WO2014146202A1 - Bactérie pour prévenir, traiter et diagnostiquer un cancer du sein - Google Patents

Bactérie pour prévenir, traiter et diagnostiquer un cancer du sein Download PDF

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Publication number
WO2014146202A1
WO2014146202A1 PCT/CA2014/050292 CA2014050292W WO2014146202A1 WO 2014146202 A1 WO2014146202 A1 WO 2014146202A1 CA 2014050292 W CA2014050292 W CA 2014050292W WO 2014146202 A1 WO2014146202 A1 WO 2014146202A1
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Prior art keywords
bacteria
breast
subject
breast cancer
unclassified
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PCT/CA2014/050292
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English (en)
Inventor
Gregor Reid
Camilla Teresa URBANIAK
John Mark TANGNEY
Joanne CUMMINS
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London Health Sciences Centre Research Inc.
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Publication of WO2014146202A1 publication Critical patent/WO2014146202A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/335—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Lactobacillus (G)

Definitions

  • the present invention relates to bacteria-based compositions useful for the prevention and/or treatment of breast cancer and to methods of using those bacterial-based compositions for the same, to uses of the bacteria-based compositions and to methods of diagnosing breast cancer based on bacteria profile of breast tissues and /or the presence of a certain bacterium in breast tissue.
  • the present invention provides methods and uses useful for assessing the risk of, the treatment, prevention, diagnosis and/or prognosis of breast cancer and protection of DNA damage.
  • the present disclosure provides for a method of breast cancer diagnosis.
  • the method includes, in one embodiment, determining the bacterial profile in a test breast sample obtained from a subject, wherein the subject is diagnosed with cancer, if the bacterial profile of the test breast sample is relatively different to the bacterial profile of a normal breast, or relatively similar to the bacterial profile of breast cancer.
  • the present invention provides for a method of prognosis of breast cancer.
  • the method includes, in one embodiment, comparing the bacterial profile of a test breast sample with the bacterial profile of different control samples representing different stages of breast cancer or comparing the bacterial profile of a test sample with the bacterial profile of different control samples that are associated with various prognoses.
  • the test breast sample is suspected of containing tumor cells.
  • test breast sample is a tissue biopsy sample.
  • test sample is obtained non-invasively from the breast of the subject.
  • test sample is obtained non-invasively from the breast of the subject with a swab, from nipple exudates including milk, or from blood or lymphatic samples.
  • the present invention provides for a method of treating, preventing or reducing the risk of breast cancer in a subject.
  • the method includes, in one embodiment, administering to the subject an effective amount of bacteria or their metabolic by-products, wherein the bacteria or their metabolic by-products are capable of increasing the levels of ceramide in a cell, and wherein the subject is a subject at risk of having breast cancer, or suspected of having breast cancer or having breast cancer, or in need thereof.
  • the bacteria are administered in a composition comprising the bacteria and a suitable carrier.
  • the bacteria are administered in a composition comprising the bacteria and a suitable carrier, and wherein the effective amount is at least about 1 x10 9 of the bacteria per milliliter or less of the suitable carrier.
  • the suitable carrier is a carbohydrate-containing medium.
  • the carbohydrate-containing medium is a dairy product.
  • the bacteria are probiotic bacteria.
  • the probiotic bacteria includes Lactobacillus rhamnosus, Lactobacillus rhamnosus GR-1, Eubacterium hallii and Bifidobacterium longum, or a combination thereof.
  • the probiotic bacteria are lactic acid bacteria. In yet another embodiment, the bacteria are breast bacteria.
  • the present disclosure provides for a method of treating, preventing or reducing the risk of breast cancer in a subject.
  • the method includes, in one embodiment, administering to the subject an effective amount of a bacteria, and wherein the subject is a subject at risk of having breast cancer, or suspected of having breast cancer or having breast cancer.
  • the bacteria are administered in a composition comprising the bacteria and a suitable carrier.
  • the bacteria are administered in a composition comprising the bacteria and a suitable carrier, and wherein the effective amount is at least about 1 x10 9 of the bacteria per milliliter or less of the suitable carrier.
  • the suitable carrier is a carbohydrate-containing medium.
  • the carbohydrate-containing medium is a dairy product.
  • the bacteria are probiotic bacteria.
  • the probiotic bacteria include Lactobacillus rhamnosus, Lactobacillus rhamnosus GR-1, Eubacterium hallii and Bifidobacterium longum, or a combination thereof.
  • the probiotic bacteria are lactic acid bacteria.
  • the present invention provides for a method of treating, reducing the risk or preventing breast cancer in a subject.
  • the method includes, in one embodiment, administering to the subject a composition for killing bacteria associated with breast cancer, or for generating an immune response against bacteria associated with cancer.
  • the present invention provides for a method of treating or reducing the risk or preventing breast cancer in a subject.
  • the method includes, in one embodiment: (a) determining a bacteria profile form the breast of the subject, (b) determining if the bacteria profile includes a bacterium or bacteria associated with breast cancer, and (c) administering to the subject a composition capable of killing the bacterium or bacteria associated with breast cancer or capable of generating an immune response to the bacterium or bacteria associated with breast cancer.
  • the subject is a subject at risk of having breast cancer, or suspected of having breast cancer or having breast cancer, or in need thereof.
  • the present invention provides for breast bacteria for use in the treatment, prevention or reducing risk of breast cancer in a subject.
  • the bacteria are capable of increasing the levels of ceramide.
  • the bacteria are lactic acid bacteria.
  • the bacteria are probiotic bacteria.
  • the bacteria are probiotic bacteria
  • the probiotic bacteria includes Lactobacillus rhamnosus, Lactobacillus rhamnosus GR-1, Eubacterium hallii and Bifidobacterium longum, or a combination thereof.
  • the present invention provides for a use of bacteria profiles of normal control breasts in the preparation of a kit for assessing the risk, the diagnosis or prognosis of breast cancer in a subject.
  • the present invention provides for breast tissue bacteria for use in the delivery of a compound to the breast of a subject in need of said compound.
  • the present invention provides for breast tissue delivery system for administration of a compound to a subject in need of such compound, wherein said breast tissue delivery system comprises a bacterium that is generally found in breast tissue.
  • the subject is a subject at risk of having breast cancer, or suspected of having breast cancer or having breast cancer, or in need thereof.
  • the present disclosure provides for a method of assessing the risk or diagnosing breast cancer. The method, in one embodiment, includes determining the expression and abundance of bacteria in a breast of a subject.
  • the subject is a subject at risk of having breast cancer, or suspected of having breast cancer or having breast cancer, or in need thereof.
  • the present invention provides for a method for protecting against DNA damage in a subject.
  • the method includes the step of administering to the subject a composition comprising a therapeutically effective amount of a probiotic and a suitable carrier.
  • the probiotic includes Lactobacillus rhamnosus, Lactobacillus rhamnosus GR-1, Eubacterium hallii and Bifidobacterium longum, or a combination thereof.
  • the present invention provides for the use of a probiotic for protecting against DNA damage in a subject.
  • the probiotic for protecting against DNA damage includes Lactobacillus rhamnosus, Lactobacillus rhamnosus GR-1, Eubacterium hallii and Bifidobacterium longum, or a combination thereof.
  • Figure 1 illustrations of the location within the breast of tissue samples collected.
  • Panels (A) and (B) illustrate the location of tissue collected from women in Canada undergoing lumpectomies or mastectomies for either malignant (A) or benign (B) tumours.
  • NB Subject 25 underwent a prophylactic mastectomy due to previous cancer in theother breast.
  • Panel (C) illustrate the location of tissue collected from women in Ireland undergoing lumpectomies or mastectomies for malignant tumours.
  • Ovals represent the location of the tumour and squares represent the location of the specimen obtained for bacterial analysis. The distance between the ovals and squares are approximate estimates of the distance between the tumour and the specimen, which was at least 5cm away from the tumour.
  • Panel (D) illustrates the location of tissue collected from women in both Canada and Ireland undergoing breast reduction surgery. Asterisks in the boxes underneath the subject number, indicate samples from Ireland. All samples were a minimum of 1 cm deep to the skin with the surgeons aiming for mid-deep rather than superficial. As shown in the panels, specimens were obtained from a variety of locations within the breast.
  • Figure 2 are graphs illustrating the percent abundance of different bacterial phyla in breast tissue identified by 16S rRNA sequencing.
  • Panel B The least abundant phyla shown in (A) were plotted on another graph with a smaller scale to allow for better visualization of percentage. In samples from both countries Proteobacteria was the most abundant phyla followed by Firmicutes (Kruskal-Wallis/Mann-Whitney U with Bonferroni correction, p ⁇ 0.001 ).
  • FIG. 3 Bray-Curtis dissimilarity principal coordinate analysis (PCoA) plots comparing bacterial profiles in breast tissue from women with and without cancer. Each tissue sample is represented as a circle on this 3D, 3-axis plane. Only the first 3 components are plotted representing (A) 44% of the variation in the Canadian samples and (B) 51 % of the variation in the Irish samples. Tissue samples collected from women without cancer are labelled “LH” or " ⁇ ", while normal-adjacent tissue collected from women with cancer are labelled "LN" or "CN”.
  • PCoA principal coordinate analysis
  • LH Prevotella
  • LN Acinetobacter, Bacillus, Comamonadaceae, Cytophaga/Flavobacterium, Enterobacteriaceae, Gammaproteobacteria, Prevotella, Propionibacterium, Pseudomonas, Staphylococcus
  • CH Janibacter
  • CN Alloiococcus otitidis, Acinetobacter, Enterobacteriaceae, Listeria welshimeri, Lysobacter, Propionibacterium, Pseudomonas, Staphylococcus.
  • Figure 4 Graph comparing Escherichia coli/Shigella amounts in tissue from women with ("CN”) and without ("CH”) cancer from Ireland. Mann Whitney U test p ⁇ 0.05. Each bar represents the mean +/- SEM.
  • FIG. 5 Graphs illustrating qPCR analysis of malignant tumour tissue (“C#T”) samples and corresponding normal adjacent tissue (“C#N”) from women recruited from Ireland ("#” represents a subject's sample number). qPCR was performed for (A) E. coli/Shigella specific 16S rRNA gene (B) Citrobacter specific 16S rRNA gene and (C) Bifidobacterium specific 16S rRNA gene. The bars represent the mean and standard deviation of 3 technical replicates. All samples were normalised to the eukaryotic gene GAPDH. Star represents samples that were shown to be
  • FIG. 7 Graph illustrating the effect of Lactobacillus on DNA damaging effects of Etoposide. Each dot on the graph represents the measurement of one individual cell. Approximately 250 cells were visualized and measured per treatment group. Stars represent significant differences between the bacterial treatment and cells treated with just Etoposide (pval ⁇ 0.01 ).
  • Figure 8 Graph illustrating JSpecies analysis comparing Bacillus cereus (S34) isolated from breast tissue to that of other Bacillus isolates with fully sequenced genomes. In addition, a comparison was also done with an isolate from breast tissue of a healthy woman. Bacillus cereus S34 was most closely related to the B.cereus ATCC 14579 strain.
  • Figure 9. Unweighted principal coordinate analysis (PCoA) plot comparing bacterial profiles in breast milk and normal adjacent tissue taken from women with breast cancer. Each symbol represents a sample, with triangles representing tissue and spheres representing milk. Distinct clusters are indicative of different bacterial communities. As show in this plot, the milk samples cluster together and the tissue samples cluster together and these clusters are distinct between the different groups. The unweighted unifrac distance matrix used for the analysis looks at differences based on presence or absence of bacteria, not abundance.
  • PCoA principal coordinate analysis
  • adjacent refers to tissue taken adjacent to a tumour and “tumour” refers to tissue taken directly from a tumour.
  • Bacteast bacteria refers to bacteria, alive or dead, that is found in breast tissue or milk.
  • food grade bacteria refers to any bacteria, alive or dead, that have no harmful effect on human health or that have a GRAS (generally recognized as safe) status. Such bacteria maybe selected from the group consisting of Lactobacilli and Bacilli.
  • “Dairy product” refers to milk or a food produced from the milk of mammals (including human milk), and includes cheese, yogurt, cream, powdered milk, butter, ice cream, and so forth.
  • probiotic as used in this document refers to bacteria, including those of food-grade, which perform beneficial functions for the subject organisms when they are present and in viable form.
  • Food production animal is used herein to describe any animal that is prepared and used for human consumption.
  • a food production animal can be, but not limited to, a ruminant animal such as beef and dairy cattle, pigs, lamb, deer, rabbits, chicken, turkey or any other fowl, or aquatic animals including shrimp, lobster or fish used for human consumption.
  • Subject or “subjects” are used herein to describe a member of the animal kingdom, including food production animals and humans.
  • the inventors rationalized that given the nutrient rich fatty composition of the female breast, the widespread vasculature and lymphatics, and the diffuse location of the lobules and ducts leading from the nipple, bacteria would be widespread within the mammary glands, irrespective of lactation.
  • the inventors used culture and 16S rRNA sequencing to analyze the breast tissue microbiota. To ensure that the results obtained were not an artifact of a single demographic, tissue was collected and processed from two countries: Canada and Ireland.
  • the inventors discovered that regardless of location sampled within the breast, presence/absence of breast malignancy, country of origin, age of the subject, history of pregnancy, and method of DNA preparation, a variety of bacteria were detected in breast tissue (see Table 7).
  • the present invention provides a method of breast cancer diagnosis or assessing the risk of developing breast cancer.
  • the method of breast cancer diagnosis or assessing the risk of developing breast cancer may include comparing the bacterial profile in a test breast sample, which may or may not be suspected of containing tumor cells obtained from a subject, with the bacterial profile of a normal breast, with the bacterial profile of a breast known to have cancer, or with both.
  • the subject may be diagnosed with cancer, if the bacterial profile of the test breast sample is relatively different to the bacterial profile of the normal breast, or if bacterial profile of the test breast sample is relatively similar to the bacterial profile of a control breast cancer tissue sample.
  • the present invention provides for a method of prognosis of breast cancer.
  • the method may include comparing the bacterial profile of a test breast sample with the bacterial profile of different control samples representing different stages of breast cancer.
  • Libraries of bacterial profiles of breast cancer tissues may be created a priori.
  • the libraries may be regionally or country based. For example, a library may be created for North America, or a library for Canada, United States and so forth.
  • the test breast sample may or may not be suspected of containing tumor cells.
  • the test sample may be obtained by any method known in the art.
  • the test sample may be a tissue sample obtained by biopsy.
  • non-invasive method may be preferred.
  • the test sample may be obtained non- invasively from the breast of the subject with a swab, or from nipple exudates including milk, or from blood or lymphatic samples.
  • the present invention relates also to the use of breast bacterial profile for diagnosing or assessing the risk of developing breast cancer.
  • a method of breast cancer diagnosis or assessing the risk of developing breast cancer may include determining the expression level and/or abundance of a bacterium and/or bacteria, in a test breast sample, which in aspects of the present invention may be suspected of containing tumor cells obtained.
  • the bacteria may be (a) over-expressed or over-represented in the test breast sample relative to normal healthy breast, (b) expressed in the test breast sample and not in the normal breast, or (c) not expressed in the test breast sample and it is expressed in the normal breast. In the latter case, the absence of certain bacterial types that have protective effects against disease, might place the test subject at higher risk of disease.
  • the subject may be diagnosed with breast cancer or of having a risk of developing breast cancer, if (a) said bacteria are overexpressed in the test breast sample relative to the expression of said bacteria in a normal healthy breast, (b) said bacteria are expressed in the test breast sample and not in the normal breast, or (c) said bacteria are not expressed in the test breast sample and it is expressed in the normal breast.
  • the present invention in another embodiment, is a method of treating or preventing or reducing the risk of breast cancer.
  • the method may include administering to a subject an antibiotic or antimicrobial agent to kill bacteria associated with breast cancer.
  • the method may also include administering to the subject a vaccine or whole bacteria or their metabolic by-products to generate an immune response against bacteria associated with breast cancer.
  • the present invention may also be a use of a vaccine or whole bacteria or their metabolic byproducts for generating an immune response against bacteria associated with breast cancer.
  • the methods of the present invention may be used alone or in combination with other known breast cancer therapies.
  • the method may include determining a bacterial profile form the breast of a subject, determining if the profile includes a bacterium or bacteria associated with breast cancer, and administering a composition capable of killing the bacteria associated with breast cancer or capable of generating an immune response to the bacteria associated with breast cancer.
  • the present invention relates also to the use of an antibiotic or antimicrobial agent against a bacteria associated with breast cancer for treating or preventing or reducing the risk of breast cancer.
  • the present invention relates also to the use of a composition, such as a vaccine, that generates an immune response against a bacteria associated with breast cancer for treating or preventing or reducing the risk of breast cancer.
  • Treatment or prevention of breast cancer in a subject may be accomplished, for example, by reducing or eliminating the amount of the carcinogenic compound carried by the subject, by administering a bacteria known to be present in the breast, which may be food-grade bacteria, or extracts thereof, to the subject.
  • a bacteria known to be present in the breast which may be food-grade bacteria, or extracts thereof
  • Another example would be the control of aberrant cell growth before it reaches the cancer stage, by up-regulating pro-apoptotic signaling molecules, such as ceramide. This could also have a role in treatment as increased ceramide levels have been shown to make chemotherapeutic drugs more effective.
  • the methods for administering bacteria may essentially be the same, whether for prevention or treatment.
  • the risk of breast cancer may be substantially reduced or eliminated by a combination of prevention and treatment.
  • Food-grade bacteria may also be used, according to another embodiment of the present invention, to feed animals that may be consumed by humans.
  • food-grade bacteria of the present invention may be added to animal feeds.
  • the present invention is a use of antibiotics, such as anti-Sac/7/ivs, anti-E. coli, or anti-H. pylori, for reducing DNA damage in breast tissue.
  • Lactobacillus isolated from human breast milk and food-grade Lactobacillus rhamnosus GR-1 were able to reduce DNA damaging effects of Etoposide, a DNA damaging chemical (see Figure 7).
  • Lactobacillus isolated from human breast milk and Lactobacillus rhamnosus GR-1 may be used for reducing DNA damage.
  • the inventors discovered that bacteria are present in breast tissue.
  • Breast tissue is an appropriate environment for these bacteria.
  • different bacterial profiles exist in tissue from women with and without cancer (see Table 7).
  • the bacteria present in breast tissue of women with cancer may be used as vectors for the delivery of anticancer therapy more effectively.
  • the bacteria used as vector will be a bacterial that will not harm the subject, such as a Lactobacillus.
  • compositions are preferred.
  • One bacterium may be administered alone or in conjunction with a second, different bacterium. Any number of different bacteria may be used in conjunction.
  • conjunction with is meant together, substantially simultaneously or sequentially.
  • the compositions may be administered in the form of tablet, pill or capsule, for example.
  • One preferred form of application involves the preparation of a freeze-dried capsule comprising the composition of the present invention.
  • Another preferred form of application involves the preparation of a lyophilized capsule of the present invention.
  • Still another preferred form of application involves the preparation of a heat dried capsule of the present invention.
  • another preferred form of application involves the preparation of a creme or solution that delivers the beneficial microorganisms.
  • amount effective is meant to be an amount of a bacterium or bacteria, high enough to significantly positively modify the condition to be treated but low enough to avoid serious side effects (at a reasonable benefit/risk ratio), within the scope of sound medical judgment.
  • An effective amount of a bacterium will vary with the particular goal to be achieved, the age and physical condition of the subject being treated, the duration of treatment, the nature of concurrent therapy and the specific Lactobacillus employed. The effective amount of a bacterium will thus be the minimum amount which will provide the desired detoxification.
  • a decided practical advantage of using food-grade bacteria or bacterial strains or their metabolic by-products, with properties attributed to anti-cancer effects, is that they may be administered in a convenient manner such as by the oral, intravenous (where non-viable), topical over the breast, or suppository (vaginal or rectal) routes.
  • the active ingredients which comprise bacteria may be required to be coated in a material to protect said organisms from the action of enzymes, acids and other natural conditions which may inactivate said organisms.
  • they should be coated by, or administered with, a material to prevent inactivation.
  • bacteria may be co-administered with enzyme inhibitors or in liposomes.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFP) and trasylol.
  • Liposomes include water-in-oil-in- water P40 emulsions as well as conventional and specifically designed liposomes which transport bacteria, such as Lactobacillus, or their by-products to an internal target of a host subject.
  • Dispersions can also be prepared, for example, in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the bacteria or their metabolic by-products, in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required.
  • dispersions are prepared by incorporating the various sterilized bacteria or their metabolic by- products, into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof. Additional preferred methods of preparation include but are not limited to lyophilization and heat-drying.
  • the active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets designed to pass through the stomach (i.e., enteric coated), or it may be incorporated directly with the food of the diet, or it may be incorporated with compounds that support and promote the growth of the bacteria being administered.
  • the food-grade bacteria may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the tablets, troches, pills, capsules, and the like, as described above, may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid, and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil or wintergreen or cherry flavoring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid, and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the organism may be incorporated into sustained-release preparations and formulations.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of the bacteria calculated to produce the desired preventive or therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention may be dictated by and may be directly depending on (a) the unique characteristics of the bacteria and the particular preventive, detoxification or therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such bacteria for the establishment and maintenance of a healthy biota in the intestinal tract.
  • the organism can be compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically or food acceptable carrier in dosage unit form as hereinbefore disclosed.
  • a unit dosage form can, for example, contain the principal active compound in an amount approximating 10 9 viable or non- viable, for example lactobacilli, per ml.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • the pharmaceutically acceptable carrier may be in the form of milk or portions thereof including yogurt. Skim milk, skim milk powder, non-milk or non-lactose containing products may also be employed.
  • the skim milk powder is conventionally suspended in phosphate buffered saline (PBS), autoclaved or filtered to eradicate proteinaceous and living contaminants, then freeze dried heat dried, vacuum dried, or lyophilized.
  • PBS phosphate buffered saline
  • substances which can serve as pharmaceutical carriers are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethycellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; calcium carbonate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, manitol, and polyethylene glycol; agar; alginic acids; pyrogen-free water; isotonic saline; cranberry extracts and phosphate buffer solution; skim milk powder; as well as other non-toxic compatible substances used in pharmaceutical formulations such as Vitamin C, estrogen and echinacea, for example.
  • sugars such as lac
  • wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, lubricants, excipients, tabletting agents, stabilizers, anti-oxidants and preservatives, can also be present. Accordingly, the subject may be orally administered a therapeutically effective amount of at least one food-grade bacteria and a pharmaceutically acceptable carrier in accordance with the present invention.
  • a preferred bacterium is a lactic acid bacterium.
  • Example 1 Microbiota of Breast Tissue
  • Sample processing Tissue samples were homogenized in sterile phosphate buffered saline (PBS) using a PolyTron 2100 homogenizer at 28000 rpm until the tissue was fully homogenized. The amount of PBS added was based on the weight of the tissue to obtain a final concentration of 0.4g/mL. Fresh homogenate was plated on different agar plates for culture dependent analysis and the remaining amount aliquotted and stored at -80°C until DNA extraction.
  • PBS sterile phosphate buffered saline
  • tissue homogenates were thawed on ice, 400 ⁇ (equivalent to 160mg of tissue) were added to tubes containing 1 .2ml of ASL buffer (QIAamp® DNA Stool Kit, Qiagen) and 400mg of 0.1 mm diameter zirconium-glass beads (BioSpec Products). Mechanical and chemical lyses were performed by bead beading at 4800rpm for 60s, then 60s on ice (repeated twice) (mini-beadbeater-1 , BioSpec Products) and then incubated at 95oC for 5min.
  • ASL buffer QIAamp® DNA Stool Kit, Qiagen
  • CBA Columbia Blood agar
  • CBA + vancomycin to select for gram negative aerobes
  • CBA+vancomycin+ kanamycin to select for gram negative anaerobes
  • MRS and MacConkey agar to detect lactic acid bacteria and conforms, respectively, and plated aerobically for 24hours at 37oC and anaerobically for 48-72hours at 37oC.
  • DNA from single colonies were then extracted using Instragene Matrix (Bio-Rad) and 10 ⁇ of instagene supernatant were then amplified using the eubacterial primers pA (5' AGAGTTTGATCCTGGCTCAG 3') and pH (5' AAGGAGGTGATCCAGCCGCA 3') which amplify the 1 .5kb 16s rRNA gene.
  • the PCR reaction was carried out in 50 ⁇ reaction containing 10 ⁇ of DNA template (or nuclease free water as a negative control), 1 .5mM MgCI2, 1 .0 ⁇ of each primer, 0.2mM dNTP, 5 ⁇ 10X PCR buffer (Invitrogen), and 0.05 Taq Polymerase (Invitrogen).
  • Thermal cycling was carried out in an Eppendorf Mastercycler under the following conditions: Initial denaturation step at 95oC for 2min, followed by 30 cycles of 94oC for 30s, 55oC for 30s and 72oC for 1 min. A final elongation step was performed at 72oC for 10min. After running 10 ⁇ of the PCR mixture on a 1 % agarose gel to verify the presence of amplicons, 40 ⁇ of the PCR mixture was then purified using the QIAquick PCR purification kit (Qiagen). The purified PCR products were then sent for Sanger sequencing to the London Regional Genomics Centre, London, Ontario, Canada. Sequences were analyzed using the GenBank 16S ribosomal RNA sequences database using the BLAST algorithm (1 1 ). Taxonomy was assigned based on the highest Max score.
  • Tissue collection Breast tissue was collected from 38 women (aged 20-85) undergoing breast surgery at Cork University Hospital or South Infirmary Victoria Hospital, Cork, Ireland. Thirty-three women underwent lumpectomies or mastectomies for cancerous tumours (taken at least 5cm away from the primary tumour site) while 5 women underwent breast reductions and had no history of breast cancer. Once collected the specimens were placed in sterile cryotubes and flash frozen in liquid nitrogen within 45 minutes of collection and then stored at - 80oC until DNA extraction. In addition to collecting non-malignant tissue adjacent to the tumour, tissue was also collected directly from the tumour itself from the same subject. Since the Canadian pathologist did not permit collection of tumour tissue, these samples were only collected from the Irish subjects.
  • DNA isolation from Irish samples Total DNA was extracted from tumour, normal and environmental swabs using Gene-Elute Mammalian Genomic DNA miniprep kit (Sigma-Aldrich) as per the manufacturer's protocol, with the exception of the elution step, where the column was eluted with 70 ⁇ of elution buffer.
  • V6-L and V6-R which amplify the V6 hypervariable region of the 16s rRNA gene.
  • the V6-L primer contained an adaptor sequence and a unique barcode. The primer sequences were as follows:
  • V6-LT 5' ccatctcatccctgcgtgtctccgactcagnnnncwacgcgargaaccttacc 3'.
  • V6-RT 5' cctctctatgggcagtcggtgatacracacgagctgacgac 3'
  • the N represents the unique barcode sequence.
  • the PCR was carried out in a 40 ⁇ reaction containing 5 ⁇ of DNA template (or nuclease free water as a negative control), 1 .5mM MgCI2, ⁇ . ⁇ of each primer, 4 ⁇ of 10x PCR Buffer (Invitrogen), 0.2mM dNTPs, 0.05U Taq Polymerase (Invitrogen) and 0.15 ⁇ 9/ ⁇ of bovine serum albumin.
  • Thermal cycling was carried out in an Eppendorf Mastercyler under the following conditions: Initial denaturation at 95oC for 2min followed by 25 cycles of 95oC for 1 min, 55oC for 1 min and 72oC for 1 min.
  • the DNA concentration was measured with the Qubit® 2.0 Fluorometer (Invitrogen) using the broad range assay. Equimolar amounts of each PCR product were then pooled together and purified using the QIAquick PCR purification kit (Qiagen). The PCR purified sample was then sent to the London Regional Genomics Center, London, Ontario, Canada for V6 16S rRNA sequencing using the Ion Torrent platform as per the Center's standard operating procedure.
  • V6 rRNA gene sequencing DNA from milk and tissue samples were run on a separate chip on different days and thus processed separately. PCR products were first thawed and the DNA concentration of the PCR reaction was measured with QuBit 2.0 using the high sensitivity assay. The concentrations were roughly the same (within 5% variance) between milk and tissue samples, so 10 ⁇ of each product (for each sample type) was pooled together and purified using the PCR purification kit (Qiagen). Samples were eluted in 50 ⁇ of buffer and then sent to the Robarts Research Institute for Ion Torrent sequencing using standard protocols set up by the facility.
  • Read processing and taxonomic assignment Custom Perl and Bash scripts were used to de-multiplex the reads and assign barcoded reads to individual samples. Reads were kept if a sequence read included a perfect match to the barcode and the V6 16s rRNA gene primers and were within the length expected for the V6 variable region, amples with more than 600 reads were kept while those with less than 600 were discarded. Reads were clustered by 97% identity into Operational Taxonomic Units (OTUs) using UCIust v. 3.0.617 (12). OTUs that represented at least 2% of the reads in any one sample were kept, while the rare OTUs were discarded to account for the high error rate intrinsic to Ion Torrent sequencing.
  • OTUs Operational Taxonomic Units
  • Taxonomic assignments for the representative OTU sequences were made by determining the lowest common taxonomy from the Ribosomal Database Project (RDP) Seqmatch tool (13). Comparisons were made with named isolates and the top 20 hits were retained for analysis. The taxonomic assignments were verified manually using BLAST against the Greengenes database with an output of 100 hits (14). Taxonomy was assigned based on hits with the highest % identities and coverage. If multiple hits fulfilled this criterion, classification was re-assigned to a higher common taxonomy.
  • RDP Ribosomal Database Project
  • the QIIME pipeline (15) was used to (i) calculate weighted UniFrac distances and Shannon's diversity index (logarithms with base 2); (ii) summarize OTUs by different taxonomic levels and (iii) generate Unweighted Pair Group Method with Arithmetic Mean (UPGMA) trees representing hierarchical clustering of samples.
  • the UniFrac distances were calculated by using a phylogenetic tree of OTU sequences built with FastTree (17) and based on an OTU sequence alignment with MUSCLE (17). Weighted UniFrac compares microbial profiles (presence/absence and abundance) between samples (i.e.
  • beta-diversity (18) while Shannon's diversity index evaluates the microbial diversity within a sample (i.e. alpha diversity). The higher the Shannon's diversity index, the more diverse a sample is and a value of zero indicates the presence of only one species (19).
  • UPGMA trees allows one to visualize the distance matrix and the robustness of what was observed was tested with jackknifing and bootstrapping. For beta-diversity analyses, the data set was rarified to the lowest read count/sample. Barplots, boxplots and stripcharts were all generated in R (20).
  • Genus-specific PCR Primers were designed to amplify specific genes within certain bacterial genera. Total genomic DNA from the tissue sample was used as template for PCR. BioMix Red (Bioline) was used for the PCR reaction. The PCR cycle was as follows (95°C x 5 min, 94°C x 30 sec, 50-55°C x 30 sec, 72°C x 30 sec for 40 cycles and final extension of 72°C for 4 min). The PCR product was run on a 1 % gel and immediately photographed on a UV transillumination. PCR primers used are listed in Table 1 .
  • Tissue was obtained from various locations within the breast, from close to the nipple to as far back as the chest wall ( Figure 1 ). Regardless of location sampled within the breast, presence/absence of breast malignancy, country of origin, age, history of pregnancy and method of DNA preparation, a variety of bacteria were detected in breast tissue (Table 7). Bacterial diversity within samples varied between individuals with Shannon's diversity indices ranging from 0.8-5.2, with an average value of 3.6. To put into perspective, using the same index, oral and gut samples, known for their diverse bacterial communities, have values between 3.9-6.5 (22-25), while vaginal samples of low bacterial diversity, generate values between 0.46-2.9 (26-28) .
  • OTUs operational taxonomic units
  • Table 7 The bacteria identified in tissue were grouped into 121 operational taxonomic units (OTUs) based on 97% sequence similarity (Table 7). These OTUs belonged to 7 different phyla: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Verrucomicrobia and Fusobacteria with Proteobacteria being the most abundant phylum followed by Firmicutes (specifically from the class Bacilli) (see Figure 2). Of the 121 OTUs identified, 57 could be classified at the genus level and 25 at the species level (Table 7).
  • Bacteria were able to be cultured from all 43 of the Canadian samples (culture analysis was not performed on the Irish samples) with amounts ranging from 75- 2000 cfu/gram of tissue, depending on the sample. Collectively, eight different strains were identified: Bacillus sp, Micrococcus luteus, Propionibacterium acnes, Propionibacterium granulosum, Staphylococcus sp, Staphylococcus saprophytics, Streptococcus oralis, Streptococcus agalactiae. No bacteria were isolated from the environmental controls.
  • ANOSIM generates the global test statistic, R, which lies between -1 and +1 .
  • Taxa information added to the PCoA plots allowed us to visualize correlations between tissue type and microbial communities (Figure 3).
  • Enterobacteriaceae, Pseudomonas, Propionibacterium, Staphylococcus and Acinetobacter were associated with tissue from women with cancer, in addition to Bacillus, Comamonadaceae, Gammaproteobacteria and Cytophaga/Flavobacterium in the Canadian samples and Listeria welshimeri, Lysobacter and Alloiococcus otitidis in the Irish ones.
  • Taxa associated with tissue from women without cancer were Prevotella in the Canadian samples and Janibacter and Gammaproteobacteria in the Irish ones.
  • PapR the PlcR regulon is the major virulence regulator for B. cereus and is regulated via bacterial density. The fact that none of the subjects had any signs of infection may be due to the low density of Bacillus in the tissue.
  • S-layer proteins are monomolecular crystalline arrays of
  • S-layer proteins proteinaceous subunits attached to peptidoglycan of gram positive bacteria. Changes in environmental conditions lead to different variations of these S-layer proteins. S-layers from Bacillaceae were found to function as adhesion sites for cell associated exo-enzymes. It is believed that the S-layer may contribute to virulence in Bacillus, as only virulent clinical isolates have S-layers. It has also been observed that strains containing S-layers adhere tomatrix proteins and are resistant to polymorphonulcear leukocytes in the absence of opsonins.
  • Chitin binding protein and chitinases Chitin is the second most abundant polysaccharide in nature next to cellulose. Chitinases are found in a variety of eukaryotic and prokaryotic species even though they do no produce endogenous chitin. It is believed that chitin binding protein and chitinases act as virulence factors in some pathogens by modulating adhesion and/or invasion into host cells. Chitinase like proteins, which are found in mammals and have the ability to bind but not cleave chitin, have been shown to enhance the adhesion of intestinal bacteria to colonic epithelial cells, promoting the pathogenesis of inflammatory bowel disease. Increased serum levels of chitinase like proteins correlate with disease severity, poorer prognosis, and shorter survival in many human cancers such as breast, colon, prostate, ovaries, brain, thyroid, lung, and liver.
  • Bacillolysin is an enzyme found in many Bacillus species. It has been shown that bacillolysin from B. megaterium has the ability to convert plasminogen to angiostatin-like fragments. Angiostatin inhibits proliferation of vascular endothelial cells, which is a fundamental process in angiogenesis. Angiostatin has been shown to suppress both in situ and metastatic tumour growth in animal models.
  • Subsequent analyses may include comparisons between this strain and the avirulent ATCC 14579 strain, as well as virulent clinical isolates.
  • Aneurinibacillus aneurinilyticus isolated from the tissue of a healthy woman, was also sequenced. This isolateshares 79% sequence identity with our Bacillus cereus isolate and was once classified in the genus Bacillus.
  • DNA damage increases the risk of developing cancer and certain bacterial strains, such as E.coli (30) and H. pylori (31 ) have been shown to induce DNA damage with the latter being linked to the development of gastric cancer. Since the Bacillus isolate that we sequenced had genes for potential toxins, we examined, using the ⁇ 2 ⁇ assay, whether this isolate had the ability to induce DNA damage. Doing a time course, we saw that the induction of DNA damage occurred as early as 30min after incubation (Figure 6).
  • Lactobacillus could protect against damage by strong DNA damaging chemicals, in this case Etoposide.
  • MDA-MB-231 cells were treated with Lactobacillus isolated from breast milk or Lactobacillus rhamnosus GR-1 for 2 hours before bacteria were washed away and Etoposide added for 4 hours. Lactobacillus isolated from both milk and GR-1 were able to reduce the DNA damaging effects of Etoposide, possibly through sequestration of this chemical (Figure 7).
  • the Inventors have also collected 50 milk samples from lactating women and analyzed the bacterial profiles in these samples. Interestingly, the predominant organisms were different than that seen in tissue with the predominant organisms being Cloacibacterium, Brevundimonas and Acinetobacter. A combination of both culture and Ion Torrent 16s rRNA sequencing have identified bacteria in milk, that have not yet been published in the literature, to be associated with breast milk (Table 3).
  • a Principal Coordinate analysis plot using an unweighted UNIFRAC distance matrix shows that bacterial profiles differ between tissue and milk (Figure 9).
  • nitrate reductase assay Some of the bacteria isolated from milk and tissue were tested for their ability to reduce nitrate to nitrite using the nitrate reductase assay. Coupling of nitrite with dietary secondary amines leads to the formation of nitrosamine, a carcinogenic compound.
  • the milk isolates Streptococcus nepalensis, Corynebacterium simulans, Staphylococcus aureus, Staphylococcus capitis, Peptostreptococcus, Staphylococcus hominis, Veillonella, Staphylocococcus pasteuri and the Bacillus tissue isolates were able to reduce nitrate to nitrite while Acinetobacter Iwoffii was able to reduce nitrate but not to something other than nitrite (Table 4).
  • Table 3 Bacteria identified in breast tissue by Ion Torrent 16s rRNA sequencing.
  • Table 5 Bacterial types detected but not previously associated with breast milk
  • Ion Torrent 16s sequencing backed by culture, provides evidence that breast tissue does indeed harbour a range of bacterial types, including many not yet reported in human milk. These include organisms known for pathogenic properties and also those reported to have mutagenic and carcinogenic potential.
  • nitrate reductase assay shows the potential of some of these breast isolates to form nitrosamines, a carcinogenic compound. We have also tested harmful effects of these bacteria such as the ability to induce DNA damage which may lead to mutations.
  • the form of administering microorganisms to prevent, reduce the risk of, or treat cancer can include fecal transplant, or multiple species of microorganisms, with and without, before or after administration of antibiotics or antimicrobial or chemotherapeutic agents.
  • antimicrobial agents and beneficial bacteria for example Lactobacillus species
  • Betaproteobactena unclassified; unclassified; Class unclassified; unclassified
  • Verrucomicrobia 73 Verrucomicrobia; Verrucomicrobiae; Verrucomicrobiales; Species Verrucomicrobiaceae; Akkermansia; muciniphila
  • DCIS ductal in situ carcinoma.
  • Lactobacillus reuteri RC-14 a randomized, double-blind, placebo-controlled trial. Can J Microbiol. 55(2): 133-138.

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Abstract

L'invention concerne des procédés d'utilisation de profils bactériens d'échantillons mammaires pour diagnostiquer et pronostiquer un cancer du sein. L'invention concerne également des procédés d'utilisation de microorganismes pour prévenir, réduire le risque et traiter un cancer du sein.
PCT/CA2014/050292 2013-03-18 2014-03-18 Bactérie pour prévenir, traiter et diagnostiquer un cancer du sein WO2014146202A1 (fr)

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CN113403244A (zh) * 2014-12-23 2021-09-17 伊利亚制药公司 伤口愈合的方法
CN114717343A (zh) * 2022-04-24 2022-07-08 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） 细菌载量作为标志物在预测鼻咽癌复发转移风险中的应用

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113403244A (zh) * 2014-12-23 2021-09-17 伊利亚制药公司 伤口愈合的方法
CN113403244B (zh) * 2014-12-23 2022-08-19 伊利亚制药公司 伤口愈合的方法
US11473091B2 (en) 2014-12-23 2022-10-18 Ilya Pharma Ab Methods for wound healing
EP3561070A4 (fr) * 2016-12-26 2020-08-05 MD Healthcare Inc. Procédé de diagnostic du cancer du sein par analyse métagénomique microbienne
WO2018229519A1 (fr) 2017-06-12 2018-12-20 Debreceni Egyetem Méthodes de diagnostic du cancer du sein
CN110982733A (zh) * 2019-11-14 2020-04-10 北京科拓恒通生物技术股份有限公司 一株防治乳腺炎的鼠李糖乳杆菌及其应用
CN111733270A (zh) * 2020-08-27 2020-10-02 中国药科大学 一种检测脂多糖感染引起动脉粥样硬化风险的荧光定量pcr引物及其应用与检测试剂盒
CN114717343A (zh) * 2022-04-24 2022-07-08 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） 细菌载量作为标志物在预测鼻咽癌复发转移风险中的应用

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