen from a non-limiting list comprising metronidazole, and carbapenems such as ertapenem, meropenem and imipenem. FosfomycinFosfomycin is the international non-proprietary name of a broad-spectrum antibiotic isolated and characterized in 1969 from Streptomyces fradiaestrains under the name phosphomycin or phosphonomycin (Hendlin et al 1969). Its structure was determined to be (-)(IR, 2S)-1,2-epoxypropylphosphonic acid (Christensen et al 1969), with the systematic (IUPAC) name [(2R,3S)-3-methyloxiran-2-yl]phosphonic acid and a formula weight of 138.1 Da. Fosfomycin is bactericidal and inhibits bacterial cell wall biosynthesis by inactivating the enzyme UDP-N-acetylglucosamine-3-enolpyruvyltransferase, also known as MurA (Brown et al 1995). This enzyme catalyzes the committed step in peptidoglycan biosynthesis, the ligation of phosphoenolpyruvate to the 3'-hydroxyl group of UDP-N-acetylglucosamine to form N-acetylmuramic acid. Fosfomycin is a phosphoenolpyruvate analogue that inhibits MurA by alkylating an active site cysteine residue. The antibiotic enters the bacterial cell via the glycerophosphate transporter. Given this mechanism of action, fosfomycin has a broad bactericidal spectrum, being active against aerobic genera such as Staphylococcus, Streptococcus, Enterococcus, Neisseria, Escherichia, Proteus (indole-negative), Serratia, Salmonella, Shigella, Pseudomonas, Haemophilus, and Vibrio, less active against indole-positive Proteus spp., Klebsiella and Enterobacter spp. Of particular relevance to the present invention, it is active against the anaerobic genus Fusobacterium, as well as the anaerobic genera Peptostreptococcus (including Peptoniphilus, Finegoldiaand Anaerococcus). However, it is inactive against the anaerobic Bacteroides fragilis group of bacteria. There is a low prevalence of bacterial resistance to fosfomycin in the community, and studies of the prevalence of resistant bacteria after the introduction of fosfomycin have shown either no increase or only a modest increase in the prevalence of resistant organisms. However, prolonged exposure to the antibiotic may enable bacteria to evolve resistance by selection of mutants that lack the glycerophosphate transporter pathway. Alternative mechanisms of resistance involve the loss of the inducible hexose phosphate transporter, a Cys-Asp mutation in MurA, or acquistion of plasmids coding for the fosfomycin inactivating enzymes fosA and fosB (in addition to the chromosomal fosX in Listeria monocytogenes). The mutant strains may, however, also show reduced pathogenicity (Karageorgopoulos et al 2012). This may explain why the emergence of bacterial resistance is seen on prolonged exposure in vitro, but much less frequently in vivo. The appearance of resistant bacterial strains in controlled clinical trials of orally or intravenously administered fosfomycin has been 3.0% overall, with a maximum of 15% for Pseudomonas aeruginosa. In general, fosfomycin is seen to be a valuable addition to the therapeutic armament against multidrug-resistant organisms. Fosfomycin has been shown to have the capacity to favor phagocytosis and act as an immunomodulator. It is accumulated by polymorphonuclear leukocytes to reach concentrations that are up to twice those of the extracellular fluid, but does not affect their cellular functions, while still exerting a bactericidal effect on Staphylococcus aureus(Höger et al 1985). With respect to its uptake into intestinal mucosal cells, Martínez et al (2013) have demonstrated the rapid uptake of fosfomycin into IPEC-J2 cells exposed to fosfomycin calcium at 580 µg / mL, to reach an intracellular concentration approaching 30 µg / mL within 15 minutes. Such high concentrations of fosfomycin are difficult to reach with standard systemic route of administration. Therefore, much higher concentrations of fosfomycin are required to kill fusobacteria in colorectal cancer cells. In accordance with the invention such concentrations are readily reached with locally administered fosfomycin solution. Fosfomycin is known to penetrate readily through tissue barriers and into bacterial biofilm (Reffert & Smith 2014), so that high, bactericidal concentrations of fosfomycin will result from the direct application of fosfomycin solution to bacterial biofilm covering and adjacent to the colorectal tumors. Fosfomycin inhibits the adhesion to epithelial cells of various bacterial species, including those involved in biofilm formation (Gobernado 2003). Fosfomycin can also break up biofilms to enhance the permeability of other antibiotics. The chief adverse effects of fosfomycin are gastric irritation from orally administered fosfomycin disodium, evidence of allergy in the form of transient rashes (0.3% of cases) and eosinophilia (0.2%), as well as transiently raised liver enzymes (0.3% of cases) from systemically administered fosfomycin (Gobernado 2003). In general, however, fosfomycin displays remarkably low toxicity, so that when high doses of fosfomycin disodium are given systemically, it is the sodium load that is the dose-limiting factor. Fosfomycin shows a considerable synergism in bactericidal effect on a large number of strains of organisms from the susceptible genera mentioned, when used in combination with a large number of antibiotics of the penicillin, cephalosporin, aminoglycoside, macrolide and lincosamide types. While early studies showed a synergistic effect on about 70-100% of tested strains for various antibiotic combinations, subsequent more extensive studies showed synergy rates of 36-74%. The remaining strains showed merely additive effects and an inhibitory effect was only seen in one or two individual antibiotic combinations on an individual bacterial strain (Gobernado 2003). The fact that fosfomycin shows synergy with many individual antibiotics and indeed abrogates the toxicity of many other antibiotics, including the nephrotoxicity and ototoxicity of the aminoglycosides, favors the use of fosfomycin in combination with other antibiotics to produce a potent bactericidal action and compensate for any development of fosfomycin resistance during more prolonged treatment. The principal forms of fosfomycin that come within the scope of this invention are:i) Fosfomycin disodium, formula weight 182.0 Da, pH of 5% solution 9.0-10.5. This salt is hygroscopic, highly soluble in water and shows a high bioavailability, but is locally irritant if un-neutralized.ii) Fosfomycin calcium monohydrate, formula weight 194.1 Da, pH of 0.4% solution 8.1-9.6. This salt is sparingly soluble in water and not hygroscopic, but is less irritating to the stomach when used for oral treatment, when its bioavailability in terms of entering the systemic circulation may be as low as 12% (Bergan 1990).iii) Fosfomycin trometamol, formula weight 259.2 Da, pH of 5% solution 3.5-5.5. This salt is highly soluble in water and is well tolerated when given orally, when it shows a bioavailability of about 40%. When the name “fosfomycin” is used herein, it refers to an inorganic or organic salt of fosfomycin as exemplified by the principal forms above, and the dose of fosfomycin refers to the amount of the free acid form of fosfomycin present in the salt. Fosfomycin trometamol shows long-term stability as a readily soluble granulate when stored in sealed sachets at room temperature. Fosfomycin disodium is presented as a freeze-dried powder in a sealed vial because of its marked hygroscopic property. In this presentation it shows long-term stability when stored at 2-8°C. Either form of fosfomycin can be used in the present compositions. Compositions according to the present invention may comprise fosfomycin such that single doses are in the range of 400 milligram to 4 gram. MetronidazoleThis semi-synthetic antibiotic, which is active against a number of anaerobic bacteria including bacteria of the B. fragilis group and Fusobacterium spp., is well known to prior art. It has been used systemically in combination with systemic fosfomycin for prophylaxis against wound infection after abdominal surgery. The intracellular penetration of metronidazole appears to be by rapid passive diffusion, so that intracellular levels approach equilibrium with extracellular levels within 15 minutes (Hand et al 1987). Compositions according to the present invention may comprise metronidazole such that single doses are in the range of 100 milligram to 1 gram. CarbapenemsThe carbapenems are members of the beta-lactam class of antibiotics, which, like the penicillins and cephalosporins, exert a bactericidal action by inhibiting bacterial cell wall synthesis by a different mechanism than that of fosfomycin. Hence the beta-lactam antibiotics, including the carbapenems, characteristically show a synergic bactericidal action with fosfomycin on most bacterial species against which both types of antibiotic are active. However, the carbapenems exhibit a broader spectrum of activity than most penicillins and cephalosporins, being active against anaerobic bacteria such as the B. fragilis group, Prevotella spp. and Fusobacterium spp. They can hence be used in place of metronidazole to exert a bactericidal action on the anaerobic bacteria that are largely insensitive to fosfomycin. Regarding the intracellular penetration of the carbapenems, bactericidal intracellular concentrations of ertapenem in a mouse macrophage cell line are achieved at extracellular concentrations as low as 15 µg / mL (Tang et al 2012), and the cellular to extracellular concentration ratios of meropenem in human macrophages were always high (range 3-12) at extracellular concentrations ranging from 0.125 to 1 µg / mL (Cuffini et al 1993). Compositions according to the present invention may comprise ertapenem, or meropenem or imipenem such that single doses are in the range of 100 milligram to 1 gram. Medical indications The primary indication for the use of the compositions described is the presence of one or more colorectal tumors arising from the colonic or rectal mucosa and / or the presence of bacterial biofilm covering the colonic or rectal mucosa whether or not the biofilm is associated with an adenocarcinoma, an adenoma or polyp or mucosal dysplasia. The indication will typically be diagnosed in relation to colonoscopy. Treatment with the compositions described can typically be administered through the endoscope during the same session or at a later procedure by the intraluminal instillation of a gel-forming solution of the composition into the region of the bowel where the mucosal pathology is observed. The initial treatment can be followed up by further treatments at the discretion of the treating clinician. An indication for treatment may also be provided detecting urinary, fecal, blood or tissue markers of pro-carcinogenic biofilm or driver organisms. Currently, such markers could be the presence of colibactin-producing E. coli, enterotoxigenic B. fragilis,F. nucleatumand Porphyromonas gingivalis, as well as other microbes, especially from the oral microbiota. Many markers are under investigation, but none has yet shown a diagnostic sensitivity or specificity quite high enough to be independently diagnostic of either CRC or colonic biofilm, so that they currently have a screening function to select subjects for colonoscopy. Among markers with a promising diagnostic sensitivity and specificity is urinary N1,N12-diacetylspermine, which, as stated above, is produced in increased amounts by CRC-associated bacterial biofilm. If it is known or suspected that a colorectal carcinoma has penetrated through the bowel wall or has metastasized to lymph nodes or remote tissues, the treating clinician may decide to supplement the local antibiotic treatment with systemic antibiotic treatment, as live cancer-promoting microorganisms may be found in metastases of colorectal carcinomas. Tumors and biofilm present in the rectum and sigmoid colon, because of their location, offer the opportunity for treatment with the compositions made up as retention enemas. In certain circumstances, larger volumes of enema containing the compositions can be used to treat regions of the colon more remote from the rectum. The use of the compositions described may also be indicated during neoadjuvant radiotherapy or chemotherapy of a colorectal cancer, e.g. with bevacizumab and / or cytotoxic drugs. Formulations In a preferred embodiment, the composition comprises fosfomycin disodium, such as fosfomycin disodium formulated for intravenous injection, supplied as freeze-dried powder in a capped glass vial. to be dissolved in a supplied pharmaceutically acceptable aqueous carrier or diluent. The carrier may be simply water or water containing pharmaceutically acceptable auxiliary substances or adjuvants, including, without limitation, buffering agents and / or tonicity adjusting agents to ensure a neutral pH and isotonicity of the fosfomycin solution, and / or one or more gelling agents to form a mucoadhesive gel on delivery into the bowel lumen. In another embodiment, the composition comprises fosfomycin trometamol supplied as a dry granulate which is similarly dissolved in a pharmaceutically acceptable aqueous carrier or diluent as described. In a further embodiment, the composition comprises fosfomycin and an additional antibiotic, such as metronidazole, or a carbapenem such as ertapenem, meropenem or imipenem, metronidazole being supplied as a stable aqueous solution for intravenous injection, and the carbapenems being supplied as a dry powder or granulate to be dissolved in the aqueous carrier. The pH value of the solutions resulting from the formulations according to the present invention may have a pH of between 4 and 8, preferably between 6.5 and 7.5 and most preferably between 7 and 7.5. The antibiotic powders or granulates and the aqueous diluents supplied may individually contain buffering agents and / or tonicity adjusting agents to ensure a neutral pH and isotonicity of the combined antibiotic solution. The solutions may also contain one or more approved biocompatible gelling or thickening agents that do not bind the active ingredients of the composition, such as hydroxypropyl methylcellulose or a large number of other biocompatible thickening agents known to the art, in order to achieve a solution of optimal viscosity for controlled delivery and adhesion to the colonic mucosa. In one embodiment, an aqueous solution is supplied which contains 0.25% weight / weight of a low-acyl gellan gum, such as Kelcogel CG-LA (CAS registration number 71010-52-1), while the antibiotics are dissolved in an equal volume of a solution containing 0.25% weight / weight of calcium chloride. When the two solutions are delivered in parallel and sprayed and mixed in the bowel lumen by means of a two-channel colonoscope, they form a mucoadhesive gel which prolongs the contact of the compositions with the bowel wall and any tumor or bacterial biofilm that is present. In one embodiment, the compositions according to the present invention may be pre-packaged as a kit of parts, wherein said kit comprises fosfomycin and metronidazole, for example in single dose units, to be dissolved or added to an aqueous solution which is also supplied pre-packaged in single dose vials, an aqueous solution containing gelling agent, also supplied pre-packaged in a single dose vial, and instructions for use according to the previous description. Administration The compositions of the present invention may be applied by administration methods conventionally used in the art for local administration in the lumen of the bowel in the region of a mucosal colorectal tumor, be it an adenocarcinoma, adenoma or region of mucosal dysplasia, or in the region of bacterial biofilm located in the colon or rectum. The preferred method of administration is by means of a colonoscope with two channels for spraying liquids into the bowel lumen, the liquids being mixed by the spraying process. The methods of administration entail that the colon and rectum have been cleansed of fecal material by means of a standard regimen of bowel preparation for colonoscopy or surgery, such as the drinking or oral administration of large volumes of liquid containing an appropriate concentration of polyethylene glycol, such as macrogol 3350 or 4000, with or without electrolytes. If the tumor or biofilm is located in the rectum or lower part of the sigmoid colon, a solution of the composition can be given as a retention enema into the bowel lumen at the level of the tumor. In certain circumstances, the composition may be given in a larger volume of enema for the treatment of pathology in more proximal parts of the colon. For tumors and biofilm located in more proximal parts of the colon, the solutions of the composition are administered at the level of the pathology during colonoscopy. If, in exceptional circumstances, a rectal or sigmoid tumor is to be treated when no bowel preparation has been carried out, the administration of a solution of the composition can be effected after first emptying the bowel by administering a laxative and then giving a colonic enema to clean the bowel; For tumors in the rectum and lower sigmoid colon, the volume of the retention enema is 50 mL to100 mL. The patient is instructed to lie down lie down for 5 minutes on each side, back and front, and to retain the enema for at least 15 minutes, if possible. When the tumor is located in more proximal parts of the colon, larger-volume enemas can be given, but the solutions of the composition should preferably be sprayed into the tumor area during colonoscopy, as stated above. Dosage While it is not intended that the invention should be prescriptive for the clinician’s choice of suitable dosages for the individual case, it is suggested that the composition be formulated such that a single unit dose of fosfomycin in the present compositions is in the range of 400 milligram to 4 gram. The single unit dose of metronidazole or a carbapenem when used as an additional antibiotic in these compositions may be in the range of 100 milligram to 1 gram. These doses may be exceeded at the discretion of the physician, for example if a large volume of enema is to be given. A typical single dose may, by the way of an example, consist of 800 mg of fosfomycin dissolved in 50 mL of aqueous diluent, i.e. constituting an approximately 1.6% weight / weight solution. If metronidazole or a carbapenem is required as an additional antibiotic, it may be added as, for example, 200 milligram in the same diluent, i.e. constituting an approximately 0.4% weight / weight solution. For mucoadhesive gel formation, the antibiotic solution contains 0.25% weight / weight of calcium chloride. A second aqueous diluent, also 50 mL in volume, contains 125 mg (0.25% weight / weight) of a low-acyl gellan gum. When the two solutions are mixed by spraying at the distal end of the two-channel colonoscope, 100 mL of mucoadhesive gel are formed, containing 8 mg / mL fosfomycin and 2 mg / mL metronidazole. This formulation of a typical single dose thus consists of two solutions of volume 50 mL each, the first containing 800 mg fosfomycin, 200 mg metronidazole and 125 mg calcium chloride, while the second solution contains 125 mg of low-acyl gellan gum. The above single dose may be augmented proportionately in dosage and volume, maintaining the same concentrations of fosfomycin and, if present, an above-mentioned additional antibiotic, to a maximum volume of 200 mL of diluent containing 4 gram of fosfomycin, and, if present, 1 gram of said additional antibiotic. If a larger volume of solution is required for a single dose, this can be prepared by diluting a single dose as described above with isotonic saline, without increasing the amount of fosfomycin to be administered above 4 gram, or the amount of additional antibiotic to be administered above 1 gram. Alternatively, the dosage of the antibiotics can be increased at the discretion of the physician, up to a maximum of 20 g of fosfomycin in one liter in any one day, while ensuring that the solution is isotonic. A dose can preferably be administered once a day, or if the treating clinician considers it necessary and practicable, twice a day such that a patient receives two typical single doses per day on two separate occasions, or even three times a day such that a patient receives three typical single doses per day on three separate occasions. Duration of the above administration regime will typically be short, ranging from 1 day to 2 days, but if the treating clinician considers an extension of treatment necessary to achieve the desired result, the dosage regime can be extended to 14 days, such as in the range of 3 days to 5 days, for example 4 days, or in the range of 5 to 7 days, for example 6 days, or in the range of 7 days to 14 days. Treatment can also be given for 1 or 2 days, followed by a pause and then repeated after one week or a fortnight. Compositions according to the present invention may comprise fosfomycin such that single doses are usually in the range of 400 milligram to 4 gram dissolved in a volume of aqueous diluent in the range of 20 mL to 200 mL.  Preferably, a single dose may contain approximately 2% weight / weight of total active ingredients in the final mixed gel or solution in the bowel lumen. Preferably, a single dose is present in isotonic solution. In some embodiments, the compositions of the invention are for administration once or several times, such as twice, three times or four times, after which the treatment is paused and only reinitiated as the attending physician requires. Methods of treatment The present invention presents methods to reduce or eliminate fusobacteria and other bacterial promoters of colorectal cancer from colorectal adenomas, polyps and adenocarcinomas, as well as from the associated bacterial biofilm, by local administration into the bowel lumen. ExamplePreoperative endoscopic treatment with fosfomycin and metronidazole in patients with right-sided colon adenoma Patients and methodsTwelve patients (6 males, 6 females, median age 71 years) with right-sided colon adenomas from two hospital centers were studied. The patients underwent an intervention colonoscopy, where pre-intervention biopsies of the healthy mucosal lining and blood samples were collected, followed by a spraying from cecum through the hepatic flexure with 100 ml gel containing 800 mg fosfomycin and 200mg metronidazole. Endoscopic mucosal resection (EMR) was performed at least five days after the intervention: post-intervention blood samples were collected and post-intervention biopsies were collected again from the healthy mucosal lining prior to the EMR procedure. The post-intervention adenoma tissue from included patients was received after routine pathological evaluation. Patients were matched 1:2 with retrospective controls in order to obtain adenoma tissue without antibiotic-exposure. Adverse events at 14 days and 12 months after the EMR were registered through interviews and entries in the patient files and graded by CTCAE version 5 to evaluate the safety of the intervention. ResultsThe patients had a significantly lower percentage of bacterial biomass after the intervention (3.0e-04% versus 9.8e-05%, p 0.025) adherent to the healthy mucosal lining, while there was no significant reduction in the percentage of bacterial biomass between colon adenomas from patients who received the intervention and the controls (8.6e-03% versus 1.1e-02%, p 0.368). There was a significant increase in the mucosal CD68+ macrophage density and the CD3+ tumor-infiltrating lymphocytes in the colon adenomas that had received the intervention (2.2 versus 3.4, p 0.030, and 524.2 versus 727.4, p 0.018, respectively) compared with the controls. No significant difference was found between CD8+ cytotoxic tumor infiltrating lymphocytes. No patients had sudden unexpected serious adverse reactions (SUSARs) or serious adverse reaction (SAR). One patient had a serious adverse event (SAE), when the patient developed fever and was readmitted after the EMR. This SAE was however deemed not related to the study drugs. All patients reported adverse events 14 days after the EMR, the most common being looser stools (10 patients). ConclusionsPreoperative local treatment with fosfomycin and metronidazole decreased the bacterial biomass adherent to the colonic mucosal lining. The intervention was considered safe.  References Bergan T (1990) Degree of absorption, pharmacokinetics of fosfomycin trometamol and duration of urinary antibacterial activity. Infection 18 Suppl 2:S65-S69. Brown ED, Vivas EI, Walsh CT, Kolter R (1995) MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli. J Bacteriol 177:4194-4197. Chen Y, Peng Y, Yu J, Chen T, Wu Y, Shi L, Li Q, Wu J, Fu X (2017). Invasive Fusobacterium nucleatum activates beta-catenin signaling in colorectal cancer via a TLR4 / P-PAK1 cascade. Oncotarget 8:31802-31814. Christensen BG, Leanza WJ, Beattie TR, Patchett AA, Arison BH, Ormond RE, Kuehl FA Jr, Albers-Schonberg G, Jardetzky O (1969) Phosphonomycin: structure and synthesis. Science 166:123-125. Chung L, Thiele Orberg E, Geis AL, Chan JL, Fu K, DeStefano Shields CE, Dejea CM, Fathi P, Chen J, Finard BB, Tam AJ, McAllister F, Fan H, Wu X, Ganguly S, Lebid A, Metz P, Van Meerbeke SW, Huso DL, Wick EC, Pardoll DM, Wan F, Wu S, Sears CL, Housseau F (2018) Bacteroides fragilis toxin coordinates a pro-carcinogenic inflammatory cascade via targeting of colonic epithelial cells. Cell Host Microbe 23:203-214. Dejea CM, Sears CL (2016) Do biofilms confer a pro-carcinogenic state? Gut Microbes 7:54-57. Drewes JL, Housseau F, Sears CL (2016) Sporadic colorectal cancer: microbial contributors to disease prevention, development and therapy. Br J Cancer 115:273-280. Dulal S, Teku TO (2014) Gut microbiome and colorectal adenomas. Cancer J 20:225–231. Flanagan L, Schmid J, Ebert M, Soucek P, Kunicka T, Liska V, Bruha J, Neary P, Dezeeuw N, Tommasino M, Jenab M, Prehn JH, Hughes DJ (2014) Fusobacterium nucleatum associates with stages of colorectal neoplasia development, colorectal cancer and disease outcome. Eur J Clin Microbiol Infect Dis 33:1381-1390. Gobernado M (2003) Fosfomycin. Rev Esp Quimioter 16:15-40. Hand WL, King-Thompson N, Holman JW (1987) Entry of roxithromycin (RU 965), imipenem, cefotaxime, trimethoprim, and metronidazole into human polymorphonuclear leukocytes. Antimicrob Agents Chemother 31:1553-1557. Hendlin D, Stapley EO, Jackson M, Wallick H, Miller AK, Wolf FJ, Miller TW, Chaiet L, Kahan FM, Foltz EL, Woodruff HB, Mata JM, Hernandez S, Mochales S (1969) Phosphonomycin, a new antibiotic produced by strains of streptomyces. Science 166:122-123. Höger PH, Seger RA, Schaad UB, Hitzig WH (1985) Chronic granulomatous disease: uptake and intracellular activity of fosfomycin in granulocytes. Pediatr Res 19:38-44. Johnson CH, Dejea CM, Edler D, Hoang LT, Santidrian AF, Felding BH, Ivanisevic J, Cho K, Wick EC, Hechenbleikner EM, Uritboonthai W, Goetz L, Casero RA Jr, Pardoll D8, White JR, Patti GJ, Sears CL, Siuzdak G (2015) Metabolism links bacterial biofilms and colon carcinogenesis. Cell Metab 21:891-897. Karageorgopoulos DE, Wang R, Yu XH, Falagas ME (2012) Fosfomycin: evaluation of the published evidence on the emergence of antimicrobial resistance in Gram-negative pathogens. J Antimicrob Chemother 67:255-268. Kostic AD, Chun E, Robertson L, Glickman JN, Gallini CA, Michaud M, Clancy TE, Chung DC, Lochhead P, Hold GL, El-Omar EM, Brenner D, Fuchs CS, Meyerson M, Garrett WS (2013) Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell Host Microbe 14:207-215. Martínez G, Pérez DS, Soraci AL, Tapia MO (2013) Penetration of fosfomycin into IPEC-J2 cells in the presence or absence of deoxynivalenol. PLoS One 8(9):e75068. Reffert JL, Smith WJ (2014) Fosfomycin for the treatment of resistant gram-negative bacterial infections. Insights from the Society of Infectious Diseases Pharmacists. Pharmacotherapy 34:845-857. Rubinstein MR, Wang X, Liu W, Hao Y, Cai G, Han YW (2013) Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin / β-catenin signaling via its FadA adhesin. Cell Host Microbe 14:195-206. Yamamura K, Baba Y, Miyake K, Nakamura K, Shigaki H, Mima K, Kurashige J, Ishimoto T, Iwatsuki M, Sakamoto Y, Yamashita Y, Yoshida N, Watanabe M, Baba H (2017) Fusobacterium nucleatum in gastroenterological cancer: Evaluation of measurement methods using quantitative polymerase chain reaction and a literature review. Oncol Lett 14:6373-6378. Yang Y, Weng W, Peng J, Hong L, Yang L, Toiyama Y, Gao R, Liu M, Yin M, Pan C, Li H, Guo B, Zhu Q, Wei Q, Moyer MP, Wang P, Cai S, Goel A, Qin H, Ma Y (2017) Fusobacterium nucleatum increases proliferation of colorectal cancer cells and tumor development in mice by activating Toll-like receptor 4 signaling to nuclear factor-κB, and up-regulating expression of microRNA-21. Gastroenterology 152:851-866. Yu T, Guo F, Yu Y, Sun T, Ma D, Han J, Qian Y, Kryczek I, Sun D, Nagarsheth N, Chen Y, Chen H, Hong J, Zou W, Fang JY (2017) Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy. Cell 170:548-563.    Claims 1. A composition comprising the antibiotic fosfomycin as an active ingredient for use in the treatment of a colorectal tumor, wherein said use comprises reducing or eliminating fusobacteria and any biofilm in and around a colorectal tumor in a subject with said tumor, wherein the composition is to be administered locally into the bowel lumen and onto the site of said tumor and biofilm associated with it, and wherein the composition is to be administered at 1 to 30 days prior to removal of the colorectal tumor, and / or after removal of the colorectal tumor at one or more intervals of up to 3 months after its removal. 2. The composition for use according to the previous claim, which further comprises one or more additional antimicrobial or antibiotic agents as active ingredients. 3. The composition for use according to the previous claim, wherein the one or more additional antimicrobial or antibiotic agents is / are chosen from a non-limiting list comprising metronidazole or a carbapenem such as ertapenem, or meropenem, or imipenem. 4 The composition for use according to the previous claim, wherein the additional antibiotic agent is metronidazole. 5. The composition for use according to any of the previous claims, wherein the active ingredients are formulated as a dry powder or granulate to be dissolved in an aqueous medium before delivery into the bowel lumen. 6. The composition for use according to any of the previous claims, wherein said colorectal tumor is selected among an adenoma, a polyp, an adenocarcinoma and a colorectal cancer tumor. 7. The composition according to any of the previous claims, wherein said composition is administered via a colonoscope. 8. The composition according to any of the previous claims, wherein said composition is formulated to form a mucoadhesive gel on being delivered into the bowel lumen. 9. The composition according to any of the previous claims, wherein the concentration of fosfomycin delivered into the bowel lumen is in the range of 4 mg / g to 16 mg / g. 10. The composition according to any of the previous claims, wherein the concentration of metronidazole delivered into the bowel lumen is in the range of 1 mg / g to 4 mg / g. 11. The composition according to any of the previous claims, wherein said composition comprises about 800 mg fosfomycin, about 200 mg metronidazole and about 125 mg calcium chloride, and wherein said composition is formulated in a first solution of about 50 mL, and wherein said treatment comprises a step of delivering said first solution in parallel with a second solution via a two-channel colonoscope, wherein said second solution comprises about 125 mg of low-acyl gellan gum, and wherein said first and second solution are to be mixed in the bowel lumen to form about 100 mL of mucoadhesive gel comprising about 8 mg / g fosfomycin and about 2 mg / g metronidazole. 12. A kit of parts for treating a colorectal tumor, wherein said kit comprisesa. a first solution comprising the composition according to any of the previous claims, andb. an aqueous second solution comprising a gelling agent. 13. The kit according to claim 12, wherein said kit comprisesc. instructions for administering the first and second solution in parallel via a two-channel colonoscope and mixing the first and second solution in the bowel lumen to form a mucoadhesive gel. 14. The kit according to claim 12-13, wherein said aqueous second solution comprises about 125 mg of low-acyl gellan gum. 15. The kit according to claim 12-14, wherein said first solution and said second solution each have a volume of about 50 mL. 16. The kit according to claim 13-15, wherein said mucoadhesive gel is about 100 mL containing about 8 mg / g fosfomycin and about 2 mg / g metronidazole. 17. A method of treating a colorectal tumor in a subject by reducing or eliminating fusobacteria and any biofilm in and around said colorectal tumor and removal of the colorectal tumor, wherein said method comprises the steps of:a. Administering a composition comprising the antibiotic fosfomycin as an active ingredient to said subject having said colorectal tumor, wherein said composition is administered locally into the bowel lumen and onto the site of said colorectal tumor and biofilm associated with it,b. removing said colorectal tumor,wherein said step a is performed 1 to 30 days prior to said step b, and / or at one or more intervals of up to 3 months after step b.     AbstractThe present invention provides compositions comprising fosfomycin for reducing or eliminating bacterial promoters of colorectal cancer from colorectal adenomas, polyps and adenocarcinomas, as well as from the associated bacterial biofilm, by local administration into the bowel lumen. 

Claims

1. A composition comprising the antibiotic fosfomycin as an active ingredient for use in the treatment of a colorectal tumor, wherein said use comprises reducing or eliminating fusobacteria and any biofilm in and around a colorectal tumor in a subject with said tumor, wherein the composition is to be administered locally into the bowel lumen and onto the site of said tumor and biofilm associated with it, and wherein the composition is to be administered at 1 to 30 days prior to removal of the colorectal tumor, and / or after removal of the colorectal tumor at one or more intervals of up to 3 months after its removal. 2. The composition for use according to the previous claim, which further comprises one or more additional antimicrobial or antibiotic agents as active ingredients. 3. The composition for use according to the previous claim, wherein the one or more additional antimicrobial or antibiotic agents is / are chosen from a non-limiting list comprising metronidazole or a carbapenem such as ertapenem, or meropenem, or imipenem. 4 The composition for use according to the previous claim, wherein the additional antibiotic agent is metronidazole. 5. The composition for use according to any of the previous claims, wherein the active ingredients are formulated as a dry powder or granulate to be dissolved in an aqueous medium before delivery into the bowel lumen. 6. The composition for use according to any of the previous claims, wherein said colorectal tumor is selected among an adenoma, a polyp, an adenocarcinoma and a colorectal cancer tumor. 7. The composition according to any of the previous claims, wherein said composition is administered via a colonoscope. 8. The composition according to any of the previous claims, wherein said composition is formulated to form a mucoadhesive gel on being delivered into the bowel lumen. 9. The composition according to any of the previous claims, wherein the concentration of fosfomycin delivered into the bowel lumen is in the range of 4 mg / g to 16 mg / g. 10. The composition according to any of the previous claims, wherein the concentration of metronidazole delivered into the bowel lumen is in the range of 1 mg / g to 4 mg / g. 11. The composition according to any of the previous claims, wherein said composition comprises about 800 mg fosfomycin, about 200 mg metronidazole and about 125 mg calcium chloride, and wherein said composition is formulated in a first solution of about 50 mL, and wherein said treatment comprises a step of delivering said first solution in parallel with a second solution via a two-channel colonoscope, wherein said second solution comprises about 125 mg of low-acyl gellan gum, and wherein said first and second solution are to be mixed in the bowel lumen to form about 100 mL of mucoadhesive gel comprising about 8 mg / g fosfomycin and about 2 mg / g metronidazole.  12. A kit of parts for treating a colorectal tumor, wherein said kit comprisesa. a first solution comprising the composition according to any of the previous claims, andb. an aqueous second solution comprising a gelling agent.  13. The kit according to claim 12, wherein said kit comprises c. instructions for administering the first and second solution in parallel via a two-channel colonoscope and mixing the first and second solution in the bowel lumen to form a mucoadhesive gel.  14. The kit according to claim 12-13, wherein said aqueous second solution comprises about 125 mg of low-acyl gellan gum. 15. The kit according to claim 12-14, wherein said first solution and said second solution each have a volume of about 50 mL.  16. The kit according to claim 13-15, wherein said mucoadhesive gel is about 100 mL containing about 8 mg / g fosfomycin and about 2 mg / g metronidazole. 17. A method of treating a colorectal tumor in a subject by reducing or eliminating fusobacteria and any biofilm in and around said colorectal tumor and removal of the colorectal tumor, wherein said method comprises the steps of:a. Administering a composition comprising the antibiotic fosfomycin as an active ingredient to said subject having said colorectal tumor, wherein said composition is administered locally into the bowel lumen and onto the site of said colorectal tumor and biofilm associated with it,b. removing said colorectal tumor, wherein said step a is performed 1 to 30 days prior to said step b, and / or at one or more intervals of up to 3 months after step b.