Compositions for the treatment of colonic neoplasia by intraluminal administration of antibiotics

Abstract

The present invention provides compositions comprising fosfomycin for reducing or eliminating bacteria that promote the development of colorectal cancer in colorectal adenomas, polyps, and adenocarcinomas, and associated bacterial biofilms, by topical administration into the intestinal lumen.

Description

Technical Field

[0001] This invention provides a composition comprising fosfomycin and other antibiotics for local application to the colonic mucosa from the luminal side to eliminate or reduce the presence of bacteria that promote the formation of adenomatous-adenocarcinoma sequences in colon tumors. These bacteria include Fusobacterium, colibacin-producing (polyketide synthase-positive) Escherichia coli (PKS+ Escherichia coli), enterotoxin-producing Bacteroides fragilis (ETBF), and other microorganisms present in bacterial biofilms associated with the initiation and progression of carcinogenic sequences. The object of this invention is to reduce the pro-cancer effects attributed to biofilms, thereby reducing the initiation and growth of colonic adenomas and their potential transformation into malignant adenocarcinomas. Therefore, this invention relates to the fields of oncology and gastroenterology, as well as other fields relating to the prevention and treatment of colorectal tumor formation. Background Technology

[0002] Colorectal cancer (CRC) develops in stages, beginning with mucosal dysplasia and aberrant crypt lesions, leading to the development of adenomatous polyps, which are considered precursors to CRC. Colonic polyps are typically classified according to their tendency to progress to malignancy (adenomatous vs. hyperplastic) and their structure (sessile, pedunculated, or flattened), shape (tubular, villous, serrated), and size (small 1mm to 5mm, medium 5mm to 10mm, large ≥10mm). Hyperplastic polyps are usually small, located in the rectum and sigmoid colon, and have little malignant potential except for certain subtypes of serrated hyperplastic polyps. Adenomatous polyps or adenomas account for approximately 70% of colonic 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 text, colonic or colorectal polyps, adenomas, and adenocarcinomas are collectively referred to as colonic or colorectal tumors or neoplasms.

[0003] The term "Fusobacterium" refers to members of the genus *Fusobacterium*, a Gram-negative anaerobic bacterium whose species (including *F. nucleatum*) are found in the oral cavity and play a role in periodontal disease. *Fusobacterium* is enriched in human colonic adenomas relative to surrounding tissues and is also enriched in fecal samples from patients with colorectal adenomas and cancers compared to healthy subjects (Kostic et al., 2013). *Fusobacterium* targets colorectal adenocarcinoma cells via its Fap2 lectin, which specifically binds to D-galactose-β(1-3)-N-acetyl-D-galactosamine (Gal-GalNAc) residues overexpressed on the surface carbohydrates of colorectal and certain other adenocarcinomas. Once inside a tumor, Fusobacterium can enhance cell proliferation (Rubinstein et al., 2013; Chen et al., 2017; Yang et al., 2017), create a tumor-friendly inflammatory environment (Kostic et al., 2013), and protect cancer cells from natural killer cells and tumor-infiltrating T cells. Nucleated Fusobacterium also promotes chemotherapy resistance in CRC (Yu et al., 2017) and, due to similar cellular mechanisms, also promotes resistance to radiotherapy. High abundance of Fusobacterium in CRC is associated with poorer disease outcomes (Flanagan et al., 2014). Meanwhile, there is a call for the development of antibacterial agents that specifically target Fusobacterium to avoid the side effects of systemic broad-spectrum antibiotics.

[0004] Fusobacteria (e.g., *Fusobacterium nucleatum*) are also present in bacterial biofilms associated with colon tumors, particularly ascending colon tumors. Among these biofilms, Bacteroides and *Prevotella* species are typically the most prominent, along with Enterobacteriaceae (e.g., *Escherichia coli*). In addition to *Fusobacterium*, *Clostridium* spp., *Actinobacteria*, and various bacilli (including *Lactobacillus*, presumed to have a protective role) are also found. Colon tumor-associated biofilms are characterized by their thickness and continuity, penetrating deeper into the mucus layer and contacting colonic epithelial cells above and adjacent to the tumor. Subsurface tissue shows reduced or altered expression of the tumor suppressor E-cadherin, increased expression of the pro-inflammatory and angiogenic cytokine IL-6 and the proliferation-associated protein Ki-67, and Stat3 activation. This suggests that biofilms have a pro-cancer effect (Dejea et al., 2016).

[0005] Bacteria present in biofilms can initiate carcinogenesis in specific ways, including through direct mutagenic effects on DNA or interference with host DNA repair. This is the case with enterotoxin-producing *Bacteroides fragilis* (ETBF), superoxide-producing *Enterococcus faecalis*, and colibacin-producing *pks+* *Escherichia coli*. Many biofilm-producing 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 aforementioned bacterial strains and *Fusobacterium nucleatum*. Local immune effects of biofilms also exist, as demonstrated by the promoting effect of ETBF on tumorigenic Th17 responses in mouse models (Chung et al., 2018). These mechanisms promoting CRC initiation may also play similar roles in CRC progression (Drewes et al., 2016). Colonic biofilms and CRC tumors, especially ascending colon tumors, appear to have entered into a symbiotic relationship, where each component can promote the formation and growth of the other, creating a vicious cycle. One factor is N... 1 N 12 The production of diacetylspermine increases, originating from both biofilms and tumors (Johnson et al., 2015). The inventors believe that the technique of locally applying fosfomycin to the colonic lumen is effective in removing pro-cancer biofilms from the colonic mucosa, which breaks the vicious cycle, reduces the size of colonic tumors, tends to normalize the immune response within and around the tumor, and reduces the occurrence of recurrent tumors. Summary of the Invention

[0006] Therefore, the present invention aims to target the bacteria that promote colorectal tumor formation and eliminate tumor-associated bacterial biofilms by providing a pharmaceutical composition and a method for treating these conditions by locally applying the composition to the intestinal lumen where the tumor and / or biofilm are located. The invention mainly includes the following items:

[0007] A composition comprising the antibiotic fosfomycin as an active ingredient, the use of the composition for treating colorectal tumors, wherein the use includes reducing or eliminating fusobacteria and any biofilms inside and around the colorectal tumor in a subject suffering from said tumor, wherein the composition is applied topically into the intestinal lumen and applied to the site where the tumor and associated biofilms are located, and wherein the composition is applied from 1 day to 30 days before the removal of the colorectal tumor, and / or at one or more time intervals within a maximum of 3 months after the removal of the colorectal tumor.

[0008] The composition according to the use described in the foregoing item further comprises one or more additional antimicrobial agents or antibiotics as active ingredients.

[0009] The composition according to the purpose described in the foregoing project, wherein the composition will be applied 1 to 30 days prior to the removal of colorectal tumors.

[0010] The composition according to the use described in the foregoing item, wherein after removal of a colorectal tumor, the composition is applied at one or more time intervals within a maximum of 3 months after its removal.

[0011] The composition according to the use described in the foregoing item, wherein the composition is applied 1 to 30 days before the removal of colorectal tumors, and applied once or multiple times at intervals for up to 3 months after the removal of colorectal tumors.

[0012] The composition for the use described in the foregoing item, wherein one or more additional antimicrobial agents or antibiotics are selected from a non-limiting list containing metronidazole or carbapenems such as ertapenem, meropenem, or imipenem.

[0013] The composition according to the use described in the foregoing item, wherein the additional antibiotic agent is metronidazole.

[0014] The composition according to the aforementioned project for use, wherein the active ingredient is formulated as a dry powder or granules to dissolve in an aqueous medium before being delivered into the intestinal lumen.

[0015] The composition according to any of the preceding items for use, wherein the colorectal tumor is selected from adenomas, polyps and cancers.

[0016] The composition according to any one of the foregoing items, wherein the composition is administered via colonoscopy.

[0017] A colonoscopy can be a dual-channel colonoscopy.

[0018] The composition according to any one of the foregoing items, wherein the composition is formulated to form a mucosal adhesive gel upon delivery into the intestinal lumen.

[0019] The composition according to any one of the foregoing items, wherein the concentration of fosfomycin delivered to the intestinal lumen is in the range of 4 mg / g to 16 mg / g.

[0020] The composition according to any one of the foregoing items, wherein the concentration of metronidazole delivered to the intestinal lumen is in the range of 1 mg / g to 4 mg / g.

[0021] A complete kit for treating colorectal tumors, wherein the kit includes...

[0022] a. A first solution comprising the composition according to any one of the foregoing items, and

[0023] b. An aqueous second solution containing a gelling agent.

[0024] The kit according to the present invention, wherein the kit comprises

[0025] c. Instructions for use in the parallel administration of the first and second solutions via a dual-channel colonoscope and in the mixing of the first and second solutions in the intestinal lumen to form a mucosal adhesive gel.

[0026] According to the kit of the present invention, the aqueous second solution contains about 125 mg of low-acyl gellan gum.

[0027] According to the kit of the present invention, the first solution and the second solution each have a volume of about 50 mL.

[0028] According to the kit of the present invention, the mucosal adhesive gel is about 100 mL and contains about 8 mg / g fosfomycin and about 2 mg / g metronidazole.

[0029] A method for treating colorectal tumors in a subject, comprising reducing or eliminating fusobacteria and any biofilms inside and around the colorectal tumor and removing the colorectal tumor, wherein the method includes the following steps:

[0030] a. Administering a composition containing the antibiotic fosfomycin as an active ingredient to a subject suffering from the colorectal tumor, wherein the composition is applied topically into the intestinal lumen and to the site of the colorectal tumor and its associated biofilm.

[0031] b. Remove the colorectal tumor.

[0032] The step a is performed 1 to 30 days before step b, and / or at one or more time intervals within a maximum of 3 months after step b.

[0033] In the following detailed description of the invention, the scope of the invention and details of its practical implementation will be given. Detailed Implementation

[0034] The object of this invention is to provide a means of reducing or eliminating oncogenic Fusobacteria and tumor-associated bacterial biofilms in and around colorectal tumors. This is achieved by directly and locally applying an appropriate antibiotic solution to the intestinal lumen, with the application level ensuring that the solution is applied to the site of the tumor and / or biofilm. Tumor colonization by Fusobacteria is associated with accelerated tumor growth and increased resistance to endogenous antitumor immune mechanisms and exogenous antitumor therapies. Some mechanisms that achieve these effects have been elucidated. Therefore, it is proposed that eliminating or significantly reducing Fusobacterium colonization in these tumors would reverse these adverse effects and improve the efficacy of other anticancer therapies in the case of cancer. Eliminating the bacterial biofilms associated with these tumors (i.e., the area covering the tumor and adjacent normal colonic epithelium) would also exert these effects, as well as remove the oncogenic effects of biofilms that can promote tumor recurrence and tumor growth.

[0035] Local administration of antibiotics via the intestinal lumen, typically via colonoscopy, produces very high antibiotic concentrations at the tumor lumen and biofilm. The antibiotics penetrate the tumor cells containing Fusobacterium and into the overlying and adjacent biofilms to achieve bactericidal concentrations, while minimal absorption into the bloodstream, thus significantly reducing any systemic side effects.

[0036] This treatment can be applied before and after tumor removal to inhibit the pro-cancer effects of any biomembranes, thereby suppressing tumor recurrence.

[0037] Quantitative polymerase chain reaction (PCR) analysis of Fusobacterium nucleatum DNA in cancerous colorectal tumors confirmed that the bacteria are concentrated in the superficial luminal portion of the tumor (Yamamura et al., 2017). Antimicrobial therapy using high-concentration antibiotics administered from the luminal surface should effectively eliminate most of the bacterial burden while minimizing the side effects associated with routine systemic administration of broad-spectrum antibiotics. The same considerations apply to eliminating overlying and adjacent bacterial biofilms.

[0038] The active ingredient of the composition of the present invention

[0039] The compositions according to the invention primarily comprise the antibiotic fosfomycin, which can be used alone or in combination with one or more additional antibiotics selected from a non-limiting list including metronidazole and carbapenems such as ertapenem, meropenem, and imipenem.

[0040] Fosfomycin

[0041] Foshomycin is the international nonproprietary name for a broad-spectrum antibiotic, isolated and identified in 1969 from *Streptomyces freundii* strains, named 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-methylepoxyethylene-2-yl]phosphonic acid and a molecular weight of 138.1 Da. Foshomycin exhibits bactericidal activity by inhibiting bacterial cell wall biosynthesis through the inactivation of the enzyme UDP-N-acetylglucosamine-3-enolpyruvyltransferase (also known as MurA) (Brown et al., 1995). This enzyme catalyzes a key step in peptidoglycan biosynthesis: the formation of N-acetylmuramic acid by the 3'-hydroxyl group of UDP-N-acetylglucosamine. Fosfomycin is a phosphoenolpyruvate analog that inhibits MurA by alkylating cysteine ​​residues at its active site. The antibiotic enters bacterial cells via a glycerophosphate transporter.

[0042] Given this mechanism of action, fosfomycin has broad-spectrum bactericidal activity, with activity against aerobic bacteria such as Staphylococcus, Streptococcus, Enterococcus, Neisseria, Escherichia, Proteus (indole-negative), Serratia, Salmonella, Shigella, Pseudomonas, Haemophilus, and Vibrio. Its activity against indole-positive Proteus spp., Klebsiella, and Enterobacter spp. is weaker. Of particular relevance to this invention is its activity against the anaerobic genus *Fusobacterium* and the anaerobic genus *Peptostreptococcus* (including *Peptoniphilus*, *Finegoldia*, and *Anaerococcus*). However, it is inactive against anaerobic *Bacteroides fragilis* bacteria.

[0043] In the community, the prevalence of bacterial resistance to fosfomycin is low, and studies on the prevalence of resistant bacteria after the introduction of fosfomycin show no or only a slight increase in the prevalence. However, prolonged exposure to antibiotics can lead to the evolution of resistance in bacteria through the selection of mutants lacking the glycerophosphate transporter pathway. Alternative resistance mechanisms involve the loss of inducible hexose phosphate transporters, the Cys-Asp mutation in MurA, or the acquisition of plasmids encoding the fosfomycin inactivating enzymes fosA and fosB (except for the chromosomal fosX in Listeria monocytogenes). However, mutant strains may also exhibit reduced pathogenicity (Karageorgopoulos et al., 2012). This may explain why bacterial resistance occurs during prolonged in vitro exposure but is less common in vivo. In controlled clinical trials of oral or intravenous fosfomycin administration, the overall incidence of resistant bacterial strains was 3.0%, with the highest rate of 15% in Pseudomonas aeruginosa. Overall, fosfomycin is considered a valuable therapeutic weapon against multidrug-resistant bacteria.

[0044] Fosfomycin has been shown to promote phagocytosis and act as an immunomodulator. It accumulates on polymorphonuclear leukocytes at twice the extracellular concentration without affecting their cellular function, while still maintaining bactericidal activity against Staphylococcus aureus (Höger et al., 1985).

[0045] Regarding the uptake of fosfomycin by intestinal mucosal cells, Martínez et al. (2013) demonstrated that IPEC-J2 cells rapidly uptake fosfomycin after exposure to 580 µg / mL of fosfomycin calcium, reaching an intracellular concentration of nearly 30 µg / mL within 15 minutes. Such high concentrations of fosfomycin are difficult to achieve via standard systemic administration. Therefore, killing Fusobacterium cloacae in colorectal cancer cells requires even higher concentrations of fosfomycin. According to the present invention, such concentrations can be easily achieved through topical application of a fosfomycin solution.

[0046] Fosfomycin is known to readily penetrate tissue barriers and enter bacterial biofilms (Reffert & Smith 2014). Therefore, direct application of fosfomycin solution to bacterial biofilms covering and adjacent colorectal tumors will produce high bactericidal concentrations of fosfomycin. Fosfomycin inhibits the adhesion of various bacterial species, including those involved in biofilm formation, to epithelial cells (Gobernado 2003). Fosfomycin can also break down biofilms to enhance the permeability of other antibiotics.

[0047] The main side effects of fosfomycin are gastric irritation caused by oral administration of fosfomycin disodium, manifested as transient skin rash (0.3% of cases) and eosinophilia (0.2%) as allergic symptoms, and transient elevation of liver enzymes caused by systemic administration of fosfomycin (0.3% of cases) (Gobernado 2003). However, overall, fosfomycin shows very low toxicity, and therefore sodium load is a dose-limiting factor when high doses of fosfomycin disodium are administered systemically.

[0048] When used in combination with a wide range of penicillins, cephalosporins, aminoglycosides, macrolides, and lincosamides, fosfomycin exhibits considerable synergistic bactericidal activity against a large number of microbial strains from these susceptible genera. While early studies indicated synergistic effects in various antibiotic combinations against approximately 70% to 100% of tested strains, subsequent, more extensive studies have shown synergistic rates of 36% to 74%. The remaining strains showed only additive effects and exhibited antagonistic effects against individual bacterial strains only in combinations of one or two separate antibiotics (Gobernado 2003). The fact that fosfomycin shows synergistic effects with many individual antibiotics and effectively eliminates the toxicity of many other antibiotics, including the nephrotoxicity and ototoxicity of aminoglycosides, is advantageous for fosfomycin in combination with other antibiotics to produce potent bactericidal effects and to compensate for any development of fosfomycin resistance during longer treatment periods.

[0049] The main forms of fosfomycin within the scope of this invention are:

[0050] i) Fosfomycin disodium, with a molecular weight of 182.0 Da, has a pH of 9.0 to 10.5 for a 5% solution. This salt is hygroscopic, highly soluble in water, and exhibits high bioavailability, but can be locally irritating if not neutralized.

[0051] ii) Fosfomycin calcium monohydrate, with a molecular weight of 194.1 Da, has a pH of 8.1 to 9.6 for a 0.4% solution. This salt is slightly soluble in water and non-hygroscopic, but it is less irritating to the stomach when used orally, in which case its bioavailability into the systemic circulation can be as low as 12% (Bergan 1990).

[0052] iii) Fosfomycin tromethamine, with a molecular weight of 259.2 Da, has a pH of 3.5 to 5.5 for a 5% solution. This salt is highly soluble in water and well tolerated orally, with a bioavailability of approximately 40%.

[0053] When the name “fosfomycin” is used in this article, it refers to the inorganic or organic salt of fosfomycin, as illustrated by the main forms described above, and the dosage of fosfomycin refers to the amount of fosfomycin in the free acid form present in the salt.

[0054] Fosfomycin tromethamine exhibits long-term stability as a readily soluble granule when stored in sealed pouches at room temperature. Fosfomycin disodium, due to its significant hygroscopicity, is present in sealed vials as a lyophilized powder. In this form, it exhibits long-term stability when stored at temperatures between 2°C and 8°C. Any form of fosfomycin can be used in the compositions of this invention.

[0055] The compositions according to the invention may contain fosfomycin, such that a single dose is in the range of 400 mg to 4 g.

[0056] Metronidazole

[0057] This semi-synthetic antibiotic, active against a variety of anaerobic bacteria including Bacteroides fragilis and Fusobacterium, is well known in the art. It has been used in combination with systemic fosfomycin for the prevention of wound infections following abdominal surgery. The intracellular permeation of metronidazole appears to occur through rapid passive diffusion, allowing intracellular levels to approach equilibrium with extracellular levels within 15 minutes (Hand et al., 1987). Compositions according to the invention may contain metronidazole such that a single dose ranges from 100 mg to 1 gram.

[0058] Carbapenems

[0059] Carbapenems are members of the β-lactam antibiotic class. Like penicillins and cephalosporins, they exert their bactericidal effect by inhibiting bacterial cell wall synthesis, but their mechanism differs from that of fosfomycin. Therefore, β-lactam antibiotics (including carbapenems) and fosfomycin characteristically exhibit synergistic bactericidal activity against most bacterial species active against both types of antibiotics. However, carbapenems exhibit a broader spectrum of activity than most penicillins and cephalosporins, showing activity against anaerobic bacteria such as Bacteroides fragilis, Prevotella, and Fusobacterium. Therefore, they can be used as an alternative to metronidazole to exert bactericidal effects against anaerobic bacteria that are largely insensitive to fosfomycin. Regarding the intracellular penetration of carbapenems, bactericidal intracellular concentrations of ertapenem are achieved in mouse macrophage lines at extracellular concentrations as low as 15 µg / mL (Tang et al., 2012), and the ratio of meropenem concentration to extracellular concentration in human macrophages remains consistently high (range 3 to 12) within an extracellular concentration range of 0.125 µg / mL to 1 µg / mL (Cuffini et al., 1993). The compositions according to the invention may comprise ertapenem, meropenem, or imipenem, such that a single dose ranges from 100 mg to 1 gram.

[0060] Medical indications

[0061] The primary indication for the use of this composition is the presence of one or more colorectal tumors originating from the colonic or rectal mucosa and / or the presence of bacterial biofilms covering the colonic or rectal mucosa, regardless of whether the biofilm is associated with adenocarcinoma, adenoma, polyp, or mucosal dysplasia. This indication is typically diagnosed in connection with colonoscopy. Treatment with this composition is typically administered endoscopically by instilling a gel-forming solution of the composition into the intestinal region where mucosal lesions are observed, either during the same course of treatment or in a later procedure. Subsequent treatment may be decided at the discretion of the treating clinician after initial treatment.

[0062] Indications for treatment can also be provided by detecting urine, feces, blood, or tissue markers of pro-cancer biofilms or driving microorganisms. Currently, such markers include colibacillin-producing *Escherichia coli*, enterotoxin-producing *Bacteroides fragilis*, *Fusobacterium nucleatum*, and *Porphyromonas gingivalis*, as well as other microorganisms, particularly those from the oral microbiota. Many markers are under investigation, but none have yet demonstrated sufficiently high diagnostic sensitivity or specificity to independently diagnose CRC or colonic biofilms; therefore, they currently serve a screening function for selecting candidates for colonoscopy. Urine N 1 N 12 -Diacetylspermine is one of the biomarkers with good diagnostic sensitivity and specificity, as mentioned above, it is produced in increased amounts by CRC-associated bacterial biofilms.

[0063] If colorectal cancer is known or suspected to have penetrated the intestinal wall or metastasized to lymph nodes or distant tissues, the treating clinician may decide to supplement local antibiotic therapy with systemic antibiotic therapy, because live cancer-promoting microorganisms may be found in metastatic lesions of colorectal cancer.

[0064] Tumors and biofilms present in the rectum and sigmoid colon offer opportunities for treatment with compositions formulated as retention enemas due to their location. In some cases, larger volumes of enemas containing the composition can be used to treat colonic regions further away from the rectum.

[0065] The use of this composition may also be applicable during neoadjuvant radiotherapy or chemotherapy for colorectal cancer (e.g., in combination with bevacizumab and / or cytotoxic drugs).

[0066] preparation

[0067] In a preferred embodiment, the composition comprises disodium fosfomycin, for example, fosfomycin disodium formulated for intravenous injection, supplied as a lyophilized powder in a capped glass vial, to be dissolved in a supplied pharmaceutically acceptable aqueous carrier or diluent. The carrier may be simple water or water containing pharmaceutically acceptable excipients or adjuvants, including but not limited to buffers and / or tonicators to ensure a neutral pH and isotonicity of the fosfomycin solution, and / or one or more gelling agents to form a mucosal adhesive gel upon delivery into the intestinal lumen. In another embodiment, the composition comprises fosfomycin tromethamine, supplied as dry granules and similarly dissolved in a pharmaceutically acceptable aqueous carrier or diluent as described above.

[0068] In another embodiment, the composition comprises fosfomycin and an additional antibiotic, such as metronidazole, or a carbapenem such as ertapenem, meropenem, or imipenem, wherein the metronidazole is supplied as a stable aqueous solution for intravenous injection, and the carbapenem is supplied as a dry powder or granules to be dissolved in an aqueous carrier.

[0069] The pH of the solution obtained from the formulation according to the invention can be between 4 and 8, preferably between 6.5 and 7.5, and most preferably between 7 and 7.5. The supplied antibiotic powder or granules and aqueous diluent may each contain a buffer and / or a tonic modifier to ensure a neutral pH and isotonicity of the mixed antibiotic solution.

[0070] The solution may also contain one or more approved biocompatible gelling agents or thickeners that do not bind to the active ingredient of the composition, such as hydroxypropyl methylcellulose or a large number of other biocompatible thickeners known in the art, to achieve a solution with optimal viscosity for controlled delivery and adhesion to the colonic mucosa. In one embodiment, an aqueous solution containing 0.25% (w / w) of a low-acyl gellan gum, such as Kelcogel CG-LA (CAS Registry No. 71010-52-1), is supplied, while the antibiotic is dissolved in an equal volume of a solution containing 0.25% (w / w) calcium chloride. When the two solutions are delivered in parallel and sprayed and mixed in the intestinal lumen using a dual-channel colonoscope, they form a mucosal adhesive gel that prolongs the contact time between the composition and the intestinal wall, as well as any present tumor or bacterial biofilm.

[0071] In one embodiment, the composition according to the invention can be pre-packaged as a kit containing fosfomycin and metronidazole, for example in single-dose units, to be dissolved or added to an aqueous solution, which is also pre-packaged in single-dose vials, and an aqueous solution containing a gelling agent (also pre-packaged in single-dose vials), and instructions for use as described above.

[0072] application

[0073] The compositions of the present invention can be applied by methods conventional in the art for local application to the intestinal lumen of areas of colonic mucosal tumors (whether adenocarcinoma, adenoma, or mucosal dysplasia) or to areas of bacterial biofilms located in the colon or rectum.

[0074] The preferred method of application is to use a colonoscope with two channels to spray the liquid into the intestinal lumen, and the liquid is mixed during the spraying process.

[0075] The administration method requires that the colon and rectum have been cleared of fecal matter using standard bowel preparation protocols for colonoscopy or surgery, such as drinking or orally taking large amounts of liquid containing an appropriate concentration of polyethylene glycol (e.g., polyethylene glycol 3350 or 4000), with or without electrolytes.

[0076] If the tumor or biofilm is located in the lower rectum or sigmoid colon, a solution of the composition can be administered as a retention enema into the intestinal lumen at the level of the tumor. In some cases, a larger volume of the composition in enema form can be given to treat lesions in more proximal parts of the colon.

[0077] For tumors and biofilms located more proximal to the colon, a solution of the composition is applied at the lesion level during colonoscopy.

[0078] In special circumstances where rectal or sigmoid colon tumors are to be treated without bowel preparation, the bowels may first be emptied by administering a laxative, followed by colonic irrigation to cleanse the intestines, and then the combined solution may be administered.

[0079] For tumors in the rectum and lower sigmoid colon, the retention enema volume is 50 mL to 100 mL. Instruct the patient to lie on their left and right sides, supine and prone, for 5 minutes each, and retain the enema for at least 15 minutes if possible. When the tumor is located more proximal to the colon, a larger volume of enema may be administered; however, preferably, the solution of the composition should be sprayed onto the tumor area during colonoscopy, as described above.

[0080] dose

[0081] While this invention is not intended to prescribe appropriate dosages for individual cases by clinicians, it is recommended that compositions be formulated such that a single unit dose of fosfomycin in the compositions of this invention is in the range of 400 mg to 4 g. When used as an adjunct antibiotic in these compositions, a single unit dose of metronidazole or carbapenems may be in the range of 100 mg to 1 g. These doses may be exceeded at the physician's discretion, for example, if a large volume enema is required.

[0082] As an example, a typical single dose can consist of 800 mg of fosfomycin dissolved in 50 mL of an aqueous diluent, constituting a solution of approximately 1.6% (w / w). If metronidazole or carbapenems are required as an additional antibiotic, they can be added to the same diluent in an amount such as 200 mg, constituting a solution of approximately 0.4% (w / w). For the formation of the mucosal adhesive gel, the antibiotic solution contains 0.25% (w / w) calcium chloride. A second aqueous diluent, also in 50 mL volume, contains 125 mg (0.25% (w / w)) of low-acyl gellan gum. When the two solutions are mixed and sprayed distally through a dual-channel colonoscope, a 100 mL mucosal adhesive gel is formed, containing 8 mg / mL fosfomycin and 2 mg / mL metronidazole. Therefore, this typical single-dose formulation consists of two 50 mL solutions, the first containing 800 mg fosfomycin, 200 mg metronidazole and 125 mg calcium chloride, and the second containing 125 mg low-acyl gellan gum.

[0083] The above single dose can be increased in proportion to the dose and volume, while maintaining the concentration of fosfomycin and (if present) the above-mentioned additional antibiotics unchanged, up to a maximum volume of 200 mL of diluent containing 4 g of fosfomycin and (if present) 1 g of the above-mentioned additional antibiotics.

[0084] If a larger volume of solution is required for a single dose, it can be prepared by diluting the single dose with isotonic saline. However, the amount of fosfomycin to be administered should not be increased to more than 4 grams, nor should the amount of additional antibiotics to be administered be increased to more than 1 gram. Alternatively, the physician may increase the antibiotic dosage as appropriate, provided that the solution remains isotonic; the maximum daily dose of fosfomycin can be up to 20 g per liter of solution.

[0085] A dose may preferably be administered once a day, or twice a day if the treating clinician deems it necessary and feasible, so that the patient receives two typical single doses at two different times each day, or even three times a day, so that the patient receives three typical single doses at three different times each day.

[0086] The duration of the above-described administration regimen is typically short, ranging from 1 to 2 days. However, if the treating clinician deems it necessary to extend the treatment to achieve the desired effect, the dosage regimen may be extended to 14 days, for example, within the range of 3 to 5 days, such as 4 days; or within the range of 5 to 7 days, such as 6 days; or within the range of 7 to 14 days. Treatment may also be performed for 1 or 2 days, then paused, and then repeated after one or two weeks.

[0087] The compositions according to the invention may contain fosfomycin, such that a single dose is typically in the range of 400 mg to 4 g, dissolved in an aqueous diluent in a volume of 20 mL to 200 mL.

[0088] Preferably, the final mixed gel or solution in the intestinal lumen after a single dose may contain about 2% (by weight) of the total active ingredient. Preferably, the single dose is in an isotonic solution.

[0089] In some embodiments, the compositions of the present invention are used for one or more applications, such as two, three, or four, after which treatment is paused and resumed only when required by the attending physician.

[0090] Treatment

[0091] This invention provides a method for reducing or eliminating fusobacteria and other bacteria that promote the development of colorectal cancer in colorectal adenomas, polyps and adenocarcinomas, as well as related bacterial biofilms, by local application into the intestinal lumen.

[0092] Example

[0093] Preoperative endoscopic treatment with fosfomycin and metronidazole was performed on patients with right colonic adenomas.

[0094] Patients and methods

[0095] This study included twelve patients (6 men, 6 women, median age 71 years) with right-sided colonic adenomas from two hospital centers. Patients underwent interventional colonoscopy, during which biopsies and blood samples were collected from the healthy mucosal lining prior to intervention, followed by spraying a 100 mL gel containing 800 mg fosfomycin and 200 mg metronidazole from the cecum to the hepatic flexure. Endoscopic mucosal resection (EMR) was performed at least five days post-intervention: post-intervention blood samples were collected, and a post-intervention biopsy sample was collected again from the healthy mucosal lining prior to the EMR procedure. Post-intervention adenoma tissue was obtained from enrolled patients after routine pathological evaluation. Patients were matched 1:2 with a retrospective control group to obtain antibiotic-free adenoma tissue. Adverse events at 14 days and 12 months post-EMR were recorded via interviews and entries in patient files and graded using CTCAE version 5 to assess the safety of the intervention.

[0096] result

[0097] Following intervention, the percentage of bacterial biomass adhering to the healthy mucosal lining in patients was significantly reduced (3.0e-04% vs. 9.8e-05%, p < 0.025), while there was no significant reduction in the percentage of bacterial biomass between interventional patients and the control group (8.6e-03% vs. 1.1e-02%, p < 0.368). Compared to the control group, the density of mucosal CD68+ macrophages and CD3+ tumor-infiltrating lymphocytes in interventional colonic adenomas were significantly increased (2.2 vs. 3.4, p < 0.030; and 524.2 vs. 727.4, p < 0.018, respectively). No significant difference was found between CD8+ cytotoxic tumor-infiltrating lymphocytes.

[0098] No sudden serious unexpected adverse events (SUSAR) or serious adverse events (SAR) occurred in any patient. One patient experienced a serious adverse event (SAE) after developing fever and requiring readmission following EMR. However, this SAE was determined to be unrelated to the study drug. All patients reported adverse events within 14 days after EMR, the most common being loose stools (10 patients).

[0099] in conclusion

[0100] Preoperative local treatment with fosfomycin and metronidazole reduced the bacterial biomass adhering to the colonic mucosal lining. This intervention was considered safe.

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Claims

1. A composition comprising the antibiotic fosfomycin as an active ingredient, the composition being used for the treatment of colorectal tumors, wherein the use comprises reducing or eliminating fusobacteria and any biofilms inside and around the colorectal tumor in a subject suffering from said tumor, wherein the composition is applied topically into the intestinal lumen and to the site where the tumor and associated biofilms are located, and wherein the composition is applied 1 to 30 days prior to the removal of the colorectal tumor, and / or at one or more time intervals within a maximum of 3 months after the removal of the colorectal tumor.

2. The composition for use according to the preceding claims, wherein the composition further comprises one or more additional antimicrobial agents or antibiotics as active ingredients.

3. The composition for use according to the preceding claims, wherein the one or more additional antimicrobial agents or antibiotics are selected from a non-limiting list comprising metronidazole or carbapenems such as ertapenem, meropenem, or imipenem.

4. The composition for use according to the preceding claims, wherein the additional antibiotic agent is metronidazole.

5. A composition for use according to any one of the preceding claims, wherein the active ingredient is formulated as a dry powder or granules to dissolve in an aqueous medium before delivery into the intestinal lumen.

6. The composition for use according to any one of the preceding claims, wherein the colorectal tumor is selected from adenomas, polyps, adenocarcinomas, and colorectal cancer tumors.

7. The composition according to any one of the preceding claims, wherein the composition is administered via colonoscopy.

8. The composition according to any one of the preceding claims, wherein the composition is formulated to form a mucosal adhesive gel when delivered into the intestinal lumen.

9. The composition according to any one of the preceding claims, wherein the concentration of fosfomycin delivered to the intestinal lumen is in the range of 4 mg / g to 16 mg / g.

10. The composition according to any one of the preceding claims, wherein the concentration of metronidazole delivered to the intestinal lumen is in the range of 1 mg / g to 4 mg / g.

11. The composition according to any one of the preceding claims, wherein the composition comprises about 800 mg fosfomycin, about 200 mg metronidazole and about 125 mg calcium chloride, and wherein the composition is formulated in about 50 mL of a first solution, and wherein the treatment comprises the step of delivering the first solution and a second solution in parallel via a dual-channel colonoscope, wherein the second solution comprises about 125 mg of low-acyl gellan gum, wherein the first solution and the second solution are mixed in the intestinal lumen to form about 100 mL of a mucosal adhesive gel, the mucosal adhesive gel comprising about 8 mg / g fosfomycin and about 2 mg / g metronidazole.

12. A complete kit for treating colorectal tumors, wherein the kit comprises: a. A first solution comprising the composition according to any one of the preceding claims, and b. An aqueous second solution containing a gelling agent.

13. The kit of claim 12, wherein the kit comprises c. Instructions for use in the parallel administration of the first solution and the second solution via a dual-channel colonoscope, and for mixing the first solution and the second solution in the intestinal lumen to form a mucosal adhesive gel.

14. The kit according to claim 12 or 13, wherein the aqueous second solution comprises about 125 mg of low-acyl gellan gum.

15. The kit according to claims 12 to 14, wherein the first solution and the second solution each have a volume of about 50 mL.

16. The kit according to claims 13 to 15, wherein the mucosal adhesive gel is about 100 mL and contains about 8 mg / g fosfomycin and about 2 mg / g metronidazole.

17. A method for treating colorectal tumors in a subject, comprising reducing or eliminating fusobacteria and any biofilms inside and around the colorectal tumor and removing the colorectal tumor, wherein the method includes the following steps: a. Administering a composition containing the antibiotic fosfomycin as an active ingredient to a subject suffering from the colorectal tumor, wherein the composition is applied topically into the intestinal lumen and to the site of the colorectal tumor and its associated biofilm. b. Remove the colorectal tumor. The step a is performed 1 to 30 days before step b, and / or at one or more time intervals within a maximum of 3 months after step b.