Use of short chain fatty acids in cancer prevention

Abstract

To provide methods and kits for preventing or delaying the onset of hepatocellular cancer.SOLUTION: The present invention provides a method for preventing or delaying the onset of hepatocellular carcinoma in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising at least one short-chain fatty acid.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 106,778, filed January 23, 2015, the contents of which are incorporated by reference in their entirety. [Background technology]

[0002] 2. Background of the Invention Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second leading cause of cancer death worldwide (Ding J et al., Cancer Lett, 2014; 346(1):17-23). ​​Early stage HCC is often asymptomatic when curative approaches may be amenable, and by the time advanced disease is detected, few treatment options are available. Untreated HCC has a 5-year survival rate of less than 3%, and even with the multikinase inhibitor sorafenib, life expectancy was only increased by an average of 3 months (Peck-Radosavljevic M, Liver Cancer, 2014; 3(2):125-31). Combination therapy with sorafenib plus cytotoxic drugs extended life expectancy by approximately 1 year after diagnosis.

[0003] HCC most often occurs in a background of persistent inflammation (hepatitis) and is frequently associated with chronic hepatitis B and C virus infections (Flores et al., Clin Med Insights Oncol, 2014; 8:71-6). For hepatitis B virus (HBV), the related woodchuck hepatitis virus (WHV) model highlights the importance of chronic liver disease (CLD) for the pathogenesis of HCC (Menne S et al., World J Gastroenterol, 2007; 13(1):104-24). In this case, chronic WHV infection and CLD led to an almost 100% HCC incidence, whereas only a small percentage of woodchucks with acute hepatitis infection developed this tumor (Menne S et al., World J Gastroenterol, 2007; 13(1):104-24). Similarly, patients who are virus carriers with progressive chronic liver disease (hepatitis, fibrosis and cirrhosis) are at high risk for HCC, whereas asymptomatic carriers are at much lower risk (Beasley et al., Lancet, 1981; 2(8256):1129-33). HBV and related mammalian hepadnaviruses (including WHV) encode a small polypeptide (termed X antigen) that contributes significantly to the pathogenesis of HCC (Feitelson MA et al., Amer J Pathol, 1997; 150:1141-1157). HBV-encoded X antigen, or HBx, is a trans-regulatory protein that alters patterns of host gene expression by constitutively activating signaling pathways in the cytoplasm and by binding to complexes that regulate gene transcription in the nucleus (Tian Y et al., Mol Cell Biol, 2013; 33(15):2810-6; Feitelson MA et al., Amer J Pathol, 1997; 150:1141-1157).Integration of the HBx gene occurs in most chromosomes, and such integration events accumulate with each succession of hepatitis and regeneration, resulting in increased intracellular accumulation of HBx (Xu C et al., Cancer Lett, 2014; 345(2):216-22; Wang W et al., Hepatology, 1998; 14:29-37; Wang W et al., Cancer Res, 1991; 51:4971-4977). In the face of cell-mediated immune responses that aim to damage and kill virus-infected cells, HBx promotes cell survival and growth. Thus, there is a close association between HBx and CLD (Jin YM et al., J Viral Hepat, 2001; 8(5):322-30). In this context, HBx appears to be activated by free radicals generated by the immune response that target virus-infected hepatocytes (Wang JH et al., Biochem Biophys Res Commun, 2003; 310(1):32-9), suggesting that disease outcomes may be affected if the immune-mediated pathogenesis of HCC can be modulated.

[0004] It is known that various probiotic bacterial strains can gently promote or suppress immune response in the intestine. In fact, selected probiotic bacterial strains metabolize complex carbohydrates into short-chain fatty acids (SCFAs), which are easily absorbed through the intestinal wall and activate regulatory T cells. Recent studies have shown that SCFAs improve inflammation in a mouse model of colitis (Smith PM et al., Science, 2013; 341(6145):569-73). This may be due to the fact that SCFAs, especially butyrate, can change the pattern of gene expression in target cells by inhibiting histone deacetylase activity (HDACi) (Tan J et al., Adv Immunol, 2014; 121:91-119). HBx has been shown to activate HDAC activity (Yoo et al., Oncogene, 2008; 27:3405-13), suggesting that administration of selected probiotic bacteria or SCFAs produced by these bacteria to HBx transgenic mice that develop HCC may provide a facile novel method to partially block the tumorigenesis-promoting ability of HBx.

[0005] There is a need in the art for effective treatments to prevent or slow the progression of hepatitis to hepatocellular carcinoma. The present invention addresses this need. Summary of the Invention [Means for solving the problem]

[0006] The present invention provides a method for preventing or delaying the onset of hepatocellular carcinoma in a subject, in one embodiment, the method comprises administering to the subject a therapeutically effective amount of a composition comprising at least one short chain fatty acid.

[0007] In one embodiment, the short chain fatty acid is selected from the group consisting of formic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, isocaproic acid, caproic acid, lactic acid, succinic acid and pyruvic acid.

[0008] In one embodiment, the composition further comprises a pharma- ceutically acceptable excipient.

[0009] In one embodiment, the composition is administered in combination with another therapeutic agent.

[0010] In one embodiment, the composition is administered orally.

[0011] In one embodiment, the composition is administered with a food or drink.

[0012] The present invention also provides a method for preventing or delaying the onset of hepatocellular carcinoma in a subject, in one embodiment, the method comprising administering to the subject a therapeutically effective amount of a composition comprising at least one probiotic bacteria.

[0013] In one embodiment the probiotic bacteria is selected from the group consisting of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei, Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus sasei, Lactobacillus salivarius, Pediococcus pentosaceus, Streptococcus thermophiles, Bacillus subtilis, Bacillus coagulans, Enteroccous faecium, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum and Bifidobacterium infantis.

[0014] In one embodiment, the composition further comprises a pharma- ceutically acceptable excipient.

[0015] In one embodiment, the excipient comprises at least one prebiotic.

[0016] In one embodiment, the composition is administered in combination with another therapeutic agent.

[0017] In one embodiment, the composition is administered orally.

[0018] In one embodiment, the composition is administered with a food or drink.

[0019] The present invention also provides a kit for preventing or delaying the onset of hepatocellular carcinoma in a subject, hi one embodiment, the kit comprises a composition comprising at least one short chain fatty acid.

[0020] The present invention also provides a kit for preventing or delaying the onset of hepatocellular carcinoma in a subject, hi one embodiment, the kit comprises a composition comprising at least one probiotic bacteria. [Brief description of the drawings]

[0021] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Figure 1] 1A-1C show graphs depicting alanine aminotransferase (ALT) levels in mice treated with Synbiotic 2000™ (Δ) or PBS (■) at the indicated ages. [Diagram 2]Figures 2A-2C show graphs comparing the incidence of different liver pathologies. This is shown for 3-month-old mice evaluated at 6 months for evidence of steatosis and dysplasia (Figure 2A), for 6-month-old mice evaluated at 9 months for evidence of dysplasic nodules and early HCC (Figure 2B), and for 9-month-old mice evaluated at 12 months for evidence of large HCC nodules (Figure 2C). Grey bars are test mice fed Synbiotic 2000™ for 3 months from the ages indicated. White bars are control mice fed PBS. HBx transgenic mice were generated using the HBx enhancer / promoter region immediately upstream of the X gene. This enhancer / promoter becomes active in mature hepatocytes such that HBx is undetectable at birth but accumulates in hepatocytes with age (Yu DY et al., J Hepatol, 1999; 31:123-132). From an immunological standpoint, HBx is recognized as a foreign body and causes an inflammatory response (hepatitis) and steatosis (fatty liver) by 4-5 months of age. This progresses to dysplasia (preneoplastic lesions) by 6-7 months of age, dysplastic nodules and micro-HCCs by 9-10 months of age, and finally to large HCCs by 10-12 months of age. [Diagram 3] Figures 3A-3D show HBx staining in the liver of 6-month-old (Figure 3A) and 9-month-old (Figure 3C) mouse livers after treatment with PBS (Figures 3A and 3C) or Synbiotic 2000™ (Figures 3B and 3D). Note that in both cases, after treatment with Synbiotic 2000™, the lobular distribution of HBx becomes more diffuse, suggesting that HBx levels may be reduced with treatment. [Figure 4]Figure 4 shows a graph of a limited microarray analysis showing expression of select markers of oncogenic mediators (columns 1-20) and immune mediators (columns 21-27) in HBx transgenic mice treated with Synbiotic 2000™ at the indicated ages compared to HBx transgenic mice treated with placebo in parallel. Differential expression values ​​were normalized to GAPDH. Other controls included genomic DNA contamination (MGDC), RNA quality (RTC) and general PCR performance (PPC). [Diagram 5] Figures 5A-5D show livers from four SCFA-fed mice obtained at 12 months of age. In Figure 5A, the number and size of observable tumors on the surface of the liver lobule were then estimated. Examples of these tumors are shown on the livers of mice treated for 3 months with PBS (Figures 5A and 5B) or SCFA (Figure 5C) from 9 months of age. Arrows indicate tumor nodules. Results are representative of mice from each group. Figure 5D is an overview of the tumor characteristics of both mouse groups. [Figure 6] Figures 6A-6E show H&E stained liver sections and bar graphs showing tumor characteristics in SCFA-fed mice. HBx mice were treated with SCFA or PBS for 3 months starting at 9 months of age. Figure 6A shows an example of a small tumor in a SCFA-treated mouse (x40). Figure 6B shows an example of a medium-sized tumor nodule in a SCFA-treated mouse (x40). Figure 6C shows an example of a large tumor from a PBS-treated mouse. Figure 6D shows a higher magnification of an HCC nodule from a PBS-treated mouse (x100). The tumor (T) is on the left and the non-tumor (NT) liver is on the right. Arrows indicate the tumor nodule. Figure 6E shows the results of treating 9-month-old mice with SCFA (+) or PBS (-) for 3 months. Formalin-fixed tissues were sectioned and stained by H&E. S = small tumor (<0.5 cm in diameter); M = medium-sized tumor (0.5-1.0 cm in diameter); L = large tumor (>1 cm). [Figure 7] FIG. 7 shows a table showing liver pathology in HBx mice treated with Synbiotic 2000™ from 3-6 months. [Figure 8] FIG. 8 shows a table depicting liver pathology in HBx mice treated with Synbiotic 2000™ from 6-9 months. [Figure 9] FIG. 9 shows a table depicting liver pathology in HBx mice treated with Synbiotic 2000™ from 9-12 months of age. [Figure 10] FIG. 10 shows a table showing the results of intrahepatic HBx staining in Synbiotic 2000™ treated and control mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description The present invention is based in part on the discovery that short chain fatty acids are effective as a chemopreventive therapeutic approach for liver cancer. The results presented herein demonstrate that administration of short chain fatty acids to subjects with inflammation, hepatitis and precancerous lesions in the liver is effective in preventing or slowing the progression of liver disease to hepatocellular carcinoma.

[0023] definition It should be understood that the figures and description of the present invention have been simplified for clarity to show elements relevant to a clear understanding of the present invention while excluding many other elements found in a typical microscope device. One of ordinary skill in the art may recognize that other elements and / or steps are desirable and / or necessary to practice the present invention. However, because such elements and steps are well known in the art and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein covers all variations and modifications to such elements and methods known to those of ordinary skill in the art.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0025] As used herein, each of the following terms has the meaning associated with it in this section.

[0026] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0027] As used herein, "about" when referring to a measurable value, such as an amount, a time period, or the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate.

[0028] The term "abnormal" when used in reference to an organism, tissue, cell, or component thereof, refers to an organism, tissue, cell, or component thereof that differs in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from an organism, tissue, cell, or component thereof that exhibits the "normal" (expected) respective characteristic. A normal or expected characteristic for one cell or tissue type may be abnormal for a different cell or tissue type.

[0029] A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which a patient experiences such sign or symptom, or both, are reduced.

[0030] The term "anti-tumor effect" as used herein refers to a biological effect that can be demonstrated by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be demonstrated by the ability of the composition of the present invention in preventing early stage tumor development.

[0031] A "disease" is an animal's health condition in which the animal cannot maintain homeostasis and, if the disease is not improved, the animal's health will continue to deteriorate.In contrast, a "disorder" in an animal is a health condition in which the animal can maintain homeostasis, but the animal's health condition is lower than in the absence of the disorder.If left untreated, the disorder does not necessarily cause further deterioration of the animal's health condition.

[0032] As used herein, the term "inhibit" means to suppress or block an activity or function by at least about 10% compared to a control value. Preferably, the activity is suppressed or blocked by 50%, more preferably 75%, and even more preferably 95% compared to a control value.

[0033] The term "liver disease" includes diseases and conditions of the liver, including cirrhosis, alcoholic and non-alcoholic fibrosis, and liver diseases or changes associated with obesity, diabetes, and metabolic syndrome. Other examples of liver disease include hepatitis, fatty liver, toxic liver failure, cirrhosis, diabetes-related liver disease, hepatic steatosis, liver fibrosis, cirrhosis, chronic hepatitis, etc.

[0034] The term "probiotic organisms" includes live microorganisms that have a beneficial effect on the health of the host. Benefits to the host's health include, but are not limited to, improved gut microbial balance. Other beneficial effects on the host include, for example, enhancing the immune system, stimulating phagocytic activity, stimulating interferon, reducing hypertension, reducing cancer risk, increasing antibacterial activity and immunomodulatory effects, reducing hypercholesterolemia, and treating cancer.

[0035] The terms "treatment", "treating" and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents a disease or its symptoms, and / or it may be therapeutic, in that it partially or completely cures a disease and / or adverse effects resulting from a disease. The term "treatment" as used herein encompasses any treatment of a disease in a subject, including (a) prevention of a disease associated with an undesirable immune response occurring in a subject who may be predisposed to the disease; (b) inhibition of the disease (i.e., preventing its progression), or (c) remission of the disease (i.e., causing regression of the disease).

[0036] The terms "effective amount" and "pharmacologically effective amount" refer to a sufficient amount of an agent to provide a desired biological result. That result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. One of ordinary skill in the art can determine, using routine experimentation, the appropriate effective amount in any individual case.

[0037] "Therapeutically effective amount" refers to an amount that provides a therapeutic effect for a given condition and administration regimen. In particular, "therapeutically effective amount" means an amount effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated, which may be a human or non-human animal. Determination of a therapeutically effective amount is within the skill of one of ordinary skill in the art.

[0038] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound of the present invention with other chemical components and entities, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients. Pharmaceutical compositions facilitate the administration of a compound to an organism. In the art, there are multiple techniques for administering a compound, including, but not limited to, intravenous administration, oral administration, aerosol administration, parenteral administration, ocular administration, pulmonary administration and topical administration.

[0039] "Pharmaceutically acceptable" refers to properties and / or substances that are acceptable to a patient from a pharmacological / toxicological standpoint and acceptable to a pharmaceutical scientist from a physical / chemical standpoint with regard to composition, formulation, stability, patient acceptability and bioavailability. A "pharmaceutically acceptable carrier" refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered.

[0040] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier (e.g., liquid or solid fillers, stabilizers, dispersing agents, suspending agents, diluents, excipients, thickeners, solvents, or encapsulating materials) involved in carrying or transporting a compound useful within the invention into or to a patient so that it can perform its intended function. Typically, such constructs are carried or transported from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation containing the compound useful within the invention and not harmful to the patient. Some examples of materials which may function as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations. As used herein, "pharmacologically acceptable carriers" also include any and all coatings, antibacterial and antifungal agents, absorption delaying agents, and the like, that are compatible with the activity of the compounds useful within the present invention and are physiologically acceptable to the patient. Supplementary active compounds can also be incorporated into the compositions. "Pharmaceutically acceptable carriers" can further include pharma-ceutically acceptable salts of the compounds useful within the present invention.Other additional ingredients that may be included in pharmaceutical compositions used to practice the invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference.

[0041] The term "nutritional composition" may be a food product for human consumption, such as a drink, a beverage, a bar, a snack, an ice cream, a dairy product, such as a refrigerated or shelf-stable dairy product, a fermented dairy product, a beverage, such as a dairy drink, an infant formula, a growing milk, a confectionery product, a chocolate, a cereal product, such as a breakfast cereal, a sauce, a soup, an instant drink, a frozen product for consumption after heating in a microwave or oven, a ready-to-eat product, a fast food or a nutritional formulation.

[0042] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof suitable for the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0043] The phrase "biological sample" is used herein in its broadest sense. A sample may be of any biological tissue or fluid from which the biomarkers of the present invention may be detected, extracted, isolated, characterized or measured. Examples of such samples include, but are not limited to, blood, lymph, urine, gynecological fluids, biopsies, amniotic fluid and smears. Samples that are liquid in nature are referred to herein as "body fluids". Biological samples may be obtained from a patient by a variety of techniques including, for example, scraping or wiping an area or using a needle to aspirate a body fluid. Methods for collecting various biological samples are well known in the art. In many cases, the sample will be a "clinical sample", i.e., a sample from a patient. Such samples include, but are not limited to, body fluids (which may or may not contain cells), such as blood (e.g., whole blood, serum or plasma), urine, saliva, tissue or fine needle biopsy samples, as well as archived samples with known diagnostic, treatment and / or outcome histories. Biological samples also include tissues, such as frozen sections taken for histological purposes. Samples also encompass any material obtained by processing a biological sample. The obtained material includes, but is not limited to, cells (or their progeny) isolated from the sample, proteins or nucleic acid molecules extracted from the sample. Processing of a biological sample may include one or more of filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc.

[0044] As used herein, the term "container" includes any receptacle for holding a pharmaceutical composition. For example, in one embodiment, the container is a packaging containing a pharmaceutical composition. In another embodiment, the container is not a packaging containing a pharmaceutical composition, i.e., the container is a receptacle (e.g., a box or a vial) containing a packaged or unpackaged pharmaceutical composition and instructions for use of the pharmaceutical composition. Packaging techniques are also well known in the art. It should be understood that instructions for use of the pharmaceutical composition can be included in the packaging containing the pharmaceutical composition, and the instructions themselves form a highly functional relationship with the packaged product. However, it should be understood that the instructions can contain information regarding the ability of the compound to perform its intended function (e.g., treat or prevent a disease in a subject).

[0045] The term "instructional material" as used herein includes publications, records, diagrams, or any other medium of expression that can be used to communicate the usefulness of the components of the present invention in the kit for identifying or alleviating or treating the various diseases or disorders listed herein. Optionally or alternatively, the educational material may describe one or more methods of identifying or alleviating a disease or disorder in a cell or tissue of a subject. The educational material of the kit may, for example, be attached to a container containing the composition of the present invention or may be shipped together with a container containing the composition of the present invention. Alternatively, the educational material may be shipped separately from the container, with the intention that the recipient will use the educational material and the compound in a coordinated manner.

[0046] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, 6, and any whole and partial increments therebetween. This applies regardless of the breadth of the range.

[0047] explanation The present invention is based in part on the discovery that the introduction of SCFA-producing probiotic bacteria or the introduction of SCFA alone delays the pathogenesis of HCC.The results presented herein demonstrate that the introduction of SCFA-producing probiotic bacteria and the corresponding SCFA alone (without probiotic treatment) suppresses the appearance of dysplastic nodules and HCC in HBx transgenic mice.Therefore, the present invention includes compositions and methods that use the introduction of SCFA-producing bacteria or the introduction of SCFA alone as a simple approach for cancer chemoprevention.The present invention is a novel application for liver cancer and other tumor types that are anatomically located distal to the large intestine.

[0048] composition In one embodiment, the invention provides a short chain fatty acid or a combination of short chain fatty acids. In one embodiment, the invention provides a probiotic bacteria or a combination of probiotic bacteria. In various embodiments, the invention includes compositions for preventing or delaying the onset of hepatocellular carcinoma in a subject, cell, tissue or organ in need thereof. Compositions of the invention include compositions for treating or preventing hepatitis, liver disease, precancerous lesions, and the like.

[0049] Short-chain fatty acids In various embodiments, the present invention includes compositions and methods for preventing or delaying the onset of hepatocellular carcinoma. In various embodiments, the present invention includes compositions and methods for preventing or delaying the onset of hepatocellular carcinoma by treating or preventing liver inflammation, liver disease and precancerous lesions. In one embodiment, a composition for preventing or delaying the onset of hepatocellular carcinoma includes a short chain fatty acid or a combination of short chain fatty acids.

[0050] In one embodiment, the present invention provides a general concept for administering short chain fatty acids as a treatment to prevent or delay the onset of hepatocellular carcinoma. In one embodiment, the composition of the present invention comprises short chain fatty acids. In one embodiment, the short chain fatty acids are selected from the group including, but not limited to, formic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, isocaproic acid, caproic acid, lactic acid, succinic acid, pyruvic acid, octanoic acid and dodecanoic acid. In one embodiment, the combination of short chain fatty acids comprises at least 10 mM, at least 20 mM, at least 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM or more of the equivalent amount of sodium acetate, sodium propionate and sodium butyrate.

[0051] For example, biologically active derivatives of short chain fatty acids having substituents on the carbon chain, such as O, S, N, methyl, ethyl, halogens, and other groups which do not interfere with biological activity, can also be used to form the compositions of the present invention.

[0052] Probiotic bacteria In various embodiments, the present invention includes compositions and methods for preventing or delaying the onset of hepatocellular carcinoma. In various embodiments, the present invention includes compositions and methods for preventing or delaying the onset of hepatocellular carcinoma by treating or preventing liver inflammation, liver disease and precancerous lesions. In one embodiment, a composition for preventing or delaying the onset of hepatocellular carcinoma includes a probiotic bacteria or a combination of probiotic bacteria.

[0053] In one embodiment, the present invention provides a general concept for administering probiotic bacteria as a treatment to prevent or delay the onset of hepatocellular carcinoma. In one embodiment, the composition of the present invention comprises probiotic bacteria. In one embodiment, the probiotic bacteria is selected from the group including, but not limited to, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei, Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus sasei, Lactobacillus salivarius, Pediococcus pentosaceus, Streptococcus thermophiles, Bacillus subtilis, Bacillus coagulans, Enteroccous faecium, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum and Bifidobacterium infantis.

[0054] The probiotic bacteria of the present invention may also include mutants, variants and genetically engineered mutants of probiotic bacterial strains whose genetic and / or phenotypic properties have been altered compared to the parent strain. Naturally occurring variants of probiotic bacterial strains include spontaneous alterations of selectively isolated target properties, whereas deliberate alterations of properties of the parent strain are achieved by conventional genetic engineering techniques, such as gene disruption, conjugative transfer, etc.

[0055] The general state of probiotic bacteria is in the form of live cells or lyophilized cells (which were used to generate the data herein). However, it can also be extended to non-living cells, such as killed cultures, or compositions containing beneficial factors expressed by probiotic bacteria. This can include microorganisms that have been thermally killed, or that have been killed by exposure to pH changes or pressure. With non-living cells, the preparation of the product is more convenient, the cells can be easily incorporated into pharmaceutical products, and storage requirements are much less restrictive than with living cells.

[0056] In one embodiment, the following bacterial compositions and dosages were used to generate the preliminary data presented herein: 10 Lactobacillus plantarum 2362, 10 10 Lactobacillus paracasei subsp paracsei 19, 10 10 Leuconostoc mesenteroids 32-77:1e, and 10 10 of Pediococcus pentosaceus 5-33:3 (a bioactive plant fiber species, having a mixture of 2.5 g inulin, 2.5 g pectin, 2.5 g beta-glucan, and 2.5 g resistant starch (Synbiotic 2000™)). The composition and dosage was administered daily for three months.

[0057] Treatment In one embodiment, the invention provides a method for treating, inhibiting, preventing or reducing hepatocellular carcinoma using a probiotic or a combination of probiotics, hi another embodiment, the invention provides a method for treating, inhibiting, preventing or reducing hepatocellular carcinoma using a short chain fatty acid or a combination of short chain fatty acids.

[0058] The present invention provides a method for preventing or inhibiting the development of hepatocellular carcinoma. Liver inflammation (e.g., hepatitis) causes early lesions that lead to liver cell damage, regeneration, and progression to precancerous nodules and hepatocellular carcinoma nodules. Treating hepatitis and other precancerous lesions can prevent or delay the development of hepatocellular carcinoma. Methods for detecting liver disease and inflammation will be apparent to those skilled in the art and / or described herein.

[0059] The general approach to reduce hepatitis according to the present invention is to provide cells with short-chain fatty acids.In one embodiment, short-chain fatty acids can be delivered directly.In another embodiment, short-chain fatty acids can be delivered indirectly through the metabolism of complex carbohydrates by probiotic bacteria (prebiotics).

[0060] To inhibit hepatitis, short chain fatty acids must be delivered to cells. One delivery mechanism is by any of the above methods of physically or chemically permeabilizing the cell membrane. Another embodiment of the invention for delivering short chain fatty acids to cells can include particle bombardment. This method relies on the ability to accelerate microprojectiles carrying short chain fatty acids to high velocities, allowing them to penetrate the cell membrane and enter the cell without killing it. Several devices have been developed for accelerating small particles. One such device uses a high voltage discharge to generate an electric current, which provides the motive force. The microprojectiles used are made of biologically inert materials, such as tungsten or gold beads.

[0061] In a further embodiment of the invention, the short chain fatty acid can be encapsulated in a liposome. A liposome is a vesicular structure characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of a closed structure, encapsulating water and dissolved solutes between the lipid bilayers.

[0062] The compositions of the present invention and pharmaceutical compositions containing the compounds can be administered orally, and therefore can be formulated in a form suitable for oral administration (i.e., as solid or liquid preparations).Suitable solid oral formulations include tablets, capsules, pills, granules, pellets, etc.Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils, etc.When formulated in the form of a capsule, the compositions of the present invention contain a hard gelling capsule in addition to the active compound and an inert carrier or diluent.

[0063] The compositions of the present invention and pharmaceutical compositions containing the compounds may further be administered intranasally, i.e., by inhalation, and therefore may be formulated in a form suitable for intranasal administration (i.e., as an aerosol or liquid preparation).

[0064] The compositions of the invention may also be formulated as suppositories, e.g., containing conventional suppository bases for use in human or veterinary medicine, or as pessaries, e.g., containing conventional pessary bases.

[0065] One aspect of the present invention provides a method for treating or preventing hepatitis, liver disease, and precancerous lesions using the compositions of the present invention. In one embodiment, the compositions of the present invention can be used to treat or prevent hepatitis, liver disease, and precancerous lesions, thereby inhibiting the development of hepatocellular carcinoma.

[0066] The following are non-limiting examples of liver diseases that may be treated by the disclosed methods and compositions: liver fibrosis, liver disease associated with obesity, liver disease associated with metabolic syndrome, liver cirrhosis, alcoholic cirrhosis, non-alcoholic cirrhosis, fatty liver, cirrhosis associated with diabetes, inherited liver disease, hepatic steatosis, or chronic hepatitis. This includes chronic liver disease associated with other viruses, such as HIV, where an estimated 40% of individuals on long-term antiretroviral therapy develop chronic liver disease.

[0067] In addition to hepatocellular carcinoma, cancers that may be treated or vaccinated against (i.e., prophylactically treated) by administering to a subject a composition of the invention may be a cancer selected from the group consisting of B cell lymphoma, T cell lymphoma, myeloma, leukemia, hematopoietic neoplasms, thymoma, lymphoma, sarcoma, lung cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, uterine cancer, adenocarcinoma, breast cancer, pancreatic cancer, lung cancer, kidney cancer, bladder cancer, prostate cancer, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancer, ductosarcoma, angiosarcoma, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, uterine cancer, cervical cancer, gastrointestinal cancer, and any other cancer now known or identified in the future (see, e.g., Rosenberg (1996) Ann. Rev. Med. 47:481-491, the entire contents of which are incorporated herein by reference). Further immunogens contemplated within the scope of the present invention are infectious agent immunogens, which may include any immunogen suitable for protecting a subject against infectious disease, including, but not limited to, microbial, bacterial, protozoal, parasitic, fungal and viral diseases. In addition to inflammation-associated cancers, SCFAs may also be useful in the treatment of inflammation-associated autoimmune or autoaggressive diseases (e.g., rheumatoid arthritis).

[0068] In some embodiments of the methods for inhibiting cancer in an individual in need thereof, a second agent, e.g., an anti-neoplastic agent, is administered to the individual. In some embodiments, the second agent comprises a second metastasis inhibitor, e.g., a plasminogen antagonist or an adenosine deaminase antagonist. In other embodiments, the second agent is an angiogenesis inhibitor.

[0069] The compositions of the present invention can be used to prevent, reduce, minimize, control and / or shrink tumor metastasis in humans and animals. The disclosed compounds can also be used to slow down the rate of primary tumor growth. The disclosed compounds, when administered to a subject in need of treatment, can be used to stop the spread of cancer cells. Thus, the compounds disclosed herein can be administered as part of a combination therapy with one or more drugs or other pharmaceuticals. When used as part of a combination therapy, the reduction in metastasis and the reduction in primary tumor growth provided by the disclosed compounds allows for more effective and efficient use of any pharmaceutical or drug therapy used to treat the patient. In addition, the control of metastasis by the disclosed compounds gives the subject a greater ability to focus the disease in one place.

[0070] The following are non-limiting examples of cancers that may be treated by the disclosed methods and compositions: acute lymphoblastic; acute myeloid leukemia; adrenal cortical carcinoma; pediatric adrenal cortical carcinoma; appendix cancer; basal cell carcinoma; extrahepatic bile duct cancer; bladder cancer; bone cancer; osteosarcoma and malignant fibrous histiocytoma; pediatric brain stem glioma; adult brain tumor; brain tumor, brain stem glioma (pediatric); brain tumor, central nervous system atypical teratoid / rhabdoid tumor (pediatric); central nervous system embryonal tumor; cerebellar astrocytoma; cerebral astrocytoma / malignant glioma; Craniopharyngioma;Ependymoblastoma;Ependymoma;Medulloblastoma;Medulloepithelioma;Intermediate pineal parenchymal tumor;Supratentorial primitive neuroectodermal tumor and pineoblastoma;Visual pathway and hypothalamic glioma;Brain and spinal tumors;Breast cancer;Bronchial tumor;Burkitt's lymphoma;Carcinoid tumor;Gastrointestinal carcinoid tumor;Central nervous system atypical teratoid / rhabdoid tumor;Central nervous system embryonal tumor;Central nervous system lymphoma;Cerebellar astrocytoma;Cerebral astrocytoma / malignant glioma (pediatric);Cervical cancer;Pediatric chordoma;Chronic lymphoma Lymphatic leukemia;Chronic myelogenous leukemia;Chronic myeloproliferative disorder;Colon cancer;Colorectal cancer;Craniopharyngioma;Cutaneous T-cell lymphoma;Esophageal cancer;Ewing family tumors;Extragonadal goblet cell tumor;Extrahepatic bile duct cancer;Eye cancer, intraocular melanoma;Eye cancer, retinoblastoma;Gallbladder cancer;Gastric (Stomach) cancer;Gastrointestinal carcinoid tumor;Gastrointestinal stromal tumor (GIST);Extracranial goblet cell tumor;Extragonadal goblet cell tumor;Ovarian goblet cell tumor;Gestational trophoblastic tumor;Glioma;Pediatric Brain stem glioma;Childhood cerebral astrocytoma glioma;Childhood visual pathway and hypothalamic glioma;Hairy cell leukemia;Head and neck cancer;Hepatocellular (liver) cancer;Langerhans cell histiocytosis;Hodgkin's lymphoma;Hypopharyngeal cancer;Hypothalamic and visual pathway glioma;Intraocular melanoma;Islet cell tumor;Kidney (renal cell) cancer;Langerhans cell histiocytosis;Laryngeal cancer;Acute lymphoblastic leukemia;Acute myeloid leukemia;Chronic lymphocytic leukemia;Chronic myeloid leukemia;Hairy cell leukemia;Lip and oral cancer (Lip and Oral Cavity Cancer);Liver Cancer;Non-Small Cell Lung Cancer;Small Cell Lung Cancer;AIDS-Related Lymphoma;Burkitt's Lymphoma;Cutaneous T-Cell Lymphoma;Hodgkin's Lymphoma;Non-Hodgkin's Lymphoma;Primary Central Nervous System Lymphoma;Waldenstrom's Macroglobulinemia;Malignant Fibrous Histiocytoma and Osteosarcoma of Bone;Medulloblastoma;Melanoma;Intraocular (Eye) Melanoma;Merkel Cell Carcinoma;Mesothelioma;Metastatic Squamous Neck Cancer of Unknown Primary;Oral Cancer;Multiple Endocrine Neoplasia Syndrome (Childhood);Multiple Myeloma / Plasma Cell Neoplasms;Mycosis Fungoides;Myelodysplastic Syndromes;Myelodysplastic / Myeloproliferative Disorders;Chronic Myeloid Leukemia;Adult Acute Myeloid Leukemia;Childhood Acute Myelogenous Leukemia;Multiple Myeloma;Chronic Myeloproliferative Disorders;Nasal and Paranasal Sinus Cancer;Nasopharyngeal Cancer;Neuroblastoma;Non-Small Cell Lung Cancer;Oral Cancer;Oral Cavity Cancer Cancer);Oropharyngeal cancer;Osteosarcoma and malignant fibrous histiocytoma of bone;Ovarian cancer;Ovarian epithelial cancer;Ovarian goblet cell tumor;Ovarian low malignant potential tumor;Pancreatic cancer;Pancreatic cancer, islet cell tumor;Papillomatosis;Parathyroid cancer;Penile cancer;Pharyngeal cancer;Pheochromocytoma;Intermediate pineal parenchymal tumor;Pineoblastoma and supratentorial primitive neuroectodermal tumor;Pituitary tumor;Plasma Cell Neoplasms Neoplasm) / multiple myeloma;pleuropulmonary blastoma;primary central nervous system lymphoma;prostate cancer;rectal cancer;renal cell (kidney) cancer;transitional cell carcinoma of the renal pelvis and ureter;airway cancer involving the NUT gene on chromosome 15;retinoblastoma;rhabdomyosarcoma;salivary gland cancer;sarcoma, Ewing family tumors;Kaposi's sarcoma;soft tissue sarcoma;uterine sarcoma;Sezary syndrome;skin cancer (non-melanoma);skin cancer (melanoma);Merkel cell skin cancer;small cell lung cancer;small Intestinal cancer; soft tissue sarcoma; squamous cell carcinoma, metastatic squamous cell cervical cancer of unknown primary; Stomach (Gastric) cancer; supratentorial primitive neuroectodermal tumor; cutaneous T-cell lymphoma; testicular cancer; throat cancer; thymoma and thymic carcinoma; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic tumor; urethral cancer; uterine cancer (endometrium); uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom's macroglobulinemia; and Wilms' tumor.

[0071] In one embodiment, the invention provides a method for treating cancer comprising treating a subject with a complementary cancer therapy, such as surgery, chemotherapy, chemotherapeutic agents, radiation therapy or hormonal therapy, or a combination thereof, prior to, simultaneously with or after treatment with a composition of the invention.

[0072] In another embodiment, the invention provides a method for treating cancer comprising treating a subject with a complementary cancer therapy, such as surgery, chemotherapy, chemotherapeutic agents, radiation therapy or hormonal therapy, or a combination thereof, prior to, simultaneously with or after treatment with a composition of the invention.

[0073] Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine sodium phosphate, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alpha-2a recombinant, paclitaxel, teniposide, and streptozocin), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylesulfonic acid). acid), alkylating agents (e.g., asaril, AZQ, BCNU, busulfan, bisulfan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis-platinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, dianhydrogalactitol, fluorodopan, hepsulfame, hycanthone, ifosphamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, pol filomycin, spirohydantoin mustard, streptozotocin, teroxylon, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, halichondrin M, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, tritylcysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mithramycin, mitomycin, daunorubicin,VP-16-213, VM-26, navelbine and taxotere), biologics (e.g., alpha interferon, BCG, G-CSF, GM-CSF and interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantron, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCl, daunorubicin, dexamethasone, cyclosporine ... oxidoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26, and VP-16) and synthetic agents (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-diamminedichloroplatinum, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel, and porfimer sodium).

[0074] Antiproliferative agents are compounds that reduce cell proliferation. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, miscellaneous drugs, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., tamoxifen citrate and its analogs, toremifene, droloxifene, and roloxifene). Further examples of specific antiproliferative agents include, but are not limited to, levamisole, gallium nitrate, granisetron, sargramostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron.

[0075] The compositions of the present invention may be administered alone or in combination with other anti-tumor agents, including cytotoxic / antineoplastic agents and anti-angiogenic agents. Cytotoxic / antineoplastic agents are defined as agents that attack and kill cancer cells. Some cytotoxic / antineoplastic agents are alkylating agents that alkylate genetic material in tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, verstine, uracil mustard, chlomaphazin, and dacabazine. Other cytotoxic / antineoplastic agents are tumor cell antimetabolites, such as cytosine arabinoside, fluorouracil, methotrexate, mercaptopuirine, azathioprime, and procarbazine. Other cytotoxic / antineoplastic agents are antibiotics, such as doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mytomycin C, and daunomycin. There are numerous liposomal formulations of these compounds available commercially. Further, other cytotoxic / antineoplastic agents are mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Miscellaneous cytotoxic / antineoplastic agents include taxol and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacitidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0076] Antiangiogenic agents are well known to those skilled in the art. Suitable antiangiogenic agents for use in the methods and compositions of the present disclosure include anti-VEGF antibodies (including humanized and chimeric antibodies), anti-VEGF aptamers and antisense oligonucleotides. Other known angiogenesis inhibitors include angiostatin, endostatin, interferon, interleukin 1 (including α and β), interleukin 12, retinoic acid and tissue inhibitor of metalloproteinase-1 and -2 (TIMP-1 and -2). Small molecules that contain topoisomerase (e.g., razoxane, a topoisomerase II inhibitor with antiangiogenic activity) can also be used.

[0077] Other anti-cancer drugs that may be used in combination with the disclosed compounds include, but are not limited to, acivicin; aclarubicin; acodazole hydrochloride; acronine; adzelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizeresin; bleomycin sulfate; blequina sodium;bropirimine;busulfan;cactinomycin;calsterone;caracemide;carbetimer;carboplatin;carmustine;carubicin hydrochloride;carzelesin;cedefingol;chlorambucil;ciloremycin;cisplatin;cladribine;crisnatol mesylate;cyclophosphamide;cytarabine;dacarbazine;dactinomycin;daunorubicin hydrochloride;decitabine;dexormaplatin;desaguanine;desaguanine mesylate;diazicon;docetaxel;doxorubicin;doxorubicin hydrochloride;droloxifene ;droloxifene citrate;dromostanolone propionate;duazomycin;edatrexate;eflornithine hydrochloride;elsamitrucin;enloplatin;enpromate;epipropizine;epirubicin hydrochloride;elbrozole;esorubicin hydrochloride;estramustine;estramustine sodium phosphate;etanidazole;etoposide;etoposide phosphate;etoprine;fadrozole hydrochloride;fazarabine;fenretinide;floxuridine;fludarabine phosphate;fluorouracil;fluorocitabine (fluor ocitabine;foskidone;fostriecin sodium;gemcitabine;gemcitabine hydrochloride;hydroxyurea;idarubicin hydrochloride;ifosfamide;irmofosine;interleukin II (including recombinant interleukin II or rIL2);interferon alpha-2a;interferon alpha-2b;interferon alpha-n1;interferon alpha-n3;interferon beta-Ia;interferon gamma-Ib;iproplatin;irinotecan hydrochloride;lanreotide acetate;letrozole;leuprolide acetate;liarozole hydrochloride;Lometrexol sodium; Lomustine; Losoxantrone hydrochloride; Masoprocol; Maytansine; Mechlorethamine hydrochloride; Megestrol acetate; Melengestrol acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate sodium; Metoprine; Meturedepa; Mitindomide; Mitocalcine; Mitochromine; Mitogillin; Mitomarcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole; Noga ramycin;ormaplatin;oxisuran;paclitaxel;peguasparagase;periomycin;pentamustine;peplomycin sulfate;perfosfamide;pipobroman;piposulfan;piroxantrone hydrochloride;plicamycin;promestane;porfimer sodium;porfiromycin;prednimustine;procarbazine hydrochloride;puromycin;puromycin hydrochloride;pirazofurin;ribopurin;logretimide;safingol;safingol hydrochloride;cetamide Mustine;Simtrazene;Sparfosate sodium;Sparsomycin;Spirogermanium hydrochloride;Spiromustine;Spiroplatin;Streptonigrin;Streptozocin;Sulofenur;Tallysomycin;Tecogalan sodium;Tegafur;Teroxantrone hydrochloride;Temoporfin;Teniposide;Teroxylon;Testolactone;Thiamiprine;Thioguanine;Thiotepa;Tiazofurin;Tirapazamine;Toremifene citrate;Trestorone acetate;Triciribine phosphate; Trimetrexate; Trimetrexate glucuronide; Triptorelin; Tubrozole hydrochloride; Uracil mustard; Uredep; Vapreotide; Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Vinepidine sulfate; Vinglisinate sulfate; Vinleurosine sulfate; Vinorelbine tartrate; Vinrocidine sulfate; Vinzolidine sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin hydrochloride; Other anti-cancer drugs include, but are not limited to, 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil;abiraterone;aclarubicin;acylfulvene;adecypenol;adozelesin;aldesleukin;ALL-TK antagonists;altretamine;ambamustine;amidox;amifostine;aminolevulinic acid;amrubicin;amsacrine;anagrelide;anastrozole;andrographolide;angiogenesis inhibitors;antagonist D;antagonist G;antarelix;anti-dorsalized morphogenetic protein-1;antiandrogens (prostate cancer);antiestrogens;anti-neoplastic agents;antisense oligonucleotides;aphidicolin glycine salt;apoptotic gene modulators;apoptotic regulators;apurinic acid;ara-CDP-DL-PTBA;arginine deaminase;aslac Phosphorus;Atamestane;Atrimustine;Axinastatin 1;Axinastatin 2;Axinastatin 3;Azasetron;Azatoxin;Azatyrosine;Baccatin III derivatives;Balanol;Batimastat;BCR / ABL antagonists;Benzochlorins;Benzoylstaurosporine;β-lactam derivatives;β-arretin;Betaclamycin B;Betulinic acid;bFGF inhibitors;Bicalutamide;Bisantrene;Bisaziridinylspermine;Bisnafide;Bistratin A;Bizelesin;Brefrath;Bropirimine;Budotitanium;Buthionine sulfoximine;Calcipotriol;Calfostin C;Camptothecin derivatives;Canarypox IL-2;Capecitabine;Carboxamido-amino-triazoles;Carboxamidotriazoles;CaRest M3;CARN700;cartilage-derived inhibitor;carzelesin;casein kinase inhibitor (ICOS);castanospermine;cecropin B;cetrorelix;chlorin;chloroquinoxaline sulfonamide;cicaprost;cis-porphyrin;cladribine;clomiphene analogues;clotrimazole;collismycin A;collismycin B;combretastatin A4;combretastatin analogues;conagenin;crumbesidin 816;crisnatol;cryptophycin 8;cryptophycin A derivatives;curacin A;cyclopentathraquinone;cycloplatam;sipemycin;cytarabine ocfosfate;cytolytic factor;cytostatin;dacliximab;decitabine;dehydrodidemnin B;deslorelin;dexamethasone;dexifosfamide;dexrazoxane;dexverapamil;diaziquone;didemnin B;didox;diethylnorspermine;dihydro-5-azacytidine;9-dihydrotaxol, 9-);Dioxamycin;Diphenylspiromustine;Docetaxel;Docosanol;Dolasetron;Doxifluridine;Droloxifene;Dronabinol;Duocarmycin SA;Ebselen;Ecomustine;Edelfosine;Edrecolomab;Eflornithine;Elemene;Emitefur;Epirubicin;Epristeride;Estramustine analogs;Estrogen agonists;Estrogen antagonists;Etanidazole;Etoposide phosphate;Exemestane;Fadrozole;Fazarabine;Fenretinide;Filgrastim;Finasteride;Flavopiridol;Flazelastine;Fluasterone;Fludarabine;Fluorodaunornithine hydrochloride (fluorod aunorunicin);forfenimex;formestane;fostriecin;fotemustine;gadolinium texaphyrin;gallium nitrate;galocitabine;ganirelix;gelatinase inhibitors;gemcitabine;glutathione inhibitors;hepsulfame;heregulin;hexamethylene bisacetamide;hypericin;ibandronate;idarubicin;idoxifene;idramantone;ilmofosine;ilomastat;imidazoacridone;imiquimod;immunostimulating peptides;insulin-like growth factor-1 receptor inhibitors;interferon agonists;interferon;interleukin;iobenguane;iododoxorubicin; 4-Ipomeanol;Iropract;Irosogladine;Isobengazole;Isohomohalichondrin B;Itasetron;Jasplakinolide;Kahalalide F;Lamellarin-N triacetate;Lanreotide;Leinamycin;Lenograstim;Lentinan sulfate;Leptolstatin;Letrozole;Leukemia inhibitory factor;Leukocyte alpha interferon;Leuprolide + estrogen + progesterone;Leuprorelin;Levamisole;Liarozole;Linear polyamine analogs;Lipophilic disaccharide peptides;Lipophilic platinum compounds;Lissoclinamide 7;Lobaplatin;Lombricine;Lometrexol;Lonidamine;Losoxantrone;Lovastatin;Loxoribine;Lultotecan;Lutetium texaphyrin; Lysofylline;lytic peptides;maitansine;mannostatin A;marimastat;masoprocol;maspin;matrilysine inhibitors;matrix metalloproteinase inhibitors;menogaril;merbarone;meterelin;methioninase;metoclopramide;MIF inhibitors;mifepristone;miltefosine;mirimostim;mismatched double-stranded RNA;mitoguazone;mitolactol;mitomycin analogues;mitonafide; Mytotoxin fibroblast growth factor-saporin; mitoxantrone; mofalotene; molgramostim; monoclonal antibodies; human chorionic gonadotrophin; monophosphoryl lipid A + myobacterium cell wall sk; mopidamol; multidrug resistance gene inhibitors; multiple tumor suppressor 1-based therapy; mustard anticancer drugs; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N -Acetyldinaline;N-substituted benzamides;Nafarelin;Nagrestip;Naloxone + pentazocine;Napavin;Nafterpine;Nartograstim;Nedaplatin;Nemorubicin;Neridronic acid;Neutral endopeptidase;Nilutamide;Nisamycin;Nitric oxide modulators;Nitroxide antioxidants;Nitrullin;O6-benzylguanine;Octreotide;Oxenone;Oligonucleotides; Onapristone; Ondansetron; Ondansetron; Oracin; Oral cytokine inducers; Ormaplatin; Osateron; Oxaliplatin; Oxaunomycin; Paclitaxel; Paclitaxel analogs; Paclitaxel derivatives; Palauamine; Palmitoyl rhizoxin; Pamidronic acid; Panaxytriol;Panomyphen;Parabactin;Pazeliptin;Pegaspargase;Perdecin;Pentosan polysulfate sodium;Pentostatin;Pentrozole;Perflubron;Perphosphamide;Perillyl alcohol;Phenazinomycin;Phenyl acetate;Phosphatase inhibitors;Picibanil;Pilocarpine hydrochloride;Pirarubicin;Piritrexim;Placetin A;Placetin B;Plasminogen activator inhibitors;Platinum complexes;Platinum compounds;Platinum-triamine complexes;Porfimer sodium;Porfiromycin;Prednisone;Pla lopyrbis-acridone;prostaglandin J2;proteasome inhibitors;protein A-based immune modulators;protein kinase C inhibitors;protein kinase C inhibitors (microalgae);protein tyrosine phosphatase inhibitors;purine nucleoside phosphorylase inhibitors;purpurins;pyrazoloacridines;pyridoxylated hemoglobin polyoxyethylene conjugates;raf antagonists;raltitrexed;ramosetron;ras farnesyl protein transferase inhibitors;ras inhibitors;ras-GAP inhibitors;demethylated reterliptin;rhenium etidronate Re186 (rhenium Re 186 etidronate; rhizoxin; ribozyme; RII retinamide; logretimide; rohitukin; romurtide; roquinimex; rubiginone B1; ruboxil; safingol; saintpin; SarCNU; sarcophytol A; sargramostim; Sdi1 mimetic; semustine; senescence derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction modulator; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate (sodium borocaptate);sodium phenylacetate;solverol;somatomedin binding protein;sonermin;sparfosic acid;spicamycin D;spiromustine;splenopentin;spongistatin 1;squalamine;stem cell inhibitors;stem cell division inhibitors;stypiamide;stromelysin inhibitors;sulfinosine;superactive vasoactive intestinal peptide antagonists;suradista;suramin;swainsonine;Synthetic glycosaminoglycans;Talimustine;Tamoxifen methiodide;Tauromustine;Tazarotene;Tecogalan sodium;Tegafur;Terlapyrylium;Telomerase inhibitors;Temoporfin;Temozolomide;Teniposide;Tetrachlorodecaoxide;Tetrazomine;Taliblastine;Thiocoraline;Thrombopoietin;Thrombopoietin mimetics;Thymalfasin;Thymopoietin receptor agonists;Thymotrinan;Thyroid-stimulating hormone;Ethyl etiopurinse;Tirapazamine;Titanocene dichloride;Topsentin;Toremifene;Totipotent stem cell factor;Translation inhibitors;Tre These include thinoin, triacetyluridine, triciribine, trimetrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists, vapreotide, variolin B, vector systems, erythrocyte gene therapy, veraresol, veramine, verudin, verteporfin, vinorelbine, vinxaltine, vitaxin, vorozole, zanoteron, zeniplatin, zilascorub and zinostatin stimalamer. In one embodiment, the anticancer drug is 5-fluorouracil, taxol or leucovorin.

[0078] Pharmaceutical Compositions The present invention includes pharmaceutical compositions comprising one or more compositions of the present invention.The formulation of pharmaceutical compositions described herein can be prepared by any method known or developed in the field of pharmacology.In general, such preparation methods include combining active ingredient with carrier or one or more other accessory ingredients, and then, if necessary or desired, shaping or packaging the product into a desired single- or multiple-dose unit.

[0079] The compositions may contain further medicinal agents, pharmaceutical agents, carriers, buffers, adjuvants, dispersing agents, diluents, etc. depending on the intended use and application.

[0080] Examples of suitable pharmaceutical carriers, excipients and / or diluents are well known in the art and include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc or mixtures thereof.

[0081] Pharmaceutically acceptable carriers for liquid preparations are aqueous or non-aqueous solutions, suspensions, emulsions or oils.Examples of non-aqueous solvents are propylene glycol, polyethylene glycol and injectable organic esters, such as ethyl oleate.Examples of oils are those of animal origin, vegetable origin or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, turmeric oil, fish liver oil, other marine oils, or fats derived from milk or eggs.

[0082] Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media such as phosphate buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions containing such carriers can be formulated by well-known conventional methods. Suitable carriers can include any material that retains biological activity when combined with the biologically active compounds of the present invention. Preparations for parenteral administration can include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), etc. Preservatives and other additives may also be present including, for example, antibacterial agents, antioxidants, chelating agents, and inert gases and the like, and in addition, the pharmaceutical composition of the invention may include a proteinaceous carrier such as, for example, serum albumin or immunoglobulin, preferably of human origin.

[0083] The pharmaceutical compositions provided herein may also be administered as controlled release compositions, i.e., compositions in which the active ingredient is released over a period of time after administration. Controlled release or sustained release compositions include formulations in lipophilic depots (e.g., fatty acids, waxes, oils). In another embodiment, the composition is an immediate release composition, i.e., a composition in which all of the active ingredient is released immediately after administration.

[0084] Furthermore, the pharmaceutical compositions of the present invention and described herein in various embodiments, or compositions comprising said compounds, may be administered by mixing them into food, functional foods, beverages, medical foods.

[0085] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all kinds of animals. Modifications of pharmaceutical compositions suitable for administration to humans to make the compositions suitable for administration to various animals are well understood, and an ordinarily skilled veterinary pharmacologist can design and perform such modifications, if necessary, with no more than routine experimentation. Subjects to which the pharmaceutical compositions of the present invention are contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals, such as non-human primates, cows, pigs, horses, sheep, cats and dogs.

[0086] Pharmaceutical compositions useful in the methods of the invention may be prepared, packaged, or sold in a formulation suitable for ocular, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intratumoral, epidural, intracerebral, intraventricular, or another route of administration. Other contemplated formulations include ejected nanoparticles, liposomal preparations, resealed red blood cells containing the active ingredient, and immunological-based preparations.

[0087] The pharmaceutical composition of the present invention can be prepared, packaged or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dosage of active ingredient administered to a subject, or a convenient fraction of such a dosage, such as 1 / 2 or 1 / 3 of such a dosage.

[0088] The relative amounts of active ingredient, pharma- ceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is to be administered. By way of example, a composition may contain from 0.1% to 100% (w / w) active ingredient.

[0089] In addition to the active ingredient, the pharmaceutical compositions of the present invention may further comprise one or more additional pharma- ceutical active agents.

[0090] Controlled or sustained release formulations of the pharmaceutical compositions of the invention may be made using conventional techniques.

[0091] The composition of the present invention may also include a prebiotic component. "Prebiotics" include substances or compounds that are fermented by the intestinal flora of a pet, thus promoting the growth or development of lactic acid bacteria in the gastrointestinal tract of a pet, taking advantage of pathogenic bacteria. The result of this fermentation may be the release of fatty acids, particularly short-chain fatty acids, in the colon. This release may have the effect of decreasing the pH value in the colon. Non-limiting examples of suitable prebiotics include oligosaccharides, such as fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, or inulin and its hydrolysis products, commonly known as oligoderivatives of starch (e.g., pectin, β-glucan, and resistant starch). Prebiotics may be provided in any suitable form. For example, prebiotics may be provided in the form of plant material containing fiber. Suitable plant materials include asparagus, artichoke, onion, wheat or chicory, or residues of these plant materials. Alternatively, the prebiotic fiber may be provided as an inulin extract, for example an extract from chicory is suitable. A suitable inulin extract may be obtained from Orafti SA, Tirlemont 3300, Belgium, under the trade name "Raftiline". For example, the inulin may be provided in the form of Raftiline (g) ST, which is a white fine powder containing about 90 to about 94% by weight of inulin, up to about 4% by weight of glucose and fructose, and about 4 to 9% by weight of sucrose. Alternatively, the fiber may be in the form of fructooligosaccharides, for example as obtained from Orafti SA, Tirlemont 3300, Belgium, under the trade name "Raftilose". For example, the inulin may be provided in the form of Raftilose (g) P95. Alternatively, the fructooligosaccharides may be obtained by hydrolyzing inulin, by enzymatic methods, or by using microorganisms.

[0092] Pharmaceutical compositions also include nutritional compositions, for example oral nutritional compositions for oral ingestion and optionally for enteral absorption, which nutritional compositions comprise a compound of the invention.

[0093] When the nutritional composition is formulated and administered orally, the composition can be a liquid oral nutritional supplement (e.g., an incomplete nutritional supplement) or a complete nutritional supplement. Thus, the nutritional composition can be administered in any known form, including, for example, tablets, capsules, liquids, chewables, soft gels, sachets, powders, syrups, liquid suspensions, emulsions, and solutions in a convenient dosage form.

[0094] A nutritional formulation includes any nutritionally complete or supplemental formulation (e.g., nutritional supplement). As used herein, "nutritionally complete" is preferably a nutritional product that contains sufficient variety and levels of macronutrients (protein, fat and carbohydrate) and sufficient micronutrients to be the sole source of nutrition for the subject to which it is administered. A patient may obtain 100% of their nutritional requirements from such a complete nutritional composition. According to one embodiment, the nutritional formulation is a supplemental formulation that provides supplemental nutrients. A "supplemental formulation" may not be nutritionally complete, but preferably contains specific nutrients that are supportive and / or address specific or additional needs of a subject, for example, in combination with the beneficial effects of physical exercise and the present invention.

[0095] The nutritional formulation may be a generally applicable nutritional formulation, for example adapted for subjects of a certain age (e.g. a children's formula), but it may also be a formulation for elderly patients, a formulation for intensive care patients, or a formulation specially adapted for example for patients suffering from a certain disease. Any nutritional formulation may be, for example, a ready-to-drink, reconstitutable (i.e. present in a substantially dry state, for example in powder form) or in the form of a liquid formulation.

[0096] Kits of the Invention The invention also includes kits containing compounds useful in the methods of the invention and instructional materials describing, for example, how to administer short chain fatty acids as described elsewhere herein or how to administer probiotic bacteria as described elsewhere herein. Formulations of pharmaceutical compositions suitable for parenteral administration include the active ingredient in combination with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, for example, in ampoules, or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further include one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0097] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients, such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable preparations may be prepared, for example, using a non-toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono- or diglycerides. Other parenterally administrable formulations that are useful include those that contain the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharma-ceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts. EXAMPLES

[0098] Experimental Example The present invention will be described in more detail with reference to the following experimental examples.These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified.Therefore, the present invention should not be interpreted as being limited to the following examples in any way, but rather should be interpreted as embracing any and all variations that become evident as a result of the teachings provided herein.

[0099] Without further description, it is believed that one of ordinary skill in the art can, using the above description and the following illustrative examples, make and utilize the compounds of the present invention and practice the methods described in the claims. Accordingly, the following examples specifically illustrate preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0100] Example 1: Commensal bacteria provide chemoprevention against hepatitis B virus-mediated hepatocellular carcinoma in hepatitis B x transgenic mice Chronic infection with Hepatitis B Virus (HBV) is associated with the progressive development of chronic liver disease (CLD) and the emergence of hepatocellular carcinoma (HCC). HCC is a cancer with worldwide prevalence and few treatment options. Considering that HCC develops decades after infection and most often appears on a background of chronic inflammation, experiments were designed to test the hypothesis that selected probiotic bacteria known to suppress inflammation could be used as a simple and inexpensive means to prevent or delay the emergence of HCC. To test this, Hepatitis B x (HBx) transgenic mice, which develop progressive liver lesions leading to HCC, were treated with a mixture of probiotic bacteria (Synbiotic 2000™). The results showed a significant reduction in the number and size of dysplastic and HCC nodules compared to control transgenic mice. Microarray analysis of selected immune and cancer-related markers showed a strong decrease in expression in the livers of mice treated with Synbiotic 2000™ compared to control mice. Since the bacteria used metabolize complex carbohydrates to short chain fatty acids (SCFAs), which are known to have anti-inflammatory properties in other systems, in parallel experiments, HBx transgenic mice were fed a combination of SCFAs produced by Synbiotic 2000™ (acetate, proprionate, butyrate) in the absence of bacteria. The results again showed a strong reduction in the number and size of dysplastic and HCC nodules. These results indicate that Synbiotic 2000™ or their metabolic by-products in the form of SCFAs attenuate the pathogenesis of HCC and may be useful as a cancer chemoprevention approach not only for HCC but also for other cancers that often develop on the background of chronic inflammation.

[0101] The materials and methods used in these experiments are described below.

[0102] Materials and Methods mouse To study the pathogenesis of HCC and evaluate new treatment approaches, an HBx transgenic mouse model has been created (Yu DY et al., J Hepatol, 1999; 31:123-132). At birth, these HBx transgenic mice have little or no HBx expression in the liver and no pathology. By 3-4 months of age, they develop detectable HBx associated with hepatitis / steatosis. By 6-7 months of age, the presence, frequency, and distribution of intrahepatic HBx becomes substantial, which is associated with the appearance of dysplastic nodules and micro-HCC. By 9-10 months of age, extensive HBx staining is associated with the appearance of macroscopic HCC nodules. Given that this sequence of events is similar to that of chronic human infection, this animal model was used in this study.

[0103] To test the hypothesis that Synbiotic 2000™ or its SCFA metabolites would delay or prevent the pathogenesis of HBV-associated HCC, HBx transgenic mice were used (Arzumanyan A et al., Cancer Res, 2012; 72(22):5912-5920). The HBx gene was used with its enhancer / promoter complex to generate transgenic mice in a C57Bl6 background, so that HBx was expressed only in differentiated hepatocytes. This resulted in an age-dependent increase in HBx expression associated with the progression of lesions in the liver. These mice were provided by Dr. Dae-Yeul Yu (Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea) and were crossed with CBA mice. A colony of C57Bl6 / CBA mice was then generated by sibling mating. The latter had a higher incidence of animals with advanced lesions compared to the original C57B16 transgenic mouse strain.

[0104] Probiotic bacteria and short chain fatty acids (SCFAs) Synbiotic 2000™ was provided by Medipharm (Des Moines, Iowa). It contains a mixture of four lactic acid producing bacilli (1010 Lactobacillus plantarum 2362, 1010 Lactobacillus paracasei subsp paracasei 19, 1010 Leuconostoc mesenteroides 32-77:1e, and 1010 Pediococcus pentosaceus 5-33:3) per packet, and a mixture of four bioactive plant fiber species (2.5 g inulin; 2.5 g pectin; 2.5 g β-glucan, and 2.5 g resistant starch). It was administered daily by gavage at 0.05 g / dose (2.5 g / 30 ml water, 0.6 ml dose / mouse) for three months.

[0105] Short-chain fatty acids consisting of sodium acetate, sodium propionate, and sodium butyrate were purchased from Acros Organics (Geel, Belgium) through Fisher Scientific (Fairlawn, NJ) and were administered by gavage at 0.2 ml (containing 150 mM SCFA (50 mM each SCFA)) / day for 30 days.

[0106] Protocol HBx transgenic mice were tested for the presence of the HBx gene by tail snip analysis and real-time PCR amplification. HBx protein was evaluated by immunohistochemical staining of sections cut from formalin-fixed paraffin-embedded liver tissue as previously described (Arzumanyan A et al., Cancer Res, 2012; 72(22):5912-5920). For this study, groups of 10 HBx transgenic mice at 3, 6 and 9 months were gavaged daily for 3 months with freshly reconstituted Synbiotic 2000™. Control groups included age-sex-matched HBx transgenic mice gavaged with PBS instead of Synbiotic 2000™. Groups of age-sex-matched HBx negative littermates were gavaged with Synbiotic 2000™ or PBS. All mice were periodically bled retroorbitally for alanine aminotransferase (ALT) measurements (ALT / GPT 50, Sigma Chemical Co., St. Louis, MO) and euthanized after 3 months of treatment. Mice were weighed immediately before each blood draw, and liver weights were measured after euthanasia. Tumor nodules visible on the surface of each liver were counted. Liver samples from each lobe were then embedded, and sections were stained with H&E. Two individuals independently examined each liver slide by light microscopy under a cord and recorded various lesions. The remaining liver tissues of all mice were snap frozen in liquid nitrogen and stored at −80°C. All animal protocols for this study were approved by the Temple University Institutional Animal Care and Use Committee.

[0107] RNA isolation and cDNA synthesis All mouse frozen liver tissue samples were homogenized in lysis buffer (RTL) using a handheld rotor-stator homogenizer (TissueRuptor, Qiagen) with an autoclaved non-disposable probe. Total RNA from each sample was then extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Contaminating DNA was removed using an RNase-Free DNase kit (Qiagen). RNA concentration was measured by reading the absorbance of 1 μl of each sample at 260 and 280 nm with a Nanodrop UV-Vis Spectrophotometer (Thermo Scientific). A typical yield of 50–1800 ng / μl was obtained in elution buffer (final volume of 50 μl) from an initial tissue sample of 30 mg. Samples were then aliquoted to a final concentration of 50 ng (ηg) / μl and stored at -80°C.

[0108] Reverse transcription was accomplished using 500 ng (ηg) of total RNA from each of these samples using the RT2 First Strand Kit (Qiagen) according to the manufacturer's instructions provided. Samples were then stored at -20°C until use in the qPCR array.

[0109] qPCR array assay A microwell-formatted Custom RT2 Profiler PCR-array (SA Biosciences, Qiagen, Izasa) contained a panel of genes that matched the specific research interests of this study. Amplification of cDNA was performed using the RT2 SYBR Green Rox qPCR Mastermix (Qiagen). Each reverse-transcribed sample was diluted 1:3 and 51 μl was added to 550 μl of master mix. From this reaction mixture, 10 μl of each mixture was loaded into each well.

[0110] statistical analysis Chi-square test was used to evaluate the relationship between various liver lesions in treated mice compared to control mice. Significance was obtained when p<0.05. Student's t-test was used to evaluate the difference in tumor size between treated and control mice. Significance was obtained when p<0.05.

[0111] Next, the results of the experiment are described.

[0112] Effect of Synbiotic 2000 on chronic liver disease and HCC Considering that the pathogenesis of HCC is immune-mediated (Feitelson MA et al., Cancer Lett, 2009; 286(1):69-79) and that the probiotic bacteria in Synbiotic 2000™ may have anti-inflammatory properties, an experiment was designed to test the hypothesis that feeding HBx transgenic mice with Synbiotic 2000™ may delay or prevent the onset of CLD and its progression to HCC. Thus, 10 mice per group were gavaged for 3 months starting at 3, 6 and 9 months of age. Mice were bled retro-orbitally immediately before the start of treatment and at monthly intervals until the animals were euthanized. The liver was then removed and samples from each lobe were formalin fixed and paraffin embedded, while the remaining liver samples were snap frozen.

[0113] Given that HBx transgenic mice develop hepatitis, serial serum samples were tested for ALT enzyme activity. The mean ALT values ​​of most HBx mice treated with PBS were significantly higher than those of age- and sex-matched HBx mice treated in parallel with Synbiotic 2000™ (Figure 1). There was no difference in mean ALT values ​​between 3- and 6-month-old mice (Figure 1A), but the mean difference between 6- and 9-month-old mice was significantly different (t=6.78, P<0.001) (Figure 1C), as was the mean difference between treated mice aged 9- and 12-months (t=14.18, P<0.001) (Figure 1B). These findings suggest that the more severe and progressive the liver disease with age, the greater the difference in mean ALT values ​​for Synbiotic 2000™-treated mice compared to controls. However, it should be emphasized that in many of these cases, although the mean ALT values ​​in Synbiotic 2000™ treated mice were statistically different compared to PBS treated mice, the ALT elevations were small, with many of the differences being less than 60 units.

[0114] The effect of Synbiotic 2000™ on the progression of chronic liver disease and onset of HCC was then evaluated in HBx mice treated for 3 months from 3, 6, or 9 months of age. Among 3-month-old mice euthanized at 6 months of age, 5 of 10 PBS-treated mice (50%) had periportal hepatitis, whereas this was observed in 4 of 10 Synbiotic 2000™-treated mice (Figure 7). There was a trend for Symbiotic-treated mice to have fewer and generally milder lesions compared to PBS-treated mice, but these differences were not statistically significant (Figure 2A). In contrast, among 6-month-old mice, all 10 PBS controls had evidence of extensive metaplasia by the time their livers were evaluated at 9 months, whereas only 40% of Synbiotic 2000™-treated mice had evidence of metaplasia. The Synbiotic 2000™ treated group generally had fewer dysplastic cells and dysplastic nodules compared to control mice. Importantly, half of the mice treated with Synbiotic 2000™ had histologically normal livers (Figure 2B, Figure 8). In a parallel experiment with 9-month-old mice, 90% of the PBS-treated mice had HCC nodules, whereas only 40% of the Synbiotic 2000™-treated mice had HCC (Figure 2C). Many of these mice had multiple lesion types (e.g., hepatitis, dysplasia, and / or HCC), characterized by multiple nodules of dysplastic cells and multinodular HCC (Figure 9). As expected, all HBx-negative mice treated in parallel did not show significant lesions in their livers (data not shown). Thus, treatment with commensal bacteria appeared to result in qualitative and quantitative changes in the liver lesions present between mice of different ages.

[0115] To determine whether the above observations with Synbiotic 2000™ were related to toxicity, all mice were weighed at the end of the experiment. Livers were also weighed. There were no statistical differences between body weight or liver weights in all groups of mice treated with Synbiotic 2000™ compared to placebo. For example, among the 9-month-old groups fed for 3 months, the mean body weight of placebo-treated mice was 45.3 grams, whereas the mean body weight of Synbiotic 2000™-treated mice was 49.2 grams (t=0.879; P>0.3). The mean liver weights were 2.62 and 2.68 grams, respectively (t=0.102; P>0.9). Similar results were observed in younger mice (data not shown). These results indicate that there was no apparent toxicity associated with 3 months of Synbiotic 2000™ treatment.

[0116] Previous studies from this and other laboratories have shown a direct correlation between intrahepatic HBx expression and the severity of chronic liver disease (Jin YM et al., J Viral Hepat, 2001; 8(5):322-30; Feitelson MA et al., J Hepatol, 1993; 17(Suppl. 3):S24-S34; Wang W et al., Hepatology, 1998; 14:29-37; Wang W et al., Cancer Res, 1991; 51:4971-4977). To test whether this was the case here and whether there was an inverse correlation with Synbiotic 2000™ treatment, liver sections from each group of treated and control mice were evaluated for HBx expression by immunohistochemical staining. Among 3-month-old mice, cytoplasmic HBx was weak to moderate (+1 and +2) in scattered hepatocytes or clusters of hepatocytes (Figure 10). The presence, frequency and distribution of intrahepatic HBx increased in livers of older animals (Figure 10). Treatment of HBx mice with Synbiotic 2000™ reduced HBx staining compared to control mice (Figure 10, Figure 3).

[0117] Partial expression profiles of selected markers related to tumorigenicity and immunity Given that HCC arises in the context of chronic inflammation and that HBx promotes the development of this tumor type, a limited PCR array analysis was performed to determine whether Synbiotic 2000™ has an effect on the expression of select tumor-associated signaling pathways and / or cytokines that may contribute to HCC pathogenesis. Three-month-old mice were treated with Synbiotic 2000™ for three months and the expression profile of selected genes was compared to that of PBS-treated animals, revealing a 1.5- to 3-fold upregulation of markers associated with tumorigenesis (Figure 4A). When 6-month-old mice were treated with Synbiotic 2000™ for three months, the expression of most tumor-associated markers tested was neither upregulated nor downregulated compared to control mice (Figure 4B). A notable exception was EGFR (which stimulates growth), which was downregulated by more than eightfold in Synbiotic 2000™. In contrast, when 9-month-old mice were fed Synbiotic 2000™ for 3 months, most markers associated with tumor formation were strongly downregulated in the study compared to placebo-treated animals (Figure 4C). These markers included signaling molecules in the Hedgehog pathway, Gli1 and 2, several Notch receptors, TGFβ-1 and 2 and TGFβR1 (which normally negatively regulate cell growth), Tcf3 (which is important for β-catenin signaling), Akt1 (which is often constitutively activated in carcinogenesis), and MMP-9 and -10 (which promote metastasis) (Figure 4C). Regarding immune-mediated markers in 3-month-old mice euthanized at 6 months, most immune markers were not elevated or suppressed within 2-fold in the study compared to placebo treatment (Figure 4A). Similar results were obtained in 6-month-old mice euthanized at 9 months (Figure 4B).However, among 9-month-old mice euthanized at 12 months, all immune response-related markers were decreased in the study compared to control mice (Figure 4C), suggesting that a shift in the immune response to HBx and / or the nature of HBx induced changes in the liver with disease progression. When this analysis was performed in livers from age-sex-matched transgene-negative littermates, there were no statistically significant differences in the levels of these markers, suggesting that the differences were related to the increased effects of HBx on the liver and not due to age-related changes (data not shown).

[0118] Short-chain fatty acid (SCFA) treatment The lactic acid-producing bacteria in Synbiotic 2000™ are provided with a rich source of prebiotic nutrients that are metabolized to SCFAs. Because SCFAs are known to be anti-inflammatory and HCC occurs in a background of chronic liver disease with an inflammatory component, experiments were designed to determine whether SCFAs have the same impact as Synbiotic 2000™ on the pathogenesis of HCC. Since the greatest impact of Synbiotic 2000™ was observed in 9-12 month old mice that had developed HCC, 9 month old mice were fed SFCA by gavage for 3 months and then their livers were examined for the presence, frequency and distribution of lesions. When livers were removed from 12 month old SCFA-treated mice, tumors on the liver surface were more numerous and larger in PBS-treated mice compared to SCFA-treated mice (Figure 5). Microscopic sections were prepared from each lobe and tumor size was again assessed, revealing that 52% of the tumors present were small (diameter <0.5 cm), whereas 32% were large (diameter >1 cm). In contrast, in placebo-treated mice, only 29% were small, whereas 50% were considered large (Figure 6; X2=4.59, P<0.05). This is also reflected in the ratio of large:small tumors in the two groups of mice. In placebo-treated mice, the ratio was 1.75, whereas in SCFA-treated mice, the ratio shifted to 0.62, suggesting that SCFA partially prevented the development of large tumors. In all mice, tumor morphology was characteristic of undifferentiated HCC, independent of tumor size (Figure 6D).

[0119] HCC is a major public health problem, especially in developing countries where HBV is endemic. This tumor type is difficult to treat because it is diagnosed at a late stage in most cases. This provides a strong rationale for the development and application of intervention strategies that can be used to treat patients with early cancer or precancerous lesions. The results herein demonstrate that even a 3-month treatment with selected lactic acid bacteria or a mixture of their SCFA metabolites significantly reduces the number and size of HCC nodules appearing in HBx transgenic mice.

[0120] HBx transgenic mice experience a progressive development of liver lesions with age, eventually resulting in the appearance of HCC by 10 months of age (Yu DY et al., J Hepatol, 1999; 31:123-132). Treatment of mice with Synbiotic 2000™ from 6-9 months of age significantly reduced the incidence of dysplasia (Figure 2B), while mice treated from 9-12 months of age had a significantly reduced incidence of HCC (Figure 2C). Considering that dysplasia develops during the 6-9 month age period and HCC develops between 9-12 months of age, these findings suggest that Synbiotic 2000™ prevents the progression of chronic liver disease to preneoplastic and tumor nodules. This interpretation is also consistent with the observation that steatosis and dysplasia well established by 9 months of age are not affected by Synbiotic 2000™ treatment initiated at that age (Figure 2C).

[0121] In HBx transgenic mice, intrahepatic levels of HBx increase with age and the severity of the underlying liver pathology (Figures 3A and 3C). This is consistent with the strong association between X protein expression and progressive chronic liver disease in both human and woodchuck carriers of chronic liver disease (Jin YM et al., J Viral Hepat, 2001; 8(5):322-30; Feitelson MA et al., J Hepatol, 1993; 17(Suppl. 3):S24-S34). HBx activity is enhanced in the presence of reactive oxygen species (ROS) provided by the cellular immune response within the hepatic inflammatory infiltrate (Wang JH et al., Biochem Biophys Res Commun, 2003; 310(1):32-9; Lim W et al., J Mol Med (Berl), 2010; 88(4):359-69). Under these circumstances, HBx transactivates its own enhancer / promoter (which is also part of the transgene in these HBx transgenic mice), leading to increased levels of HBx expression. Small amounts of HBx are also known to associate with mitochrondria, where it damages the electron transport chain, leading to further accumulation of ROS (Fatima G et al., J Gen Virol, 2012; 93(Pt 4):706-15). In this context, lactic acid-producing bacteria and their SCFA metabolites are known to stimulate regulatory T cells in and outside the gut, which can limit inflammatory responses and ROS generation (Smith PM et al., Science, 2013; 341(6145):569-73), thereby reducing the activity and intrahepatic levels of HBx (Figure 3). This is consistent with limited microarray analysis, which showed that several immune system markers were downregulated in mice treated with Synbiotic 2000™ (Figure 4).Previous studies have shown that intrahepatic ROS was increased in HBV carriers and in HBx transgenic mice with progressive chronic liver disease (Ha HL et al., World J Gastroentero, 2010; 16(39):4932-7). When a sampling of immune markers was assessed in mice with progressive aging, Synbiotic 2000™ treatment was most effective (i.e., downregulation of these immune markers) in the oldest mice with the highest levels of intrahepatic ROS (Figure 4). Since HBx levels and activity are partially ROS-dependent and HBx drives tumor development in these animals, this may explain the correlation between reduced HBx expression in Synbiotic 2000™-treated mice (Figures 3, 10) and reduced incidence of dysplasia and HCC (Figures 8 and 9, 2).

[0122] HBx activates the expression of genes in multiple pathways that contribute significantly to liver carcinogenesis. Their suppression in 9-month-old mice treated with Synbiotic 2000™ supports the hypothesis that this intervention partially blocks the tumorigenesis-promoting ability of HBx (Figure 4). For example, HBx-mediated activation of Hedgehog signaling through upregulation of Gli1 and 2 (Arzumanyan A et al., Cancer Res, 2012; 72(22):5912-5920) is strongly downregulated by Synbiotic 2000™ by more than 40-fold and more than 7-fold, respectively (Figure 4C). Treatment of HBx transgenic mice with the standard hedgehog inhibitor GDC-0449 also reduced the number and size of tumors appearing in HBx transgenic mice (Arzumanyan A et al., Cancer Res, 2012; 72(22):5912-5920), highlighting the importance of hedgehog signaling for HCC. Notch signaling, which is also upregulated by HBx (Wang F et al., Cancer Lett, 2010; 298(1):64-73), was also reduced by more than 10-fold on average after Synbiotic 2000™ treatment (Figure 4C). Since Notch contributes to cell fate during embryogenesis, its reactivation in carcinogenesis may also mediate the process of converting normal cells into tumor cells. A similar argument can be made for Nodal, which is downregulated by more than 20-fold in 9-month-old mice treated with Synbiotic 2000™. In Synbiotic 2000™-treated mice that developed HCC, HBx activation of β-catenin, which is important for liver carcinogenesis, was not activated ( FIG. 4C ). Considering that β-catenin is a “stemness”-associated protein and that HBx promotes HCC, at least in part through up-regulation expression of “stemness” markers, inhibition of β-catenin activation by Synbiotic 2000™ may block the expansion of hepatic stem cells and / or cancer stem cells in the liver.TGFβ signaling, which is also upregulated by HBx, is essentially abolished by Synbiotic 2000™ treatment beginning in 9-month-old mice ( FIG. 4C ). Given that HBx shifts TGFβ signaling from a negative to a positive regulator of hepatocyte growth, inhibition of this pathway would partially prevent HBx from promoting tumor development. In addition, the fact that Synbiotic 2000™ treatment strongly inhibited MMP-9 (15-fold) and MMP-10 (>65-fold) (FIG. 4C), which are otherwise upregulated by HBx (Liu LP et al., Cancer Invest, 2010; 28(5):443-51; Sze KM et al., Hepatology, 2013; 57(1):131-9) and promote metastatic cancer spread, suggests additional pathways by which this treatment approach may prevent liver lesions from progressing to dysplasia and HCC.

[0123] The role of NF-κB activated by HBx in the presence of ROS appears to overlap with many of the observed effects of Synbiotic 2000™ on liver pathology and associated molecular changes. For example, in liver carcinogenesis, downregulation of Notch signaling is accompanied by downregulation of NF-κB activity (Luo J et al., Int J Oncol, 2013; 42(5):1636-43). Inhibition of Notch also leads to inhibition of β-catenin activity (Sun Q et al., Int J Oncol, 2014), suggesting crosstalk between these pathways in the development of HCC. HBx activation of NF-κB also upregulates the expression and activity of MMP-9, which promotes tumor metastasis (Liu LP et al., Cancer Invest, 2010; 28(5):443-51). Interestingly, HBx upregulation of IL-6 is MyD88 (and NF-κB) dependent (Xiang WQ et al., J Hepatol, 2011; 54(1):26-33). Binding of IL-6 to its receptor activates (phosphorylates) STAT3, which in turn activates various genes (including STAT3 itself). Subsequent binding of unphosphorylated STAT3 to NF-κB alters the expression of selected additional cellular genes that contribute to HCC (Yang J et al., Genes Dev, 2007; 21(11):1396-408). Additionally, inflammatory mediators such as TLR3, IL-18, TNFα, TGFβ, MyD88, and IRF3 also signal downstream through NF-κB, suggesting that the anti-inflammatory environment created by Synbiotic 2000™ treatment would be expected to reduce expression and / or signaling through these molecules, which is what appears to be happening in these mice (Figure 4C).TLR3 and IL-18 levels are reduced 6-fold, TNFα and MyD88 are reduced 3-fold, TGFβ is reduced more than 5-fold, and IRF3 is reduced almost 4-fold.These findings suggest that reduction of ROS and subsequent inactivation of NF-κB may be an important strategy for cancer chemoprevention.

[0124] SCFAs are major metabolic products of lactic acid-producing bacteria that promote T cell differentiation into both effector and regulatory T cells, promoting either immunity or tolerance (Park J et al., Mucosal Immunol, 2014). In this study, tolerance appears to prevail. SCFA treatment recapitulated the results of Synbiotic 2000™ treatment in preventing HCC (Figures 5 and 6). Administration of butyrate helped resolve chemically induced colitis (Smith PM et al., Science, 2013; 341(6145):569-73; Celasco G et al., Biomed Rep, 2014; 2(4):559-563) and partially prevented DEN-induced HCC (Kuroiwa-Trzmielina J et al., Int J Cancer, 2009; 124(11):2520-7; de Conti A et al., J Nutr Biochem, 2012; 23(8):860-6), while proprionate has been shown to reduce the growth of established tumors in mouse models (Bindels LB et al., Br J Cancer, 2012; 107(8):1337-44). Thus, SCFAs may be beneficial in preventing tumor initiation and progression. The anti-inflammatory properties of SCFAs may reflect their function by binding to G protein-coupled receptors (GPCRs) and HDACi (Tan J et al., Adv Immunol, 2014; 121:91-119). Considering that HBx activation of Hedgehog and Wnt signaling occurs through GPCR-associated pathways (where GPCRs are smoothened and frizzled) (Dorsam RT et al., Trends Pharmacol Sci, 2013; 34(4):226-32), it is possible that alterations in GPCR signaling by SCFAs may alter or partially block signaling activated by HBx, as in other cancers.In addition, the finding that HBx activates HDAC expression (Tian Y et al., Mol Cell Biol, 2013; 33(15):2810-6) and SCFAs act as HDACi suggests that important pathways that contribute to carcinogenesis may be blocked by SCFAs. Finally, it is important to consider that the effects of Synbiotic 2000™ and SCFA treatment may go well beyond the above mechanisms, as the reduction in HBx expression by treatment (Figure 3) may also suppress the ability of HBx to mediate other epigenetic gene expression changes (e.g., DNA and protein methylation, protein phosphorylation, ubiquitination, sumoylation, and other post-translational modifications that contribute to carcinogenesis) (Mann DA, Hepatology, 2014).

[0125] The disclosures of each and every patent, patent application and publication cited herein are hereby incorporated by reference in their entirety.

[0126] While the present invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and modifications of the present invention may be devised without departing from the true spirit and scope of the invention, and it is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. A method for preventing or delaying the onset of hepatocellular carcinoma in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising at least one short chain fatty acid.

2. 2. The method of claim 1, wherein the short chain fatty acid is selected from the group consisting of formic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, isocaproic acid, caproic acid, lactic acid, succinic acid and pyruvic acid.

3. The method of claim 1 , wherein the composition further comprises a pharma- ceutically acceptable excipient.

4. The method of claim 1 , wherein the composition is administered in combination with another therapeutic agent.

5. The method of claim 1 , wherein the composition is administered orally.

6. The method of claim 5 , wherein the composition is administered with a food or beverage.

7. 1. A method for preventing or delaying the onset of hepatocellular carcinoma in a subject, comprising administering to the subject a therapeutically effective amount of a composition comprising at least one probiotic bacterium.

8. 8. The method of claim 7, wherein the probiotic bacteria is selected from the group consisting of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei, Leuconostoc mesenteroides, Lactobacillus bulgaricus, Lactobacillus sasei, Lactobacillus salivarius, Pediococcus pentosaceus, Streptococcus thermophiles, Bacillus subtilis, Bacillus coagulans, Enteroccous faecium, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum and Bifidobacterium infantis.

9. The method of claim 7, wherein the composition further comprises a pharma- ceutically acceptable excipient.

10. 10. The method of claim 9, wherein the excipient comprises at least one prebiotic.

11. The method of claim 7 , wherein the composition is administered in combination with another therapeutic agent.

12. The method of claim 7 , wherein the composition is administered orally.

13. The method of claim 12, wherein the composition is administered with a food or beverage.

14. 13. A kit for preventing or delaying the onset of hepatocellular carcinoma in a subject, the kit comprising a composition comprising at least one short chain fatty acid.

15. 13. A kit for preventing or delaying the onset of hepatocellular carcinoma in a subject, the kit comprising a composition comprising at least one probiotic bacterium.

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